2015 Research results

1. Preliminary detection method for added rice- and sugarcane-derived brewer's alcohol in bulk samples of sake by measurement of hydrogen, oxygen, and carbon isotopes

Author: Tomokazu Hashiguchi, Fumikazu Akamatsu, Hanae Izu, Tsutomu Fujii
Abstract: The δD and δ18O of sake without ethanol extraction as well as the δ13C of ethanol were determined in order to detect rice-derived brewer's alcohol. Brewing tests demonstrated that the δD decreases upon addition of brewer's alcohol (both rice- and sugarcane-derived), while the δ18O has little response to either.
Source: Bioscience, Biotechnology, and Biochemistry, 79, 1018-1020 (2015)

2. Comparison of the neurotoxicities between volatile organic compounds and fragrant organic compounds on human neuroblastoma SK-N-SH cells and primary cultured rat neurons

Author: Yasue Yamada, Kohei Ohtani, Akinori Imajo, Hanae Izu, Hitomi Nakamura, Kohei Shiraishi
Abstract: These are many volatile organic compounds (VOCs) that are synthesized, produced from petroleum or derived from natural compounds, mostly plants. Fragrant and volatile organic compounds from plants have been used as food additives, medicines and aromatherapy. Several clinical and pathological studies have shown that chronic abuse of VOCs, mainly toluene, causes several neuropsychiatric disorders. Little is known about the mechanisms of neurotoxicity of the solvents. n-Octanal, nonanal, and 2-ethyl-1-hexanol, which area used catalyzers or intermediates of chemical reactions, are released into the environment. Essential oils have the functions of self-defense, sterilization, and antibiosis in plants. When volatile organic compounds enter the body, there is the possibility that they will pass through the blood-brain barrier (BBB) and affect the central nervous system (CNS). However, the direct effects of volatile organic compounds on neural function and their toxicities are still unclear. We compared the toxicities of n-octanal, nonanal and 2-ethyl-1-hexanol with those of five naturally derived fragrant organic compounds (FOCs), linalool, cis-3-hexen-1-ol, isoamyl alcohol, n-propyl alcohol and n-phenethyl alcohol. MTT assay of human neuroblastoma SK-N-SH cells showed that the IC50 values of linalool, cis-3-hexen-1-ol, isoamyl alcohol, n-propyl alcohol and phenethyl alcohol were 1.33, 2.3, >5, >5, and 2.39 mM, respectively, and the IC50 values of toluene, n-octanal, nonanal and 2-ethyl-1-hexanol were 850, 37.2, 8.31 and 15.1 μM, respectively. FOCs showed lower toxicities than those of VOCs. These results indicate that FOCs are safer than other compounds.
Source: Toxicology Reports, 2, 729-736 (2015)

3. Mitogen-activated protein kinases MpkA and MpkB independently affect micafungin sensitivity in Aspergillus nidulans

Author: Yoshimi Akira, Fujioka Tomonori, Mizutani Osamu, Marui Junichiro, Hagiwara Daisuke, Abe Keietsu
Abstract: The transcriptional regulation of the MAPK mpkA and cell wall-related genes in Aspergillus nidulans differs from that of their counterparts in Saccharomyces cerevisiae. The A. nidulans MAPK MpkB is putatively orthologous to the yeast MAPKs Kss1p and Fus3p. To investigate MpkB and its contribution to cell wall integrity in A. nidulans, we constructed mpkB-disruptant (mpkB_△) strains. We previously showed that mpkA_△ strains exhibited reduced colony growth and increased sensitivity to the β-1,3-glucan synthase inhibitor micafungin. Like mpkA_△ strains, mpkB_△ strains exhibited slight growth retardation and increased sensitivity to micafungin. Although MpkB-dependent signaling modulated the transcription of some cell wall-related genes, the sugar composition of cell wall fractions was similar among wild-type, mpkA_△, and mpkB_△ strains. To elucidate the relationship between MpkA and MpkB pathways, we compared conditional mutants of mpkB with those with mpkA deletion. Sensitivity testing suggested that MpkA and MpkB additively contribute to micafungin activity in A. nidulans.
Source: Bioscience, Biotechnology, and Biochemistry, 79, 836-844 (2015)

