National Research Institute of Brewing
独立行政法人 酒類総合研究所

2019 Research results

1. Glutelin subtype-dependent protein localization in rice grain evidenced by immunodetection analyses.

Author
Kei Takahashi, Hiromi Kohno, Tomomichi Kanabayashi, and Masaki Okuda
Abstract

Key message

GluA and GluB-4/5 glutelin subfamilies are mainly localized to outer region of the endosperm, particularly in its ventral side, in rice grain, but GluC is localized to throughout the endosperm.

The major seed storage protein in rice (Oryza sativa) is glutelin, which forms a vacuole-derived protein body type-II. Glutelins are encoded by multiple genes, and generally comprise four protein subfamilies, namely, GluA, GluB, GluC, and GluD: however, the localization pattern of glutelin in rice grains remains obscure. In this study, we investigated the localization pattern of five subtypes of the glutelin protein in rice grains using glutelin-subtype specific antibodies. Immunoblot analysis against sequentially polished rice flour fractions from three crop years and seven japonica rice varieties revealed that GluA was strongly localized in the outer region of the endosperm, including the subaleurone layer, whereas GluC was distributed throughout the endosperm. Among the glutelin subtypes, GluA was mostly found in the outer region of the rice grain, followed by GluB-4/5, GluB-1, GluD, and GluC. Immunofluorescence labeling microscopy analysis using immature rice seeds clearly revealed that the localization pattern of GluC and GluD was completely different from that of GluA and GluB. Expression levels of all glutelins, particularly GluA, GluB-1, and GluB-4/5, were stronger on the ventral than dorsal side in rice grains. These results provide strong and consistent evidence that glutelins localize to the rice grain in a subfamily-dependent manner.

Source
Plant Molecular Biology, 100, 3, 231-246 (2019)

2. Anti-diabetic effect of S-adenosylmethionine and α-glycerophosphocholine in KK-Ay mice

Author
Hanae Izu, Mayumi Okuda, Sachi Shibata, Tsutomu Fujii, and Kiminori Matsubara
Abstract
Six-week-old male KK-Ay mice received drinking water with S-adenosylmethionine (SAM), α-glycerophosphocholine (GPC), or SAM+GPC for 10 weeks. The serum glucose of SAM+GPC at 15 weeks old, total cholesterol of GPC at 12 weeks old, and triglyceride of GPC at 15 weeks old and of SAM at 16 weeks old were reduced. SAM+GPC reduced serum leptin and food intake.
Source
Biosci Biotechnol Biochem. 83(4), 747-750 (2019)

3. Analysis of Sake Components Presented to Sake Contest in 2018

Author
Tsutomu FUJII, Akiko FUJITA, Sachiko IIZUKA, Hanae IZU, Ryoko KANDA, Toru KISHIMOTO, Nami GOTO-YAMAMOTO
Source
Report of the Research Institute of Brewing, 191, 1-17 (2019) in Japanese

4. Review of the 41st Honkaku Shochu and Awamori Contest in 2018

Author
Nobuhiko MUKAI, Atsuko ISOGAI, Muneyoshi KANAI, Masayuki TAKAHASHIO, Yukio OSAFUNE, Akinori MUTO, Toshihiro YOSHIMURA, and Nami GOTO-YAMAMOTO
Source
Report of the Research Institute of Brewing, 191, 18-36 (2019) in Japanese

5. Analysis of Beer Components Presented to Craft Beer Contest of Japan Brewers Association in 2019

Author
Shinichi ITO, Kazutaka KUSAKA
Source
Report of the Research Institute of Brewing, 191, 37-43 (2019) in Japanese

