2017 Research results

1. 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)

2. 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)

3. 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)

4. 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)

5. 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

6. 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)

7. 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)

8. 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)

9. 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)

10. Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Author: Kazuya Tomimoto, Takeshi Akao, Hisashi Fukuda
Abstract: Screening of drug-resistant mutants of sake yeast strains has been a major method for creation of superior strains. We attempted to create a valproic acid (VPA)-resistant mutant strain from sake yeast Kyokai No. 7 (K7). VPA is a branched-chain fatty acid and is an inositol synthesis inhibitor in mammals and yeast. We succeeded in isolating a mutant of strain K7 that can survive long-term in a VPA-containing medium. This strain, K7-VPA^LS, is significantly more resistant to not only VPA-induced cell death but also ethanol in comparison with the parent strain. Further experiments showed that the new strain is likely to have a deficiency in inositol and/or phosphatidylinositol synthesis. The major characteristics of sake brewed by strain K7-VPA^LS (compared with K7) were lower amino acidity, higher isoamyl acetate content without an increase in the isoamyl alcohol level and changes in constituent organic acids, particularly higher malate and succinate but lower acetate concentrations. In addition, taste sensor analysis revealed that K7-VPA^LS-brewed sake has milder sourness and higher saltiness or richness than K7-brewed sake does. High isoamyl acetate production may be related to a deficiency in phosphatidylinositol because this compound directly inhibits alcohol acetyltransferase, an enzyme responsible for isoamyl acetate synthesis. Strain K7-VPA^LS grew more rapidly than the parental strain did in a medium containing acetate as a sole carbon source, indicating that K7-VPA^LS effectively assimilates acetate and converts it to malate and succinate through the glyoxylate cycle. Thus, strain K7-VPA^LS shows improved characteristics for brewing of high-quality sake.
Source: Journal of the Institute of Brewing, 123, 252-258 (2017)

11. A correlation analysis of the compounds in Awamori and sweet potato shochu

Author: Hisashi Fukuda, Jinshun Han, Osamu Mizutani, Muneyoshi Kanai, Osamu Yamada
Abstract: Eighty-four volatile compounds of Awamori and sweet potato shochu were analyzed by head space solid phase micro extraction (SPME) and direct head space analysis, and the correlation between the compounds was analyzed. A high correlation between the medium-chain fatty acid esters and between higher fatty acid esters was generally shown, and the similarity of the chemical structure and biosynthesis was speculated to this contribute to correlation. In monoterpene alcohol and low boiling point ester, the compounds showed a high correlation and a synthesis path was expected. Surprisingly, furfural correlated with some of the aldehyde compounds. In addition, in Awamori and sweet poatato shochu, the compounds showing a high correlation were given individual consideration.
Source: J. Brew. Soc. Japan, 112, 273-314 (2017) in Japanese

12. Comparison the volatile composition of shochu exhibited in the Honkaku Shochu Contontest by used koji

Author: Hisashi Fukuda, Jinshun Han, Osamu Yamada
Abstract: Shochu exhibited in the Honkaku Shochu Contest was classified according to kinds of used koji, and 84 volatile compounds in it were attempted to be compared by koji category. Though there was a significant difference in some volatile compounds of shochu between used koji, it was expected to be due to materials. Then, as sample bias of sweet potato shochu among shochu exhibited was small compared to it of the other shochu, volatile compounds of sweet potato shochu were examined the effect of used koji. In sweet potato shochu, there was a significant difference in 13 volatile compounds between white koji products and black koji products. In a stepwise discriminant analysis procedure, white koji products and black koji products were classified by 6 volatile compounds (1-hexanol, 1-octen-3-ol, isobutyl caproate, ethyl myristate, α-bisabolol, and S-methyl thioacetate). Estimating the accuracy of the discriminant analysis by cross validation, the ratios of appropriate classification were 83.0%.
Source: J. Brew. Soc. Japan, 112, 343-349 (2017) in Japanese

13. Characteristics of the volatile composition of Japanese rum

Author

Hisashi Fukuda, Jinshun Han

Abstract

To classify Japanese rum (n=18) and imported rum (n=32), we analyzed 78 volatile compounds by solid phase micro extraction (SPME), and 6 volatile compounds by direct head space analysis. There was a significant difference in 34 volatile compounds between Japanese rum and rum made in Caribbean countries and bordering the Indian Ocean. In a stepwise discriminant analysis procedure using 6 volatile compounds (1-hexanol, guaiacol, diethyl succinate, 2-methylbutyl acetate, acetaldehyde and furfural), all of 50 samples were rightly classified. Estimating the accuracy of the discriminant analysis by cross validation, the ratios of appropriate classification were 100 %.