4. Classification of rums by volatile composition and comparison with kokuto shochu

Author: Hisashi Fukuda, Jinshun Han
Abstract: To classify the compounds of different rums, 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 7 volatile compounds (iosoamylalcohol, ethyl linoleate, 1-hexanol, β-phenethyl acetate, citronellol, S-methylthioacetate and 2,5-dimethylpyrazine) between agricol rum and industrial rum. In a stepwise discriminant analysis procedure, 48 of 50 samples were rightly classified. Estimating the accuracy of the discriminant analysis by cross validation, the ratios of appropriate classification were 94%. In the case of white rum and gold rum, there was a significant difference in 11 volatile compounds. Fifty of 52 samples were rightly classified by 14 volatile compounds (ethyl acetate, ethyl caproate, citronellol, geraniol, nonanal, propyl caprate, isoamyl caprate, ethyl enanthate, benzaldehyde, vanillin, DMDS, ethyl valerate, diacetyl and 2-ethyl-5(6)-methylpyrazine). Estimating the accuracy of the discriminant analysis, the ratios of appropriate classification were 88%. In rum and kokuto shochu, there was a significant difference in 38 volatile compounds (β-phenethyl alcohol, isoamyl caprate, ethyl crotonate and the other 35 volatile compounds). All of samples were rightly classified by β-phenethyl alcohol, isoamyl caprate and ethyl crotonate. Estimating their accuracy, all of the rums and kokuto shochu were appropriately classified into two groups.
Source: J. Brew. Soc. Japan, 110, 261-275 (2015) in Japanese

5. Determination of 2,4,6-Trichloroanisole (TCA) in shochu by combination with headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS), and the investigation of TCA contamination in shochu

Author: Nobuhiko Mukai, Jinshun Han, Osamu Yamada, Haruyuki Iefuji
Abstract: Among various alcoholic beverages such as wine, beer, and sake, 2,4,6-trichloroanisole (TCA) is known as a main contaminator which causes a musty/muddy off-flavor. In shochu, this musty/muddy smell is also known as one of off-flavors, but there have been no analytical reports of TCA in shochu. In this study, we determined TCA in shochu by combination with headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS), with a precision limit of detection of 3 ng/L and a quantitation limit of 9 ng/L, as a result of optimizing the selection of fibers and the extraction condition. Next, contaminated TCA in shochu was investigated in shochu which were exhibited at a traditional shochu contest. In 354 samples exhibited to the contest in 2008, 9 samples contained TCA at more than the quantitation limit. Also, in 325 samples exhibited at the contest in 2009, 9 samples also contained TCA at more than the quantitation limit. Among the samples which contained high TCA, some samples were evaluated as having the musty/muddy off-flavor by many contest judges, but other samples were indicated as having the musty/muddy off-flavor by few judges in spite of the high content of TCA in the samples. These results suggest that the musty/muddy off-flavor is also caused by the contamination of TCA in shochu, but the flavor of TCA might be masked by other flavors.
Source: J. Brew. Soc. Japan, 110, 453-458 (2015) in Japanese

6. Transfer of inorganic element from rice to Japanese sake

Author: Masaki Okuda, Midori Joyo, Hisashi Fukuda, Nami Goto-Yamamoto
Abstract: The transfer of inorganic elements from rice to sake was investigated in this study. The elemental concentration of sake was determined by using ICP-AES and ICP-MS methods. The concentrations of 30 elements including Li, B, Al, Ti, Cr, Co, Ni, Ga, Ge, As, Se, Rb, Mo and Pb, which have not been reported in the previous study, were detected in the sake. Although the concentrations of most elements were higher in polished rice grain than those in water for sake mashing, the concentrations of Li,Ca,Si,Ba,Na and Sr in water for sake mashing were relatively high compared with those in polished rice grains. The transfer ratios of Na, Ca, Cs, Li, Mn, B, Si and Ga from polished rice to sake were higher than those to sake cake, and the transfer ratios of K, Rb, Mg, Sr, Ba, P, N, S, Mo, Cu, Zn, Cd, Ge, Se, As, Ti and V from polished rice to sake cake were higher than those to sake.
Source: J. Brew. Soc. Japan, 110, 357-367 (2015) in Japanese

7. Changes in mineral concentrations during sake making

Author: Masaki Okuda, Midori Joyo, Hisashi Fukuda
Abstract: The behaviors of elemental concentrations during sake making were examined by using ICP-AES, ICP-MS and Duma methods. The sakes were brewed using 100 kg of polished rice grains. The patterns of elemental concentrations in sake during mash differed among elements. The effects of washing and steeping rice, digestion of steamed rice by koji enzyme and absorption of sake yeast on the element concentrations were also examined. These steps during sake making affected the transfer ratio of elements from rice grains to sake and the sake cake.
Source: J. Brew. Soc. Japan, 110, 431-443 (2015) in Japanese