6. Characteristics of the starch and protein composition of rice grains used for awamori

Author
Masaki Okuda, Midori Joyo, Hisashi Fukuda, Takako Tamamura
Abstract
Recently, the properties of Thai non-broken rice for the production of awamori have varied significantly among shipments imported from Thailand, and these variations make the production of awamori difficult. In the present study, we investigated the starch and protein properties of Thai rice used for awamori production compared with Nipponbare, which is used as a cultivar for sake making, because these properties have the possibilities of affecting the water absorption ratio and enzyme digestibility of steamed rice grains. The results of individual alkali solubility testing and DSC (= Differential Scanning Calorimeter) analyzing showed that the shipments of Thai rice have mixtures of different properties in gelatinization temperature and alkali solubility. The Thai rice of high gelatinization temperature had a lower content of short-chain (DP6-8 = Degree of polymerization ) of amylopectin structure than that of low temperature gelatinization and Nipponbare. This result indicated that the difference of the amylopectin structure was due to the starch synthase IIa activity from a previous report. The starch in Thai rice with a high gelatinization temperature retrogradated faster than that with a low gelatinization temperature. The analysis of protein composition showed no clear difference between the Thai rice with high gelatinization temperature and that with low gelatinization temperature although intense 60kDa protein bands were observed in Thai rice. From the above results, the mixture of cultivars in Thai rice may influence treatments of materials for the production of awamori.
Source
J. Brew. Soc. Japan, 114,585-595 (2019)

7. Effect of organic acids in alcoholic drinks on the umami of food paired with alcoholic drinks

Author
Akiko FUJITA, Kenichi TOMIHARA, Yuri HISATSUNE, Yukio HAMADA, Tsutomu FUJII
Abstract
While cheese is widely known to pair well with wine, it is presently also consumed as a complement to sake in Japan. Cheese contains high levels of umami constituents such as amino acids and peptides. Its rich taste decreases sensorially in pairing with wine in comparison with sake. In order to confirm this fact, the intensity of the aftertaste of some kinds of food in paring with alcoholic drinks was measured by instrumental taste sensor analysis. Intensity of the umami aftertaste of cheese, miso soup, noodle soup, monosodium glutamate, and a kokumi seasoning was lower in paring with white wines than with sake. Among the major chemical constituents that distinguish wine from sake, organic acids such as tartaric acids and malic acids reduced umami aftertaste intensity. In paired comparison tests conducted for a pair of sake samples with and without tartaric acid, more panelists selected the sake without tartaric acid as the sample which left a stronger umami aftertaste of cheese. These results suggest that organic acids in alcoholic drinks reduce umami aftertaste derived from amino acids and peptides of food in food and alcohol pairings.
Source
J. Brew. Soc. Japan, 114(8), 522-529 (2019)

8. Nutritional characterization of sake cake (sake-kasu) after heat-drying and freeze-drying.

Author
Hanae IZU, Sayo YAMASHITA, Hideyuki ARIMA, Tsutomu FUJII
Abstract
Sake cake contains rice-derived components, as well as cell components and metabolites of Aspergillus oryzae and Saccharomyces cerevisiae. In this study, the effect of food processing on sake cake (sake-kasu) ingredients was investigated. Sake cake, obtained through brewing liquefied rice, was heat-dried (HD) or freeze-dried (FD) and analyzed. There were no differences in the amounts of proteins, lipids, carbohydrates, vitamin B6, choline, betaine, nicotinic acid, β-glucan and resistant proteins in HD and FD. There was also no difference in the amount of hydrolyzed amino acids in HD and FD, but many free amino acids were observed in HD. S-adenosyl methionine (SAM) was found to be abundant in FD. Meanwhile, nucleic acid-related components were found to be increased in HD, which seems to be due to the degradation of microbial metabolites. When considering the health benefits of sake cake, it is necessary to pay attention to the effects of processing method.
Source
Biosci Biotechnol Biochem. 83(8), 1477-1483 (2019)

9. Sake cake (sake-kasu) ingestion increases branched-chain amino acids in the plasma, muscles, and brains of senescence-accelerated mice prone 8