To test the robustness of a discrimination coefficient obtained, a discrimination analysis of Japanese rum and different imported rum (n=40) provided the ratios of appropriate classification at 92.5 %. We believe that Japanese rum can be distinguished from imported rum with relatively high precision by the discriminant analysis. In addition, the production process of Japanese rum speculated from compounds was given consideration.

Source

J. Brew. Soc. Japan, 112, 423-431 (2017) in Japanese

14. Factors affecting dimethyl trisulfide formation in wine

Author: Nahoko Nishibori, Asami Kuroda, Osamu Yamada, Nami Goto-Yamamoto
Abstract: Dimethyl trisulfide (DMTS), which develops in wine during storage, creates unpleasant aromas. However, mechanisms of DMTS formation in the wine-making process are poorly understood. Here we attempted to identify several factors that affect DMTS development during storage. Results show that the death of yeast cells followed by the leakage of cell contents into must enhances DMTS formation, but this effect differs with the kind of grapes. Oxidation of must as well as insufficient clarification of grape juice can also lead to DMTS development during storage. DMTS-P1 and methanethiol, precursor compounds of DMTS in other beverages, made little contribution to DMTS formation in wine. When synthetic grape must was fermented instead of grape juice, less DMTS developed, although almost yeast cells were dead. Results indicated that unknown compounds contained in grape juice are involved in DMTS formation and that key compounds involved in DMTS formation in wine originate from grapes.
Source: Food Science and Technology Research, 23, 241-248 (2017)

15. Analysis of Sake Components Presented to Sake Contest in 2016

Author: Tsutomu Fujii, Atsuko Isogai, Sachiko Iizuka, Hanae Izu, Ryoko Kanda, Nami Goto-Yamamoto
Source: Report of the Research Institute of Brewing, 189, 1-16 (2017) in Japanese

16. Review of the 39th Honkaku Shochu Contest in 2016

Author: Osamu Yamada, Nobuhiko Mukai, Muneyoshi Kanai, Osamu Mizutani, Nahoko Nishibori, Akinori Muto, Takeshi Akao, Maki Kamimoto, Nami Goto-Yamamoto
Source: Report of the Research Institute of Brewing, 189, 17-30 (2017) in Japanese

17. Anasysis of Beer Components Presented to Craft Beer Contest of Japan Brewers Association in 2016

Author: Kazutaka Kusaka, Akinori Muto
Source: Report of the Research Institute of Brewing, 189, 31-36 (2017) in Japanese

18. Shochu Sensory Assessmen

Author: Nobuhiko Mukai, Jinshun Han, Nahoko Nishibori, Muneyoshi Kanai
Source: Report of the Research Institute of Brewing, 189, 37-43(2017) in Japanese

19. Effects of low dose of ethanol on the senescence score, brain function and gene expression in senescence-accelerated mice 8 (SAMP8)

Author: Akiko Kimoto, Hanae Izu, Churan Fu, Sofya Suidasari, Norihisa Kato
Abstract: Accumulating epidemiological evidence suggests light to moderate alcohol intake reduces risk of several chronic diseases. However, there is limited information regarding the effects of low alcohol intake in animal studies. This study investigated the effect of low ethanol dosage on senescence-accelerated mouse (SAMP8), an animal model of aging and neurodegenaration. Male SAMP8 mice (11 weeks old) had free access to a commercial stock diet with drinking water containing 0%, 1% or 2% (v/v) ethanol for 15 weeks. The total grading score of senescence in the 1%-ethanol group was, in large part, the lowest among the three groups. Analysis using the open-field test revealed a significant elevation (+77%, P < 0.05) in the rearing activity (index of seeking behavior) in the 1%-ethanol group, but not in the 2%-ethanol group. In addition, 2% ethanol elevated spontaneous locomotor activity (+75%, P < 0.05), whereas 1% ethanol did not. Scrutiny of serum parameters indicated intake of 1% ethanol significantly decreased serum insulin levels (-13%, P < 0.05), whereas 2% did not. Intake of 2% ethanol significantly elevated (2.5-fold, P < 0.05) S100a8 mRNA (an inflammatory signal) in the brain, but that of 1% ethanol did not. Intriguingly, 1% ethanol intake remarkably elevated (10-fold, P < 0.05) mRNA of brain alcohol dehydrogenase 1 (Adh1), which metabolizes lipid-peroxidation products and is involved in the synthesis of retinoic acid, a neuroprotective factor. Of note, 2%-ethanol intake did not exert this effect. Taken together, intake of 1% ethanol is likely to be beneficial for SAMP8 mice.
Source: Experimental and Therapeutic Medcine, 14, 1433-1440 (2017)

