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

2020 Research results

1. 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.

2. 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)

3. 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)

4. 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)

5. 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)

6. Evaluating the influence of temperature on proanthocyanidin biosynthesis in developing grape berries (Vitis vinifera L.)

Author
Puspa Raj POUDEL, Kazuya KOYAMA, Nami GOTO-YAMAMOTO
Abstract

The variability in grape (Vitis vinifera L.) proanthocyanidin content is largely attributable to viticultural and environmental conditions. However, the particular effect temperature has on proanthocyanidin biosynthesis is poorly understood. The aim of the present study was to ascertain the magnitude of the effect of temperature on proanthocyanidin biosynthesis in Cabernet Sauvignon grape berries cultured in vitro. In addition, the effects of temperature on global gene transcription were evaluated, and the microarray data were later validated by quantitative real-time PCR (qPCR). The grape berries used in this research were sampled 3-4 weeks after full bloom and cultured in vitro either under a low (20 ℃) or high (30 ℃) temperature treatment for 15 days (d) with sampling occurring every five days. The proanthocyanidin content was higher in the skin and seeds of grape berries cultured at a low temperature compared with a high temperature. However, overall proanthocyanidin composition between the treatments was not significantly affected. Microarray data revealed a total of 1298 genes with ≥ 3.5-fold expression differences under high temperature conditions. High temperature also inhibited the expression level of key genes involved in proanthocyanidin biosynthesis, anthocyanidin reductase (ANR) and leucoanthocyanidin reductase-1 (LAR-1) within the berry skin. However, the transcriptomic accumulation of transcription factors, such as VvMybPAs, VvMyb5a and VvMyb5b, was barely influenced during the peak expression of ANR and LAR-1. Thus, the present study revealed that temperature has a significant effect on proanthocyanidin biosynthesis in grape during berry development through enhancing the expression of key biosynthetic genes.

Source
Molecular Biology Reports, 47, 3501-3510 (2020)

7. Separation and Purification of Glucose in Sake for Carbon Stable Isotope Analysis

Author
Fumikazu AKAMATSU, Yukari IGI, Akiko FUJITA
Abstract

An existing procedure for the separation and purification of glucose in sake for compound-specific carbon stable isotope analysis was examined with a view to reducing the sample amount required and process complexity, with a focus on the freeze-drying, solid-phase extraction (SPE), and high-performance liquid chromatography (HPLC) steps. In the revised procedure, the carbon stable isotopic composition, as indicated by δ13C values, of glucose was not significantly affected by glucose concentration in the range 0.25-1.00 mg mL-1, solvent (water or 80% aqueous acetonitrile), or HPLC procedure. Glucose freeze-dried after separation and purification by SPE and HPLC exhibited procedure-induced carbon isotopic discrimination of ≤ 0.1‰. The isolation procedure achieved the same accuracy in glucose δ13C values as that achieved with traditional ion-exchange procedures while requiring a sample volume (1 mL) only 4% that of the usual volume. The improved method enables cost-effective, labor-efficient, small-scale, compound-specific carbon stable isotope analyses of glucose in sake.

Source
Food Analytical Methods, 13 (4), 885-891 (2020)

8. Characteristic analysis of the fermentation and sporulation properties of the traditional sake yeast strain Hiroshima no.6

Author
Risa Yamasaki, Tetsuya Goshima, Kenji Oba, Atsuko Isogai, Ritsushi Ohdoi, Dai Hirata, Takeshi Akao
Abstract

General sake yeasts (e.g., Kyokai no.7, K7) show high fermentation ability and low sporulation frequency. Former is related to stress-response defect due to the loss-of-function of MSN4 and RIM15. Later is mainly caused by low IME1 expression, leading to difficulty in breeding and genetic analysis. Sake yeast Hiroshima no.6 (H6), which had been applied for sake fermentation, has sporulation ability. However, its detailed properties have not been unveiled. Here we present that the fermentation ability of H6 is suitable for sake brewing, and the precursor of dimethyl trisulfide in sake from H6 is low. MSN4 but not RIM15 of H6 has the same mutation as K7. Our phylogenetic analysis indicated that H6 is closely related to the K7 group. Unlike K7, H6 showed normal sporulation frequency in a partially RIM15-dependent manner, and IME1 in H6 was expressed. H6 possesses excellent properties as a partner strain for breeding by crossing.