Author
Hanae IZU, Sachi SHIBATA, Tsutomu FUJII, and Kiminori MATSUBARA
Abstract
To examine metabolic effects of sake cake ingestion, plasma and tissues were analyzed in senescence-accelerated mice prone 8 (SAMP8) fed a sake cake diet. As a result, branched-chain amino acids (BCAA) were found to be significantly higher in the plasma, gastrocnemius muscles and brains of the sake cake group than in the control group. Mice in the sake cake group showed stronger grip strength than the control group. High levels of circulating BCAA have been reported to be associated with pathological states, such as metabolic diseases, but the parameters of glucose and lipid metabolism were not affected between the two groups. Otherwise, pyridoxal was significantly higher and nicotinamide as well as 1-methylnicotinamide showed a tendency to be higher in the plasma of the sake cake group than in the control group. These findings indicate that intake of sake cake increases the levels of BCAA, vitamin B6, and vitamin B3.
Source
Biosci Biotechnol Biochem. 83(8), 1490-1497 (2019)

10. Histone deacetylases in sake yeast affect fermentation characteristics

Author
Kazuya Tomimoto, Takeshi Akao, Hisashi Fukuda
Abstract
Yeast histone deacetylases (HDAC) affect the production of alcoholic beverages. In this study, we evaluated the sake fermentation characteristics when using HDAC gene-disrupted yeast strain Kyokai No. 701. Flavor components of the sake product were significantly changed. RPD3 or HDA1 disruption increased twofold the amount of isoamyl acetate, and isoamyl alcohol levels also increased in the rpd3△ strain. To determine the contribution of Rpd3L and Rpd3S complexes to sake characteristics, a gene responsible for Rpd3L and/or Rpd3S formation was also disrupted. Disruption of DEP1 or SDS3 that is an essential component of Rpd3L led to increased isoamyl alcohol production similar to that of the rpd3△ strain, but the efficiency of isoamyl alcohol esterification was not affected. In addition, Rpd3 and Hda1 may regulate the responsiveness to oxygen in isoamyl acetate production. We conclude that HDAC genes regulate the production of flavor components during sake fermentation.
Source
Bioscience, Biotechnology, and Biochemistry, 83 (8), 1498-1505 (2019)

11. SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae

Author
Koji Masumura, Sachi Matsukami, Kumiko Yonekita, Muneyoshi Kanai, Kazunori Kume, Dai Hirata & Masaki Mizunuma
Abstract
Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.
Source
Bioscience, Biotechnology, and Biochemistry, 83(8), 1473-1476 (2019)

12. Analysis of metabolites in Japanese alcoholic beverage sake made from the sake rice Koshitanrei

Author
Eri ICHIKAWA, Shougo HIRATA, Yuko HATA, Hisashi YAZAWA, Hiroyasu TAMURA, Mitsuoki KANEOKE, Kazuhiro IWASHITA, Dai HIRATA
Abstract
In sake brewing, the steamed rice is used in two ways, added to sake-mash (as kake-mai) and making koji. The rice is an important determinant for the quality of sake, as the metabolites in sake affect its taste/aroma. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by genetically crossing two sake rice, Gohyakumangoku and Yamadanishiki. However, the metabolites in sake from KOS have not been analyzed. Here, to investigate the characteristic metabolites in sake from KOS, we performed two types of small-scale sake fermentation tests changing only the rice used for kake-mai or total rice (both kake-mai and koji) by these three rice cultivars and examined the effect of KOS on sake metabolites by the metabolome analysis method using UPLC-QTOF-MS. We identified the peaks/metabolites, whose intensity in sake from KOS was higher/lower than those from the other cultivars. The brewing properties of KOS were partially characterized by this analysis.
Source
Bioscience, Biotechnology, and Biochemistry, 83, 1570-1582 (2019)

13. Genome editing to generate nonfoam-forming sake yeast strains

Author
Shunsuke Onuki, Mao Kashima, Toshikazu Yamada, Farzan Ghanegolmohammadi, Yan Zhou, Tetsuya Goshima, Jun-ichi Maruyama, Katsuhiko Kitamoto, Dai Hirata, Takeshi Akao, Yoshikazu Ohya
Abstract
Mutations frequently occur during breeding of sake yeasts and result in unexpected phenotypes. Here, genome editing tools were applied to develop an ideal nonfoam-forming sake yeast strain, K7GE01, which had homozygous awa1△/awa1△ deletion alleles that were responsible for nonfoam formation and few off-target mutations. High-dimensional morphological phenotyping revealed no detectable morphological differences between the genome-edited strain and its parent, while the canonical nonfoam-forming strain, K701, showed obvious morphological changes. Small-scale fermentation tests also showed differences between components of sake produced by K7GE01 and K701. The K7GE01 strain produced sake with significant differences in the concentrations of ethyl acetate, malic acid, lactic acid, and acetic acid, while K701 produced sake with more differences. Our results indicated genuine phenotypes of awa1△/awa1△ in sake yeast isolates and showed the usefulness of genome editing tools for sake yeast breeding.
Source
Bioscience, Biotechnology, and Biochemistry, 83 (8), 1583-1593 (2019)