8. Awa1p on the cell surface of sake yeast inhibits biofilm formation and the co-aggregation between sake yeasts and Lactobacillus plantarum ML11-11

Author: Satoru Hirayama, Masashi Shimizu, Noriko Tsuchiya, Soichi Furukawa, Daisuke Watanabe, Hitoshi Shimoi, Hiroshi Takagi, Hirokazu Ogihara, Yasushi Morinaga
Abstract: We examined mixed-species biofilm formation between Lactobacillus plantarum ML11-11 and both foaming and non-foaming mutant strains of Saccharomyces cerevisiae sake yeasts. Wild-type strains showed significantly lower levels of biofilm formation compared with the non-foaming mutants. Awa1p, a protein involved in foam formation during sake brewing, is a glycosylphosphatidylinositol (GPI)-anchored protein and is associated with the cell wall of sake yeasts. The AWA1 gene of the non-foaming mutant strain Kyokai no. 701 (K701) has lost the C-terminal sequence that includes the GPI anchor signal. Mixed-species biofilm formation and co-aggregation of wild-type strain Kyokai no. 7 (K7) were significantly lower than K701 UT-1 (K701 ura3/ura3 trp1/trp1), while the levels of strain K701 UT-1 carrying the AWA1 on a plasmid were comparable to those of K7. The levels of biofilm formation and co-aggregation of the strain K701 UT-1 harboring AWA1 with a deleted GPI anchor signal were similar to those of K701 UT-1. These results clearly demonstrate that Awa1p present on the surface of sake yeast strain K7 inhibits adhesion between yeast cells and L. plantarum ML11-11, consequently impeding mixed-species biofilm formation.
Source: Journal of Bioscience and Bioengineering, 119, 532-537 (2015)

9. RSC Chromatin-Remodeling Complex Is Important for Mitochondrial Function in Saccharomyces cerevisiae

Author: Yuko Imamura, Feifei Yu, Misaki Nakamura, Yuhki Chihara, Kyo Okane, Masahiro Sato, Muneyoshi Kanai, Ryoko Hamada, Masaru Ueno, Masashi Yukawa, Eiko Tsuchiya
Abstract: RSC (Remodel the Structure of Chromatin) is an ATP-dependent chromatin remodeling complex essential for the growth of Saccharomyces cerevisiae. RSC exists as two distinct isoforms that share core subunits including the ATPase subunit Nps1/Sth1 but contain either Rsc1 or Rsc2. Using the synthetic genetic array (SGA) of the non-essential null mutation method, we screened for mutations exhibiting synthetic growth defects in combination with the temperature-sensitive mutant, nps1-105, and found connections between mitochondrial function and RSC. rsc mutants, including rsc1△, rsc2△, and nps1-13, another temperature-sensitive nps1 mutant, exhibited defective respiratory growth; in addition, rsc2△ and nps1-13 contained aggregated mitochondria. The rsc2△ phenotypes were relieved by RSC1 overexpression, indicating that the isoforms play a redundant role in respiratory growth. Genome-wide expression analysis in nps1-13 under respiratory conditions suggested that RSC regulates the transcription of some target genes of the HAP complex, a transcriptional activator of respiratory gene expression. Nps1 physically interacted with Hap4, the transcriptional activator moiety of the HAP complex, and overexpression of HAP4 alleviated respiratory defects in nps1-13, suggesting that RSC plays pivotal roles in mitochondrial gene expression and shares a set of target genes with the HAP complex.
Source: PLOS ONE, 10, e0130397 (2015)

10. The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts

Author: Kun Wan, Haruka Kawara, Tomoyuki Yamamoto, Kazunori Kume, Yukari Yabuki, Tetsuya Goshima, Kenji Kitamura, Masaru Ueno, Muneyoshi Kanai, Dai Hirata, Kouichi Funato, Keiko Mizuta
Abstract: The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature-sensitive fission yeast strains, rrs1-D14/22G and rrs1-L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1-84 (D22/30G) and rrs1-124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two-hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts.
Source: Yeast, 32(9), 607-614 (2015)

11. Isolation of a spontaneous cerulenin-resistant sake yeast with both high ethyl caproate-producing ability and normal checkpoint integrity