Source
Bioscience, Biotechnology, and Biochemistry, 84 (4), 842-853 (2020)

9. The sake yeast YHR032W/ERC1 allele contributes to the regulation of the tetrahydrofolate content in the folate synthetic pathway in sake yeast strains

Author
Muneyoshi Kanai, Tomoko Kawata, Tomoko Morimoto, Masaki Mizunuma, Daisuke Watanabe, Takeshi Akao, Tsutomu Fujii, Haruyuki Iefuji
Abstract
To elucidate the mechanism underlying tetrahydrofolate (THF) accumulation in sake yeast strains compared with that in laboratory yeast strains, we performed a quantitative trait locus (QTL) analysis. The results revealed that the sake yeast ERC1 allele contributes to an increase in the ratio of THF to the total folate content in sake yeast.
Source
Bioscience, Biotechnology, and Biochemistry, 84 (5), 1073-1076 (2020)

10. Efficacy of a long term feeding of α-glycerophosphocholine on aging-related phenomena in old mice

Author
Tsutomu Masataka NARUKAWA, Aya KAMIYOSHIHARA, Hanae IZU, Tsutomu FUJII, Kiminori MATSUBARA, and Takumi MISAKA
Abstract
α-Glycerophosphocholine (GPC) is a natural source of choline. It reportedly prevents aging-related decline in cognitive function, but the underlying mechanism remains unclear. Although it is understood that aging influences taste sensitivity and energy regulation, whether GPC exerts antiaging effects on such phenomena requires further elucidation. Here, we used old C57BL/6J mice that were fed a GPC-containing diet, to investigate the molecular mechanisms underlying the prevention of a decline in cognitive function associated with aging and examine the beneficial effects of GPC intake on aging-related phenomena, such as taste sensitivity and energy regulation. We confirmed that GPC intake reduces the aging-related decline in the expression levels of genes related to long-term potentiation. Although we did not observe an improvement in aging-related decline in taste sensitivity, there was a notable improvement in the expression levels of β-oxidation-associated genes in old mice. Our results suggest that the prevention of aging-related decline in cognitive function by GPC intake may be associated with the improvement of gene expression levels of long-term potentiation. Furthermore, GPC intake may positively influence lipid metabolism.
Source
Gerontology, 2020, 66: 275-285.

11. Long-term Responses to Climate Change of the Carbon and Oxygen Stable Isotopic Compositions and Gelatinization Temperature of Rice

Author
Fumikazu AKAMATSU, Masaki OKUDA, Tsutomu FUJII
Abstract
The stable isotopic compositions of carbon (δ13C) and oxygen (δ18O) in rice are often used to confirm its authenticity. The gelatinization temperature is a crucial factor in alcoholic fermentation. However, little is known about the isotopic and thermal responses of rice to climate change. We show that in sake rice grown annually in the same paddy field from 1994 to 2013, the δ13C (-27.4‰ to -25.9‰) and δ18O values (20.4‰ to 27.0‰) correlated negatively with the mean daily minimum air temperature and precipitation, and positively with the hours of sunshine during grain-filling. In contrast, of the air temperatures tested, the gelatinization temperature of the rice grains (63.2 ℃ to 70.0 ℃) correlated positively and most strongly with the mean daily mean air temperature. Thus, we identified the mean daily minimum temperature during grain-filling, a nocturnal temperature, as a major factor affecting the isotopic and thermal variations in rice grains.
Source
Food Chemistry, 315, 126239 (2020)

12. Invasive growth of Aspergillus oryzae in rice koji and increase of nuclear number

Author
Mizuki Yasui, Ken Oda, Shunsuke Masuo, Shuji Hosoda, Takuya Katayama, Jun-ichi Maruyama, Naoki Takaya and Norio Takeshita
Abstract
Background