14. Breeding of a cordycepin-resistant and adenosine kinase-deficient sake yeast strain that accumulates high levels of S-adenosylmethionine

Author
Muneyoshi Kanai, Norito Yasuda, Tomoko Morimoto, Satoko Yoshida, Nahoko Nishibori, Masaki Mizunuma, Tsutomu Fujii & Haruyuki Iefuji
Abstract
Adenosine kinase (ADO1)-deficient mutants can be obtained from cordycepin-resistant strains, and the disruption of ADO1 causes S-adenosylmethionine (SAM) accumulation. To breed a high-SAM-accumulating yeast strain without genetic manipulation for industrial purposes, we bred a cordycepin-resistant strain using sake yeast kyokai No. 9 as the parent strain with a mutation in adenosine kinase (ADO1) and acquired high-SAM-accumulating strain. In the bred strain (NY9-10), a single mutation (T258I) was present in the ADO1, and this mutation site is an ATP binding site and is highly conserved during evolution. Moreover, it was suggested that high accumulation of SAM and cordycepin resistance in NY9-10 was due to functional deficiency of ADO1 by this mutation. This strain is not a genetically-modified organism and can be employed for use in the food and medicine industry such as mass production and sake making.
Source
Bioscience, Biotechnology, and Biochemistry, 83(8), 1530-1537 (2019)

15. Investigation of relationship between sake-making parameters and sake metabolites using a newly developed sake metabolome analysis method

Author
Hisashi Yazawa, Masafumi Tokuoka, Hajime Kozato, Yutaro Mori, Miyuki Umeo, Rieko Toyoura, Ken Oda, Hisashi Fukuda, and Kazuhiro Iwashita
Abstract
We developed a sake metabolome analysis method using liquid chromatography-quadrupole/time-of-flight mass spectrometry to investigate the metabolome of various types of sakes and other alcohol beverages. Our method identified 198 compounds by comparison with standard metabolites. Using this method, we investigated the relationship between several sake-making parameters and sake metabolites by conducting combination experiments of these parameters using small-scale fermentation. The results indicated that all parameters significantly affected sake metabolites (P < 0.005) and most peaks were affected by multiple sake-making parameters. Interestingly, the effect of the rice cultivar on sake metabolites was higher for koji rice than for kake-rice. This result suggests that the rice cultivar used has a greater effect on the characteristics of Aspergillus oryzae compared to sake yeast and affects sake metabolites. In this study, we also evaluated the combined effect of several parameters. We demonstrated the different effects of each parameter on several amino acids. The results showed a new aspect of the science of sake making. For example, the amount of α-ethylglucoside, which can affect the taste of sake, was negatively correlated with α-glucosidase activity in koji (r=-0.84). In this study, various unidentified peaks were observed; detectable peaks can be increased by analyzing additional standard reagents. Investigating these unidentified peaks and accumulating datasets for sake-making parameters will give us insight into how to improve sake taste and quality.
Source
Journal of bioscience and bioengineering, 128, 183-190 (2019)