Author: Hiroyasu Tamura, Hiroki Okada, Kazunori Kume, Takayuki Koyano, Tetsuya Goshima, Ryo Nakamura, Takeshi Akao, Hitoshi Shimoi, Masaki Mizunuma, Yoshikazu Ohya, Dai Hirata
Abstract: In the brewing of high-quality sake such as Daiginjo-shu, the cerulenin-resistant sake yeast strains with high producing ability to the flavor component ethyl caproate have been used widely. Genetic stability of sake yeast would be important for the maintenance of both fermentation properties of yeast and quality of sake. In eukaryotes, checkpoint mechanisms ensure genetic stability. However, the integrity of these mechanisms in sake yeast has not been examined yet. Here, we investigated the checkpoint integrity of sake yeasts, and the results suggested that a currently used cerulenin-resistant sake yeast had a defect in spindle assembly checkpoint (SAC). We also isolated a spontaneous cerulenin-resistant sake yeast FAS2-G1250S mutant, G9CR, which showed both high ethyl caproate-producing ability and integrity/intactness of the checkpoint mechanisms. Further, morphological phenotypic robustness analysis by use of CalMorph supported the genetic stability of G9CR. Finally, we confirmed the high quality of sake from G9CR in an industrial sake brewing setting.
Source: Bioscience, Biotechnology, and Biochemistry, 79, 1191-1199 (2015)

12. Isolation and screening of biopolymer-degrading microorganisms from northern Thailand

Author: Watsana Penkhrue, Chartchai Khanongnuch, Kazuo Masaki, Wasu Pathom-aree, Winita Punyodom, Saisamorn Lumyong
Abstract: Forty agricultural soils were collected from Chiang Mai and Lampang provinces in northern Thailand. Bacteria, actinomycetes and fungi were isolated and screened for their ability to degrade polylactic acid (PLA), polycaprolactone (PCL) and poly (butylene succinate) (PBS) by the agar diffusion method. Sixty-seven actinomycetes, seven bacteria and five fungal isolates were obtained. The majority of actinomycetes were Streptomyces based on morphological characteristic, chemotaxonomy and 16S rRNA gene data. Seventy-nine microorganisms were isolated from 40 soil samples. Twenty-six isolates showed PLA-degradation (32.9 %), 44 isolates showed PBS-degradation (55.7 %) and 58 isolates showed PCL-degradation (73.4 %). Interestingly, 16 isolates (20.2 %) could degrade all three types of bioplastics used in this study. The Amycolatopsis sp. strain SCM_MK2-4 showed the highest enzyme activity for both PLA and PCL, 0.046 and 0.023 U/mL, respectively. Moreover, this strain produced protease, esterase and lipase on agar plates. Approximately, 36.7 % of the PLA film was degraded by Amycolatopsis sp. SCM_MK2-4 after 7 days of cultivation at 30°C in culture broth.
Source: World Journal of Microbiology and Biotechnology, 31, 1431-1442 (2015)

13. A study on methods for analyzing amino acid levels in sake using ethanol

Author: Akiko Fujita, Kaori Tsukamoto, Tomoko Hashimoto, Michiko Endo, Katsumi Matsumaru, Tsutomu Fujii
Abstract: For the analysis of total amino acid levels in sake, a method using deleterious formalin, namely formaldehyde aqueous solution, has been adopted in the National Tax Agency: the prescribed analysis method (NTA method). After neutral amino acids react with formaldehyde, they become acidic, then to be titrated using the alkaline solution. In recent years in Japan, formaldehyde cannot be used without local exhaust ventilation systems, such as fume hoods. Therefore, we have studied the method without formalin. When we used ethanol instead of formalin in the NTA method, the titration values showed a high correlation with the amino acid level as determined by the NTA method. Because the ethanol additional method uses almost the same instruments and operations as the NTA method, it can be expected to be utilized in sake industries.
Source: J. Brew. Soc. Japan, 110, 591-598 (2015) in Japanese

14. Analysis of Sake Components Presented to Sake Contest in 2014

Author: Tsutomu Fujii, Atsuko Isogai, Hanae Izu, Ryoko Kanda, Yasuzo Kizaki
Source: Report of the Research Institute of Brewing, 187, 1-16 (2015) in Japanese

15. Analysis of Honkaku Shochu Presented to the 37th Contest in 2014

Author: Osamu Yamada, Nobuhiko Mukai, Osamu Mizutani, Nahoko Nishibori, Genki Ono, Kazutoshi Sakamoto, Hideaki Shimizu, Yasuzo Kizaki, Yoshitsugu Iemura
Source: Report of the Research Institute of Brewing, 187, 17-26 (2015) in Japanese