'Rice koji' is a solid culture of Aspergillus oryzae on steamed rice grains. Multiple parallel fermentation, wherein saccharification of rice by A. oryzae and alcohol fermentation by the budding yeast occur simultaneously, leads to the formation of a variety of ingredients of Japanese sake. In sake brewing, the degree of mycelial invasive growth into the steamed rice, called 'haze-komi', highly correlates with the digestibility and quality of rice koji, since the hyphae growing into the rice secrete amylases and digest starch.

Results

In this study, we investigated mycelial distribution of GFP-tagged A. oryzae in rice koji made with different types of rice, such as sake rice and eating rice, with 50 or 90% polishing rate to remove abundant proteins and lipids near the surface. In addition, we compared transcriptomes of A. oryzae in the different types of rice koji. Finally, we found that A. oryzae increases the nuclear number and hyphal width in the course of 1-3 days cultivation.

Conclusions

Our imaging analyses indicate that A. oryzae hyphae grew more deeply into 50% polished rice than 90% polished rice. The increases of nuclear number may be a selectively acquired characteristic for the high secretory capacity during the long history of cultivation of this species.

Source
Fungal Biology and Biotechnology, 7, 8 (2020)

13. Development of sake yeast haploid set with diverse brewing properties using sake yeast strain Hiroshima no. 6 exhibiting sexual reproduction.

Author
Risa Yamasaki, Tetsuya Goshima, Kenji Oba, Muneyoshi Kanai, Ritsushi Ohdoi, Dai Hirata, Takeshi Akao
Abstract
Among sake yeast strains, Kyokai no. 7 (K7) and its closely related strains (K7 group) are predominantly used because of their excellent brewing properties. In the sake industrial sector, the need for various types of yeast strains is high. Although crossbreeding is an effective method for generating genetic diversity that should result in diverse characteristics, most K7 group strains lack normal sporulation ability, including the ability to undergo meiotic chromosomal recombination, which leads to difficulties in crossbreeding. Accordingly, the improvement of sake yeast strains primarily depends on mutagenesis and suitable selection in a stepwise manner. Our recent study revealed that the long-preserved sake yeast strain Hiroshima no. 6 (H6) does not belong to the K7 group despite genetically being extremely similar. In addition, H6 exhibited normal sporulation. Thus, we isolated haploid cells from H6 and mated them with previously isolated haploid cells of K7 group strains. The crossbred diploid strains had normal sporulation ability; hence, we performed tetrad analysis. The brewing characteristics of the obtained haploid set were extremely diverse. Principal component analysis based on the volatile and organic acid components measured using small-scale sake brewing tests revealed that the haploid strains derived from each diploid strain displayed a characteristic distribution. Thus, we demonstrated the availability of genetic crossbreeding using H6 with sporulation ability to facilitate both the development of novel sake yeast strains with many desirable characteristics and analyses of the function of sake yeast.
Source
Journal of Bioscience and Bioengineering, 129 (6), 706-714 (2020)

14. Analysis of Sake Components Presented to the Japan Sake Awards 2019

Author
Osamu YAMADA, Akiko FUJITA, Sachiko IIZUKA, Hanae IZU, Toru KISHIMOTO, Ryoko KANDA, Satoko TERAMOTO, Nami GOTO-YAMAMOTO
Source
Report of the Research Institute of Brewing, 192, 1-17 (2020)

15. Review of the 42nd Honkaku Shochu and Awamori Contest in 2019

Author
Hanae Nobuhiko MUKAI, Atsuko ISOGAI, Muneyoshi KANAI, Masayuki TAKAHASHIO, Yukio OSAFUNE, Yoshiharu YAMANE, Toshihiro YOSHIMURA, and Nami GOTO-YAMAMOTO
Source
Report of the Research Institute of Brewing. 192, 18-34 (2020)