20. Carbon Stable Isotope Analysis for Glucose in Sake: Simple Freeze-Dried Sake Can Substitute for Glucose Following HPLC Isolation

Author: Fumikazu Akamatsu, Tomokazu Hashiguchi, Yukari Igi, Hanae Izu, Tsutomu Fujii
Abstract: Carbon stable isotope analysis of ethanol is an established method for determining the authenticity of alcoholic beverages, but there is no accepted method for measurements of glucose carbon stable isotopic composition (δ¹³C). This study aims to establish two methods to determine accurately the δ¹³C value of glucose isolated from sake by high-performance liquid chromatography (HPLC) after three clean-up steps using ion exchange chromatography and in simple freeze-dried sake (total nonvolatile matter) and to examine two methods that yield different δ¹³C values. In the glucose isolation procedure, the δ¹³C values of glucose were not significantly affected by the glucose concentration, solvent (water or aqueous acetonitrile), drying method (freeze-dry or nitrogen-spraying), the clean-up step using ion exchange chromatography (weakly acidic cation-exchange, strongly acidic cation-exchange, and weakly basic anion-exchange resins), or HPLC isolation. Glucose nitrogen spray-dried after isolation by ion exchange chromatography and HPLC showed a carbon isotope discrimination value of <0.1 ‰. The glucose δ¹³C values ranged from -26.8 to -25.5 ‰ in authentic sake and from -27.0 to -11.0 ‰ in commercial sake samples. This HPLC isolation approach may provide a first step toward visualizing the brewing process via isotopic carbon flow during fermentation. Additionally, there was no significant difference between the δ¹³C values of glucose and total nonvolatile matter from sake. The δ¹³C measurement of simple freeze-dried sake can substitute for glucose δ¹³C measurement using HPLC isolation, thus aiding high-throughput detection of carbon derived from C₄ plants in sake.
Source: Food Analytical Methods, 10, 2792-2799 (2017)

21. Application of Carbon and Hydrogen Stable Isotope Analyses to Detect Exogenous Citric Acid in Japanese Apricot Liqueur

Author: Fumikazu Akamatsu, Takaaki Oe, Tomokazu Hashiguchi, Yuri Hisatsune, Takafumi Kawawo, Tsutomu Fujii
Abstract: Japanese apricot liqueur manufacturers are required to control the quality and authenticity of their liqueur products. Citric acid made from corn is the main acidulant used in commercial liqueurs. In this study, we conducted spiking experiments and carbon and hydrogen stable isotope analyses to detect exogenous citric acid used as an acidulant in Japanese apricot liqueurs. Our results showed that the δ¹³C values detected exogenous citric acid originating from C4 plants but not from C3 plants. The δ²H values of citric acid decreased as the amount of citric acid added increased, whether the citric acid originated from C₃ or C₄ plants. Commercial liqueurs with declared added acidulant provided higher δ¹³C values and lower δ²H values than did authentic liqueurs and commercial liqueurs with no declared added acidulant. Carbon and hydrogen stable isotope analyses are suitable as routine methods for detecting exogenous citric acid in Japanese apricot liqueur.
Source: Food Chemistry, 228, 297-300 (2017)

22. Evaluation of method bias for determining bacterial populations in bacterial community analyses

Author: Masayuki Takahashi, Yasuko Kita, Akihiro Mizuno, Nami Goto-Yamamoto
Abstract: Various methods are used for analyzing a bacterial community. We recently developed a method for quantifying each bacterium constituting the microbiota by combining a next-generation sequencing (NGS) analysis with a quantitative polymerase chain reaction (NGS-qPCR) assay. Our NGS-qPCR method is useful for analyzing a comprehensive bacterial community because it is enables the easy calculation of the amounts of each bacterium constituting the microbiota. However, it has not been confirmed whether the estimated bacterial community obtained using this NGS-qPCR method corresponds to the results obtained using conventional methods. Accordingly, we prepared model bacterial community samples and analyzed them by several methods (NGS-qPCR, species-specific qPCR, flow cytometry, total direct counting by epifluorescent microscopy [TDC], and plate count). The total bacterial cell densities determined by the PCR-based methods were largely consistent with those determined by the TDC method. There was a difference between the amounts of each bacterium analyzed by NGS-qPCR and species-specific qPCR, although the same trend was shown by both species-specific qPCR and NGS-qPCR. Our findings also demonstrated that there is a strong positive correlation between the cell densities of a specific bacterial group in craft beer samples determined by group-specific qPCR and NGS-qPCR, and there were no significant differences among quantification methods (we tested two bacterial groups: lactic acid bacteria and acetic acid bacteria). Thus, the NGS-qPCR method is a practical method for analyzing a comprehensive bacterial community based on a bacterial cell density.
Source: Journal of Bioscience and Bioengineering, 124(4), 476-486 (2017)