16. Increase in the Oxygen Stable Isotopic Composition of Water in Wine with Low Ethanol Yield

Author
Fumikazu AKAMATSU, Hideaki SHIMIZU, Aya KAMADA,Yukari IGI, Tsutomu FUJII, Nami GOTO-YAMAMOTO
Abstract
The stable isotopic composition of oxygen (δ18O) in wine is often analysed to determine the geographic origin of the wine and the amount of water dilution. However, little is known regarding the effects of two major winemaking techniques (the addition of acid (acidification) and sugar (chaptalization)) on theδ18O value of water in wine. Here we show that acidification and chaptalization have minor direct effects on theδ18O value but indirect effects based on the ethanol yield, which causes isotopic variation of up to 0.6‰. During fermentation,δ18O values increase at low ethanol yields, suggesting that yeast release water with a highδ18O value into wine when consuming sugars. Additionally, the ethanol yield is negatively correlated with the consumption of amino acids by the yeast, indicating that yeast growth decreases the ethanol yield. We therefore identify ethanol yield, which is decreased by the consumption of sugars by yeast for non-alcohol-fermentation processes as a potential factor leading to variations in theδ18O value of water during the winemaking process.
Source
Scientific Reports, 9, 11039 (2019)

17. Identification of enzymes from genus Trichoderma that can accelerate formation of ferulic acid and ethyl ferulate in collaboration with rice koji enzyme in sake mash.

Author
Toshihiko Ito, Anna Sato, Itsuki Takahashi, Takahito Ito, Kouto Takano, Koji Noge, Masaki Okuda, Katsumi Hashizume
Abstract
The enzymes responsible for acceleration of ferulic acid and ethyl ferulate formation in sake mash were studied. Ferulic acid and ethyl ferulate are formed during the sake brewing process from feruloylated glucuronoarabinoxylan. Cellulase reagent from genus Trichoderma was used instead of rice koji, because rice koji for sake brewing produces extremely low levels of xylan-degrading enzymes. A combination of the reagent with rice koji enzymes accelerated the formation of ferulic acid from a-rice powder. Addition of the reagent to sake mash increased ferulic acid and ethyl ferulate formation. The enzyme responsible for the accelerated formation was purified using a newly developed assay method and a-rice powder as a substrate. During the assay procedure, feruloylated oligosaccharide was converted to ferulic acid by feruloylesterase for HPLC analysis. Analysis of the N-terminal amino acid sequence of the purified samples was successfully conducted after pyroglutamyl aminopeptidase de-blocking. Purified enzymes were identified as members of the glycoside hydrolase family 10 (GH10) and family 11 (GH11) xylanases by BLASTP database research. The GH10 xylanase showed higher specific activity for a-rice powder and insoluble wheat arabinoxylan compared with GH11 xylanase; the GH11 xylanase showed higher specific activity for the other xylan substrates, especially glucuronoarabinoxylan.The GH10 xylanase showed higher accelerating activity than the GH11 xylanase in the sake mash. The results of this study provides useful knowledge on ferulic acid and ethyl ferulate formation in sake mash, the relative levels of these compounds and their influence on the sensory quality of sake.
Source
Journal of Bioscience and Bioengineering 128,177-182(2019)

18. Study for practical application of supported gold nanoparticles for removal of DMTS responsible for hineka in sake

Author
Atsuko Isogai, Haruno Murayama, Moemi Kimura, Takaaki Shinozaki, Eiji Yamamoto, Tsutomu Fujii, Sachiko Iizuka, Makoto Tokunaga
Abstract
Dimethyl trisulfide (DMTS) is one of the primary components responsible for "hineka", the stale aroma of Japanese sake. We have reported that gold nanoparticles supported on silica (Au/SiO2) were effective for the selective removal of DMTS by adsorption from model ethanol solutions and sake. In this study, we carried out the adsorptive desulfurization of several kinds of sake: daiginjo-shu, junmai-shu, and jukusei-shu (long aged sake) in 5 L-scales. The results of instrumental analyses and sensory evaluations suggested that the effect of Au/SiO2 was most remarkable for the daiginjo sample containing a high level of ginjo-ka components and DMTS; DMTS was significantly reduced while ginjo-ka remained. For the practical application of Au/SiO2, we established a large-scale production by optimizing the calcination conditions using a rotary electric furnace. We also confirmed that Au/SiO2 can be reused by heating at 300°C.
Source
J. Brew. Soc. Japan, 114, 779-786 (2019)