16. Analysis of Beer Components Presented to Craft Beer Contest of Japan Brewers Association in 2020

Author
Shinichi ITO, Hisashi YAZAWA, Maki KAMIMOTO and Takayuki EMURA
Source
Report of the Research Institute of Brewing. 192, 35-39 (2020)

17. Formulation of Wine Flavor Samples for Sensory Evaluation Training

Author
Akiko FUJITA, Nami GOTO-YAMAMOTO
Source
Report of the Research Institute of Brewing, 192, 40-45 (2020)

18. Food safety evaluation of Aspergillus oryzae species based on the regulation by Ministry of Agriculture, Forestry and Fisheries, Japan

Author
Sharon Marie Bahena-Garrido, Ryota Saito, Yuko Komatsu, Shiori Kodama, Ken Oda and Kazuhiro Iwashita
Abstract
Based on the tandem approach of DI-HRMS and LC-QTOF-MS/SIM（and MS/MS), we could finally conclude that no significant traces of harmful secondary metabolites or mycotoxins found in all of the 13 Aspergillus oryzae industrial strains cultivated under different culture conditions. We further confirmed the safety of these Aspergillus oryzae strains for industrial production of fermented foods and beverages.
Source
Report of the Research Institute of Brewing, 192, 46-57 (2020)

19. Effect of light on carotenoid and lipid production in the oleaginous yeast Rhodosporidium toruloides

Author
Khanh Dung Pham, Yosuke Shida, Atsushi Miyata, Takeru Takamizawa, Yoshiyuki Suzuki, Satoshi Ara, Harutake Yamazaki, Kazuo Masaki, Kazuki Mori, Sachiyo Aburatani, Hideki Hirakawa, Kosuke Tashiro, Satoru Kuhara, Hiroaki Takaku and Wataru Ogasawara
Abstract
The oleaginous yeast Rhodosporodium toruloides is receiving widespread attention as an alternative energy source for biofuels due to its unicellular nature, high growth rate and because it can be fermented on a large-scale. In this study, R. toruloides was cultured under both light and dark conditions in order to understand the light response involved in lipid and carotenoid biosynthesis. Our results from phenotype and gene expression analysis showed that R. toruloides responded to light by producing darker pigmentation with an associated increase in carotenoid production. Whilst there was no observable difference in lipid production, slight changes in the fatty acid composition were recorded. Furthermore, a two-step response was found in three genes (GGPSI, CAR1, and CAR2) under light conditions and the expression of the gene encoding the photoreceptor CRY1 was similarly affected. Light effect on carotenoid production in Rhodosporidium toruloides
Source
BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, 84, 1501-1512 (2020)

20. New urethanase from the yeast Candida parapsilosis

Author
Kazuo Masaki, Taichi Mizukure, Dararat Kakizono, Kanako Fujihara, Tsutomu Fujii, Nobuhiko Mukai
Abstract
Urethanase (EC 3.5.1.75) is an effective enzyme for removing ethyl carbamate (EC) present in alcoholic beverages. However, urethanase is not well studied and has not yet been developed for practical use. In this study, we report a new urethanase (CPUTNase) from the yeast Candida parapsilosis. Because C. parapsilosis can assimilate EC as its sole nitrogen source, the enzyme was extracted from yeast cells and purified using ion-exchange chromatography. The CPUTNase was estimated as a homotetramer comprising four units of a 61.7 kDa protein. In a 20% ethanol solution, CPUTNase had 73% activity compared with a solution without ethanol. Residual activity after 18 h indicated that CPUTNase was stable in 0%-40% ethanol solutions. The optimum temperature of CPUTNase was 43℃. This enzyme showed urethanase activity at pH 5.5-10.0 and exhibited its highest activity at pH 10. The gene of CPUTNase was identified, and a recombinant enzyme was expressed in the yeast Saccharomyces cerevisiae. Characteristics of recombinant CPUTNase were identical to the native enzyme. The putative amino acid sequence indicated that CPUTNase was an amidase family protein. Further, substrate specificity supported this sequence analysis because CPUTNase showed higher activities toward amide compounds. These results suggest that amidase could be a candidate for urethanase. We discovered a new enzyme and investigated its enzymatic characteristics, sequence, and recombinant CPUTNase expression. These results contribute to a further understanding of urethanase.
Source
Journal of Bioscience and Bioengineering, 130, 115-120 (2020)