23. Phenotypic Diagnosis of Lineage and Differentiation During Sake Yeast Breeding

Author: Shinsuke Ohnuki, Hiroki Okada, Anne Friedrich, Yoichiro Kanno, Tetsuya Goshima, Hirokazu Hasuda, Masaaki Inahashi, Naoto Okazaki, Hiroyasu Tamura, Ryo Nakamura, Dai Hirata, Hisashi Fukuda, Hitoshi Shimoi, Katsuhiko Kitamoto, Daisuke Watanabe, Joseph Schacherer, Takeshi Akao, and Yoshikazu Ohya
Abstract: Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomyces cerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies.
Source: G3:Genes,Genomes,Genetics, 7 (8), 2807-2820 (2017)

24. Effects of moisture and pulverizing of rice on the absorption rate of rice grains used for awamori: improvement of the absorption ratio of steamed rice by two steamings

Author: Masaki Okuda, Midori Joyo, Hisashi Fukuda, Takako Tamamura
Abstract: Thai non-broken rice has been mainly used for the production of awamori after 2010. It is more difficult to absorb water ideally than for Thai broken rice, which has been mainly used for a long time. This difficulty in absorption for Thai non-broken rice has the recently demanded on immediate solution. In the present study, to obtain the basic knowledge for improving the absorption of Thai non-broken rice, we examined the suitability of Thai rice used for awamori production compared with Nipponbare, which is used for sake making. The property of Thai rice starch by RVA analysis was significantly different from that of Nipponbare. The water absorption ratio of rice showed a negative correlation with the moisture of rice grains in both Thai rice and Nipponbare. However, the ratio of water absorption of Thai rice was lower than that of Nipponbare, when the moisture of the rice was same. The speed of water absorption was significantly improved by pulverizing the rice grains when the steeping time was short. Two steamings also significantly improved the ratio of water absorption of steamed rice and enzyme digestibility. From the above results, we recommend that prevention of an increase in the moisture of rice grains is important to deal with Thai rice appropriately. Also, sufficient steeping time, or two steamings are effective to achieve the ideal ratio of water absorption when using Thai rice.
Source: J. Brew. Soc. Japan 112, 629-640 (2017) in Japanese

25. Chloride-free and water-soluble Au complex for preparation of supported small nanoparticles by impregnation method

Author: Haruno Murayama, Takayuki Hasegawa, Yusuke Yamamoto, Misaki Tone, Moemi Kimura, Tamao Ishida, Tetsuo Honma, Mitsutaka Okumura, Atsuko Isogai, Tsutomu Fujii, Makoto Tokunaga
Abstract: A novel simple and easy impregnation method for preparation of small gold (Au) nanoparticle (NP) catalysts (<3 nm) deposited on various supports including silica, which is difficult to be applied for conventional methods, has been developed. Chloride-free and water-soluble precursor, Au complexes coordinated with β-alanine, were successful for the preparing Au NPs, which exhibited an average diameter less than 3 nm. Thermal behavior of the Au complex was investigated by TG-DTA and in situ XAFS. XAFS analyses and DFT calculations revealed a molecular structure of the Au complex to be square-planar coordination structure and mononuclear complex of Au³⁺. Lower decomposition and reduction temperature of the chloride-free Au complex prevented Au atoms from aggregating and from following growth of Au particles. The prepared Au/SiO2 showed high selectivity for hydrogenation of 1-nitro-4-vinylbenzene into 1-ethyl-4-nitorobenzene and good performance for removal of unpalatable aroma by means of adsorption of polysulfide molecules.
Source: Journal of Catalysis, 353, 74-81 (2017)