16. Genetic Analysis of East Asian Grape Cultivars Suggests Hybridization with Wild Vitis

Author: Nami Goto-Yamamoto, Jason Sawler, Sean Myles
Abstract: Koshu is a grape cultivar native to Japan and is one of the country's most important cultivars for wine making. Koshu and other oriental grape cultivars are widely believed to belong to the European domesticated grape species Vitis vinifera. To verify the domesticated origin of Koshu and four other cultivars widely grown in China and Japan, we genotyped 48 ancestry informative single nucleotide polymorphisms (SNPs) and estimated wild and domesticated ancestry proportions. Our principal components analysis (PCA) based ancestry estimation revealed that Koshu is 70% V. vinifera, and that the remaining 30% of its ancestry is most likely derived from wild East Asian Vitis species. Partial sequencing of chloroplast DNA suggests that Koshu's maternal line is derived from the Chinese wild species V. davidii or a closely related species. Our results suggest that many traditional East Asian grape cultivars such as Koshu were generated from hybridization events with wild grape species.
Source: PLOS ONE, 10, e0140841 (2015)

17. Complete genome sequence of Lactobacillus acetotolerans RIB 9124 (NBRC 13120) isolated from putrefied (hiochi) Japanese sake

Author: Hidehiro Toh, Hidetoshi Morita, Hiroyuki Tsujic, Kazuhiro Iwashita, Nami Goto, Jiro Nakayama, Mitsuo Sekine, Yumiko Kate, Ken-ichiro Suzuki, Nobuyuki Fujita
Abstract: Lactobacillus acetotolerans RIB 9124 (NBRC 13120) was isolated from putrefied (hiochi) Japanese Sake. Here we report the complete genome sequence of this organism. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a L. acetotolerans strain.
Source: Journal of Biotechnology, 214, 214-215 (2015)

18. Classification of tequila by volatile composition and comparison with Kokuto shochu and rum

Author: Hisashi Fukuda, Jinshun Han
Abstract: To classify the compounds of tequila, we analyzed 79 volatile compounds by head space solid phase micro extraction (SPME), and 6 volatile compounds by direct head space analysis. In a stepwise discriminant analysis, blanco and reposado were rightly classified by 10 volatile compounds (whisky-lactone, isobutyl caproate, propyl caprate, citronellol, ethyl isovalerate, vanillin, ethyl undecanoate, ethyl oleate, ethyl isobutyrate and diethyl succinate). When estimating the accuracy of the discriminant analysis by cross validation, the ratio of an appropriate classification was 95%. In the case of reposado and anejo, all of 36 samples were rightly classified by 8 volatile compounds (isobutyl caprylate, phenylacetaldehyde, 2-pentylfuran, cedrol, acetaldehyde, furfural, 2-Ethyl-5(6)-methylpyrazine and β-phenethyl alcohol). When estimating the accuracy of the discriminant analysis, the ratio of an appropriate classification was 89%. In the case of blanco and anejo, 41 of 42 samples were rightly classified by 4 volatile compounds (whisky-lactone, ethyl benzoate, ethyl caprylate and phenylacetaldehyde). When estimating the accuracy of the discriminant analysis, the ratio of an appropriate classification was 95%. In rum, kokuto shochu, and tequila, there was a significant difference in 34 volatile compounds (α-terpineol, 5-methyl-2-furaldehyde, dodecanol, ethyl benzoate, isoamyl caprate and the other 29 volatile compounds). In rum and tequila, almost all samples were rightly classified by 5 volatile compounds (α-terpineol, 5-methyl-2-furaldehyde, dodecanol, ethyl benzoate and isoamyl caprate). When estimating the accuracy of it, 111 of 114 samples were classified. When estimating the accuracy of the discriminant analysis, the ratio of an appropriate classification was 97%.
Source: J. Brew. Soc. Japan, 110, 715-727 (2015) in Japanese

19. Determination of Wheat Protein in Beer by FASPEK Wheat/Gluten (Gliadin) ELISA Kit II

Author

Takayuki Watanabe, Takashi Akiba, Ritsuko Arai, Yusuke Ishizuka, Makoto Kanauchi, Masaru Kato, Kazutaka Kusaka, Kaori Kuwahara, Tsuyoshi Miyagi, Satoko Mori, Atsushi Ohuchi, Soutaro Tatsu

Abstract

Relative repeatability standard deviation (RSDr) and relative reproducibility standard deviation (RSDR) for determination of wheat protein content using FASPEK Wheat/Gluten(Gliadin) ELISA Kit II ranged from 2.0 to 4.7 % and from 7.5 to 20.1 %, respectively, and were judged acceptable.
Recovery of wheat protein was 83.8 %, and was judged acceptable.

Source

Journal of the American Society of Brewing Chemists, 73, 370-371 (2015)