21. Variation in the mineral composition of wine produced using different winemaking techniques

Author
Hideaki Shimizu, Fumikazu Akamatsu, Aya Kamada, Kazuya Koyama, Kazuhiro Iwashita, and Nami Goto-Yamamoto
Abstract
It has been reported that the concentrations of minerals in wines can be used to discriminate their geographical origin. However, some winemaking techniques may also affect the mineral concentration of the final product. In this study, we examined the effects of various winemaking techniques, including (i) fining, (ii) aging with oak tips, (iii) maceration with grape skins, (iv) chaptalization and acidification, and (v) yeast nutrient addition for alcohol fermentation, on the concentration of 18 minerals (Li, B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba and Pb) in a total of 154 wine samples using grapes from different production areas. Among the various winemaking techniques, maceration with grape skin had the largest effect on mineral content, significantly changing the concentrations of 17 or 18 elements (B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba and Pb). Fining treatment using bentonites had the second largest effect, altering the concentrations of 14 elements (Li, B, Na, Si, P, S, Ca, Co, Ga, Rb, Sr, Mo, Ba and Pb). However, in principal component analysis using all data (n=154), the wine samples were clearly clustered according to grapes used in the experiments rather than the winemaking technique. In conclusion, some winemaking techniques significantly altered the concentration of some minerals in wine; however, the principal factor influencing wine mineral composition seems to be its geographical origin.
Source
Journal of Bioscience and Bioengineering, 130, 166-172 (2020)

22. Aspergillus oryzae acetamidase catalyzes degradation of ethyl carbamate

Author
Kazuo Masaki, Kanako Fujihara, Dararat Kakizono, Taichi Mizukure, Masaki Okuda, Nobuhiko Mukai
Abstract
Urethanase (EC 3.5.1.75) catalyzes the hydrolysis of ethyl carbamate (EC) to ethanol, carbon dioxide, and ammonia. From our recent study, we expected that an acetamidase encoded by amdS of Aspergillus oryzae may catalyze the degradation of EC because it is homologous with a Candida parapsilosis urethanase (CPUTNase) recently identified. Urethanase is a prospective candidate to reduce EC in alcoholic beverages, but knowledge of this enzyme is very limited. Recombinant AmdS was expressed to study its enzymatic properties. Purified AmdS was identified as a homo-tetramer consisting of four 60 kDa units and exhibited urethanase activity. In a 20% ethanol solution, AmdS had 65% activity compared with a solution without ethanol. Residual activity after 18 h indicated that AmdS was stable in 0%–40% ethanol solutions. The optimum temperature of AmdS was 40°C. This enzyme showed urethanase activity at pH 6.4–9.6 and exhibited its highest activity at pH 9.6. The Km value of AmdS for EC was 8.2 mM, similar to the Km value (7.6 mM) of CPUTNase. AmdS showed activity not only for EC and acetamide but also other amide compounds. In this study, we investigated the enzymatic properties of AmdS that was identified as acetamidase and showed that an amidase can be an enzymatic candidate that degrades EC.
Source
Journal of Bioscience and Bioengineering, 130, 577-581 (2020)

23. Mutagenesis, breeding, and characterization of sake yeast strains with low production of dimethyl trisulfide precursor