26. Investigation of the content of nutrients and functional ingredients in shochu distillation residue

Author: Nobuhiko Mukai, Tomoko Morimoto, Narumi Fuke, Satoko Yoshida, Tsutomu Kumazaki, Takumi Teramoto, Daiki Uesako, Ryuichi Sato, Nahoko Nishibori, Muneyoshi Kanai, Osamu Yamada, Tsutomu Fuji
Abstract: Shochu distillation residue (SDR), generated as a shochu distillery by-product, includes abundant nutrients. We collected 54 samples of SDR made from various raw materials from 30 shochu manufacturers. SDR was separated into cake and liquid parts by centrifuges. The cake ratio of SDR was 28.0% on average, and the cake of SDR contained 79.1 g/100 g of moisture on average. The cake of SDR included 32.1 g of protein, 12.7 g of lipid, 51.7 g of carbohydrate, and 3.4 g of ash, 450.1 kcal of calorific value, 381.1 mg of folate (the sum of tetrahydrofolic acid (THF) and 5-methyltetrahydrofolate (5-MTHF)), 0.72 mg of vitamin B₆, 14.5 mg of S-adenosylmethyonine (SAM) and 9.1 mg of polyamines (the sum of putrescine (PUT), spermidine (SPD) and spermine (SPM)) at an average level per 100 g (dry weight). The liquid part of SDR included 10.3 mg of folate, 0.15 mg of vitamin B₆, 3.0 mg of SAM and 1.3 mg of polyamines at an average level per 100ml. It was revealed that SDR contains relatively large amounts of folate, vitamin B₆, SAM and polyamines in comparison with other foods and drinks. These results can lead to new utilizations and value-added improvements for SDR.
Source: J. Brew. Soc. Japan 112, 695-706 (2017) in Japanese

27. Chromosomal aneuploidy improves brewing characteristics of sake yeas

Author: Masafumi Kadowaki, Yuki Fujimaru, Seiga Taguchi, Jannatul Ferdouse, Kazutaka Sawada, Yuta Kimura, Yohei Terasawa, Gennaro Agrimi, Toyoaki Anai, Hideki Noguchi, Atsushi Toyoda, Asao Fujiyama, Takeshi Akao, Hiroshi Kitagaki
Abstract: The effect of chromosomal aneuploidy on the brewing characteristics in brewery yeasts has not been studied. Here, we report that chromosomal aneuploidy in sake brewery yeast (Saccharomyces cerevisiae) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondria-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids and this effect was canceled in their petite strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number including that in chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile.

IMPORTANCE

Chromosomal aneuploidy has not been evaluated in development of sake brewing yeast strains. This is the first study to show the relationship between chromosomal aneuploidy and brewing characteristics of brewery yeast strains. High concentrations of pyruvate during sake storage give rise to α-acetolactate and in turn to high diacetyl, which is considered an off-flavor. It was demonstrated that pyruvate-underproducing sake yeast is trisomic for chromosome XI and XIV. Furthermore, sake yeast haploids with extra chromosomes produced reduced levels of pyruvate and showed metabolic processes characteristic of increased mitochondrial activity. This novel discovery will enable the selection of favorable brewery yeasts by monitoring the copy numbers of specific chromosomes through a process that does not involve generation/use of genetically modified organisms.
Source: Applied and Environmental Microbiology, 83 (24), e01620-17 (2017)

28. Characterization of amylolytic enzyme overproducing mutant of Aspergillus luchuensis obtained by ion beam mutagenesis

Author: Toshihiro Kojo, Chihiro Kadooka, Marisa Komohara, Shiori Onitsuka, Miya Tanimura, Yukiko Muroi, Shugo Kurazono, Yohei Shiraishi, Ken Oda, Kazuhiro Iwashita, Masahira Onoue, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Taiki Futagami, Kazuki Mori, Hisanori Tamaki
Abstract: Aspergillus luchuensis is a kuro (black) koji fungus that has been used as a starch degrader for the awamori- and shochu-making industries in Japan. In this study, we investigated the effect of ion beam irradiation on A. luchuensis RIB2601 and obtained a high starch-degrading mutant strain U1. Strain U1 showed reduced growth rate, whereas it showed higher α-amylase, glucoamylase, and α-glucosidase activities on a mycelial mass basis than the wild type (wt) strain both on agar plates and in rice koji. In addition, strain U1 showed higher N-acetylglucosamine content in the cell wall and higher sensitivity to calcofluor white, suggesting a deficiency in cell wall composition. Interestingly, produced protein showed higher expression of acid-labile α-amylase (AmyA) and glucoamylase (GlaA) in strain U1, although real-time RT-PCR indicated no significant change in the transcription of the amyA or glaA gene. These results suggested that the high amylolytic activity of strain U1 is attributable to a high AmyA and GlaA production level, but the elevated production is not due to transcriptional regulation of the corresponding genes. Furthermore, RNA-seq analysis indicated that strain U1 shows transcriptional changes in at least 604 genes related to oxidation-reduction, transport, and glucosamine-containing compound metabolic processes, which may be involved in the deficient cell wall composition of strain U1.
Source: The Journal of general and applied microbiology, doi10.2323/jgam.2017.02.004