19. Effect of Vineyard Altitude on Phenological Events and Berry Components of 'Chardonnay' and 'Merlot' in Yamanashi

Author
Eri INOUE, Munekazu KISHIMOTO, Manabu NEMOTO, Koki WATANABE, Hiroshi SAITO, Kazuya KOYAMA, Nami GOTO-YAMAMOTO, Hidetoshi MINO, Tomohiro SUZUKI and Tohru OKUDA
Abstract
The aim of this study was to investigate the relationships between vineyard altitude and climate classification, phenological events, and berry components. Vineyards located at an altitude of 250 m to 750 m where 'Chardonnay' and 'Merlot' grapevines are cultivated in Yamanashi Prefecture were classified into regions III to V on the basis of the Winkler Index. As the altitude increased, budburst, flowering, veraison or coloration, and harvest were delayed, whereas malic acid and anthocyanin contents increased. The difference in air temperature owing to vineyard altitude greatly affected both phenological events and berry components.
Source
Journal of ASEV Japan, 30(3), 111-120, 2019

20. Characterization of a wild sake-brewing yeast isolated from barley malt in Toyama

Author
Hiroyasu Onaka, Jun-ichi Maruyama, Shumpei Asamizu, Mayumi Kuroiwa, Katsuhiko Kitamoto, Masato Yamada, Tetsuya Goshima, Takeshi Akao
Abstract
A wild sake-brewing yeast, Toyama yeast (Toyama-Umare-no-Koubo) was isolated from the malt of six-rowed barley harvested in Takaoka, Toyama. Molecular phylogenetic analysis revealed that Toyama yeast had the highest homology with Saccharomyces cerevisiae NBRC2114 (Kotobukiya whisky No. 1) in the internal transcribed spacers (ITS) region. Small-scale sake brewing test revealed that the mash (moromi) fermentation period for Toyama yeast was 3 days more than that for K701 yeast, and the alcohol concentration reached almost the same i.e. 18%. In the composition of organic acids in sake fermented by Toyama yeast, the amounts of acetic acid, malic acid, and succinic acid were considerably different from sake fermented by K701. Considering volatile contents, sake fermented by Toyama yeast also contained more higher alcohols and less esters than K701 sake. In amino acid content, sake fermentated by Toyama yeast was much higher than K701. These properties also appeared in organoleptic evaluation. Further, Toyama yeast sake has unique and distinctive properties characterized by differences in aroma and flavor such as acid smell, umami, heavy, zatsumi, and thin as compared to K701 sake. Commercialized brewing with Toyama yeast has been done five times in Narimasa Shuzo Breweries, the current brewing sake has become rich in flavor and has a sharp finish. Also, since this yeast is not a sake yeast but a wild yeast, it has been also used for the brewing of craft beer, wine, and whiskey.
Source
J. Brew. Soc. Japan, 114 (10) 645-653 (2019)

21. Effects of Rice Wine Lees on Cognitive Function in Community- Dwelling Physically Active Older Adults: A Pilot Randomized Controlled Trial.

Author
Narumi NAGAI, Nana SHINDO, Akari WADA, Hanae IZU, Tsutomu FUJII, Takahiro AKASHI, Misato TOKUI, Kiminori MATSUBARA, Yuji WADA, and Naoki SAKANE
Abstract
BACKGROUND

Rice wine lees (RWL), a Japanese traditional fermented product, is a rich source of one-carbon metabolism related nutrients, which may have beneficial effects on cognitive function.

OBJECTIVES

We aimed to examine the effect of the RWL on cognitive function in community-dwelling physically active older adults.

Design

Double-blind, randomized, placebo-controlled study (clinical trial number: UMIN 000027158).

SETTING

Community-based intervention including assessments conducted at the University of Hyogo and a public liberal arts school in Himeji City, Japan.

PARTICIPANTS

A total of 35 community-dwelling older adults (68-80 years) who performed mild exercise before and during the trial were assigned to either the RWL (n=17) or the placebo group (n=18).

INTERVENTION

Daily consumption of 50 g RWL powder, which contained one-carbon metabolism-related nutrients, or the placebo powder (made from soy protein and dextrin) for 12 weeks. Both supplements included equivalent amounts of energy and protein.