Author
Jun Makimoto, Kou Wakabayashi, Toyohisa Inoue, Yuriko Ikeda, Ryoko Kanda, Atsuko Isogai, Tsutomu Fujii, Takashi Nakae
Abstract
Dimethyl trisulfide (DMTS) is one of the main components responsible for “hineka”, the aroma associated with deteriorated Japanese sake during storage. The molecule 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) has been previously identified as a major precursor compound of DMTS. Furthermore, it had been suggested that the yeast methionine salvage pathway is involved in the production of DMTS-P1. In sake brewing tests, DMTS-P1 and the DMTS producing potential (DMTS-pp; DMTS amount of sake after accelerated storage) were significantly reduced in mde1 or mri1 strain, which lack genes of the methionine salvage pathway. Industrial use of the gene-disrupting strains may not be accepted in the Japanese food industry. In order to obtain mde1 or mri1 mutants, we established a method to screen 5’-methylthioadenosine (MTA) non-utilizing strains using minimum culture medium containing methionine or MTA by ethyl methanesulfonate (EMS) mutagenesis with methionine-auxotrophic sake yeast haploid. As expected, mde1 and mri1 mutants were identified among the obtained mutants by an established screening method. The obtained strains had poor fermentation ability in sake brewing tests, so back-crossing was performed on the mutants to obtain mde1 or mri1 homozygous mutants. These strains had improved brewing characteristics, and DMTS-P1 and the DMTS-pp of the produced sake were significantly lower than those of the parent strains. These strains are expected to contribute to improving the maintenance of sake quality during storage.
Source
Journal of Bioscience and Bioengineering, 130, 610-615 (2020)

24. Effect of Cold Soak on Red Wine Making

Author
Hideaki SHIMIZU，Minori KONO, Kazuya KOYAMA, Kazuhiro IWASHITA, Nami GOTO-YAMAMOTO
Abstract
The effect of cold soak in pilot-scale and plant-scale vinification processes at various maceration temperatures was examined in nine wineries, and the produced wines were analyzed for chemical and sensory properties. Cold soak tended to increase total anthocyanins, color intensity, and total phenols (A280) of wine regardless of grape variety, when the temperatures for the cold soak and the subsequent maceration were controlled appropriately. The basic chemical components of wines were minimally affected by the cold soak. Total anthocyanins in the cold soak wines were higher than those in control wines, and some cold soak wines received favorable evaluation with regard to flavor, astringency, or body in the sensory analysis compared with control wines. The results indicate that cold soak carried out appropriately resulted in good coloration in many cases and had no deleterious effects.
Source
Journal of ASEV Japan, 31, 115-124 (2020)

25.Low-Dose Ethanol Has Impacts on Plasma Levels of Metabolites Relating to Chronic Disease Risk in SAMP8 mice

Author
Churan Fu, Yongshou Yang, Thanutchaporn Kumrungsee, Akiko Kimoto,　Hanae Izu and Norihisa Kato
Abstract
The effects of low-dose alcohol on experimental animals are unclear. This study examined plasma metabolites in senescence-accelerated mice 8 (SAMP8) given lowdose ethanol, and compared them with aging progress and skeletal muscle strength. Male SAMP8 mice (10-wk-old) were given drinking water containing 0% (control), 1%, 2%, or 5% (v/v) ethanol for 14 wk. Compared with the control group, only mice who consumed 1% ethanol experienced a lower senescence score at 18 and 23 wk, as well as an increased limb grip strength at 21 wk. Plasma metabolites of control, 1% and 2% ethanol groups were analyzed by capillary electrophoresis–time-of-flight mass spectrometry (CE-TOF/MS). Among the 7 metabolites affected by ethanol, notewhorthy is the positive association of the ethanol levels in drinking water with the levels of α-ketoglutarate (antioxidant and anti-inflammatory metabolite) and hippurate (antioxidant and microbial co-metabolite) (p<0.05). Intriguingly, the levels of 2-hydroxyisobutyrate (the biomarker of energy metabolism and microbial co-metabolite) were higher in the 1% ethanol group (p<0.05), but not in the 2% ethanol group as compared to the control. Furthermore, the levels of some of the metabolites affected were correlated with some variables in the grading score of senescence and muscle strength. This study provides a novel insight into how low-dose ethanol in SAMP8 mice modulates the levels of circulating metabolites relating to chronic disease risk.
Source
J Nutr Sci Vitaminol, 66, 553–560, 2020