MEASUREMENTS

Montreal Cognitive Assessment, computerized cognitive function test, and measurements of serum predictive biomarkers (transthyretin, apolipoprotein A1, and complement C3) were conducted at baseline and follow-up.

RESULTS

Visual selective attention and serum transthyretin significantly improved in the RWL group, whereas there was no significant change in the placebo group. No significant group difference was observed in the remaining cognitive performance tests.

CONCLUSIONS

RWL supplements seem to have a few effects on cognitive function in community-dwelling physically active older adults. However, the impact was limited; therefore, further studies with sufficient sample size are warranted to elucidate this issue.

Source
The Journal of Prevention of Alzheimer's Disease 2020, 7(2): 95-103.

22.Aspergillus luchuensis fatty acid oxygenase ppoC is necessary for 1-octen-3-ol biosynthesis in rice koji

Author
Ryousuke Kataoka, Taisuke Watanabe, Shigekazu Yano, Osamu Mizutani, Osamu Yamada, Takafumi Kasumi, Jun Ogihara
Abstract
Awamori is a distilled spirit produced in Okinawa Prefecture, in southern Japan. Awamori contains the volatile organic compound 1-octen-3-ol, an important flavor component. Here, using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GCMS), we demonstrate that the black koji mold Aspergillus luchuensis produces 1-octen-3-ol in rice koji. To examine the role of the fatty acid oxygenase genes ppoA and ppoC in 1-octen-3-ol biosynthesis by A. luchuensis, we constructed ppoA and ppoC disruptants, ΔppoA and ΔppoC, respectively, via protoplast-PEG transformation. No clear differences in growth and conidiation were observed between the transformants and the parent strain. Volatile compounds in rice koji prepared using these gene disruptants were analyzed by SPME-GCMS. The amount of 1-octen-3-ol contained in koji produced by the ΔppoA strain was the same as that produced by the parental strain. In contrast, although the ΔppoC strain grew on the rice koji, was not detected. These results indicate that ppoC is involved in 1-octen-3-ol biosynthesis in A. luchuensis.
Source
Journal of Bioscience and Bioengineering, 129, 192-198 (2020)

23. Characterisation and threshold measurement of aroma compounds contributing to the quality of Honkaku shochu and Awamori

Author
Yukio Osafune, Kenji Toshida, Jinshun Han, Atsuko Isogai, Nobuhiko Mukai
Abstract
Honkaku shochu and Awamori are traditional Japanese spirits. The detection and recognition thresholds of 16 compounds in these spirits were determined to understand the compounds contributing to the aroma characteristics of Honkaku shochu and Awamori. The odour activity values calculated using detection thresholds suggested that 14 compounds affect the quality of Honkaku shochu and Awamori. The odour activity values of β‐damascenone and rose oxide were much higher in sweet potato shochu and these two compounds are considered to contribute to the characteristics of sweet potato shochu. Similarly, the odour activity value of 1‐octen‐3‐ol was higher in Awamori than in the other categories, suggesting that 1‐octen‐3‐ol produces the characteristic aroma of Awamori. In addition, the sensory attribute terms of 16 compounds are summarised.
Source
Journal of The Institute of Brewing, 126, 131-135 (2020)

24. Mechanism of high folate accumulation in a sake yeast other than Kyokai yeasts

Author
Yusuke Shibata, Tasuku Yamada, Tomoko Morimoto, Tsutomu Fujii, Takeshi Akao, Tetsuya Goshima, Toshinari Takahashi, and Nobuchika Tanaka
Abstract
Folates are important vitamins in human nutrition. Pressed sake cake, a brewing by-product of sake, is a rich dietary source of folates derived from sake yeast (Saccharomyces cerevisiae). The National Research Institute of Brewing investigated 106 samples of pressed sake cake and revealed that three samples containing large amounts of folates were produced by Km67 yeast derived from the house sake yeast strain of Kiku-Masamune sake brewery. In this study, we performed sake brewing tests using Km67 and Kyokai no. 7 group strains and confirmed that Km67 yeast contributed to the production of pressed sake cake containing large amounts of folates. To elucidate the mechanisms of high folate accumulation in Km67, we performed whole-genome sequence analysis in Km67 and then screened 10 folate metabolizing genes showing different sequences in Km67 and K7 strains. By folate analysis of each gene-disrupted strain derived from strain BY4743, we also selected four genes having significant effects on folate content in yeast from 10 candidate genes. Folate analysis of gene-disrupted yeast strains complemented with either Km67-type genes or K7-type genes revealed that the Km67-type HMT1 gene was related to high folate accumulation not only in laboratory yeast but also in sake yeast. In this gene, Leu63Phe was present in the methyltransferase motif I of Hmt1p, which was essential for the methyltransferase activity of Hmt1p. Our results and previous reports suggested that the methyltransferase activity of Km67-Hmt1p was higher than that of K7-Hmt1p, leading to enhanced production and high accumulation of folates in Km67 yeast.
Source
Journal of Bioscience and Bioengineering, 129 (1) 1-5 (2020)

25. Factors affecting levels of ferulic acid, ethyl ferulate and taste-active pyroglutamylpeptides in sake

Author
Katsumi Hashizume, Toshihiko Ito, Karin Shirato, Naomi Amano, Tetsuo Tokiwano, Tsuyoshi Ohno,Sho Shindo, Seiei Watanabe and Masaki Okuda
Abstract
Factors affecting ferulic acid, ethyl ferulate and taste-active pyroglutamyl (pGlu) peptides levels in sake were analyzed using small-scale sake brewing tests on eighteen rice samples with differing cultivar variety, cropped year and area, and polishing rate. Ferulic acid concentration in sake was highly positively correlated with its content in rice (r = 0.782**, double asterisk indicates 1% significance level), feruloylesterase (FE) activity (r = 0.804**) and feruloylated saccharide forming activity (FSFA) (r =0.619**) in the rice koji. The results suggested that ferulic acid in rice induced FE activity and FSFA, and these two enzymes accelerated the formation of ferulic acid in sake mash. The concentration of bitter-tasting peptides in sake was highly positively correlated with crude protein content in rice (r = 0.786**), and negatively correlated with acid carboxypeptidase (ACP) activity to (pGlu)LFGPNVNPWH (r = -0.612**), fermentation length (r = -0.820**), and pyroglutamyl leucine ((pGlu)L) concentration in sake (r = -0.502*; 5% significance level). The observation suggested that bitter-tasting peptides are initially formed in sake mash in accordance with protein content in rice, and are then hydrolyzed to smaller peptides, such as (pGlu)L. An ACP specific to the hydrolysis of bitter-tasting peptides was suggested by the observation that ACP activity to (pGlu)LFGPNVNPWH was significantly correlated (r = -0.612**) to their formation whereas an ACP to Cbz-Glu-Tyr was not (r = -0.220). It was suggested that (pGlu) oligopeptide ethyl esters were formed during the decomposition of bitter-tasting peptides to which the ACP to (pGlu) LFGPNVNPWH might contribute.
Source
Journal of Bioscience and Bioengineering, 129,322-326 (2020)

26. Isolation methods of high glycosidase-producing mutants of Aspergillus luchuensis and its mutated genes

Author
Kazuya TOMIMOTO, Yukio Osafune, Dararat Kakizono, Jinshun HAN, Nobuhiko MUKAI
Abstract
High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, β-glucosidase and β-xylosidase activity in the mutants was ~3, ~2, ~4 and ~2.5 times higher than the parental strain RIB2604 on koji-making conditions, respectively. Citric acid production and mycelia growth of the mutants, however, approximately halved to that of the parent. Compared to the parent, the alcohol yield from rice and sweet potato shochu mash of the mutant increased ~5.7% and 3.0%, respectively. The mutant strains showed significantly low glucose assimilability despite the fructose one was almost normal, and they had a single missense or nonsense mutation in the glucokinase gene glkA. The recombinant strain that was introduced at one of the mutations, glkA Q300K, demonstrated similar but not identical phenotypes to the mutant strain. This result indicates that glkA Q300K is one of the major mutations in 2-DG resistant strains.
Source
Bioscience, Biotechnology, and Biochemistry, 84, 198-207 (2020)