2009 Research results

1. Changes in mean air temperature after heading and starch characteristics of rice grains for sake making among harvest years and areas

Author: M. Okuda, K. Hashizume, M. Joyo, M. Numata, N. Goto-Yamamoto and S. Mikami
Abstract: The relationship between mean air temperature after heading and starch characteristics of rice grains, which correlated with enzyme digestibility of steamed rice grains, was investigated. It was confirmed that the difference in the temperature during grain filling was the main factor for the variation in digestibility of steamed rice grains among harvest areas even within the same rice cultivar. When the mean air temperatures during one month after heading were higher, twenty-five rice cultivars for sake making showed higher gelatinization temperatures measured by differential scanning calorimetry (DSC). Previously, we reported that, when the gelatinization temperature was higher, the enzyme digestibility of steamed rice grains was lower. Therefore, from the previous and present studies, the enzyme digestibility of steamed rice grains can be predicted by mean air temperature after heading.
Source: J. Brew. Soc. Japan, 105, 2, 97-105 (2010)

2. Volatile compounds and the changes in their concentration levels during storage in beers containing varing malt concentrations

Author: H. Tsuji and A. Mizuno
Abstract: Volatile compounds in beers brewed with different amounts of malt were analyzed by using the stir bar sorptive extraction-gas chromatography-mass spectrometry method. We identified 90 compounds-25 esters, 17 terpenes, 14 alcohols, 11 acids, 6 furans, 6 aroma compounds, 5 carbonyls, and other compounds. Ananalysis of aged beer suggested that the concentration levels of stale flavor compounds-β-damascenone, γ-nonalactone, ethylcinnamate, and 2-methoxy-4-vinylphenol-in nonmalt beer were different from those in all-malt and standard beer. Additionally, concentrations of these compounds did not increase during storage in mostnonmalt beer analyzed in this study. Nerolidol may be a good marker candidate regardless of the malt content.
Source: Food Chemistry, 75 (1), C79-84 (2010)

3. Perception by experts of potential odor reference compounds of sake

Author: Hitoshi UTSUNOMIYA, Atsuko ISOGAI and Hiroshi IWATA
Abstract: Eighteen chemical compounds, ethyl acetate, including isoamyl acetate, ethyl hexanoate, isoamylalcohol, 2-phenylethanol, acetaldehyde, isovaleraldehyde, benzaldehyde, sotolon, ethanethiol, dimethyl sulfide (DMS), dimethyl trisulfide (DMTS), 2,4,6-Trichloroanisole (TCA), diacetyl, hexanoic acid, acetic acid, butyric acid,and isovaleric acid were selected as potential odor reference compounds for sake sensory analysis. Each compound at a 90% recognition threshold concentration was added to commercial sake. The solutions were assessed by 79 experts. It was suggested that 17 compounds -- except benzaldehyde -- were able to be used as reference standards. Sixty-one experts selected the term "Hineka" from an odor terms list in the assessment, 42.6% experts used "Hineka" for DMTS, and 23.0% used it for ethanthiol. Only one expert selected it for sotolon. The result shows that the odor impression of "Hineka" among experts is close to the odor of sulfuric compounds.
Source: J. Brew. Soc. Japan, 105, 106-115 (2010)

4. Behavioral Evaluation of Intoxication in Mice by Oral Administration of Alcoholic Beverages

Author: Hanae IZU, Kuniyasu GOTO
Abstract: We attempted to evaluate differences in intoxication in mice after the administration of various alcoholic beverages. Measurement of locomotor activity and elevated plus-maze test in mice did not show differences in the extent of intoxication after oral administration of alcoholic beverages (sake, red wine, shochu, and whiskey;approximately 15% alcohol, 1.2 g of alcohol/kg BW dosed single or twice). Changes in the concentrations of ethanol and acetaldehyde in blood and brain were very similar 0.5, 3, and 5 hours after the oral administration of alcoholic beverages (sake and shochu;15% alcohol, 2.4 g of alcohol/kg BW). There seems to be no differences in intoxication in mice after the administration of alcoholic beverages at the same doses with the same alcohol content.
Source: J. Brew. Soc. Japan, 104, 787-795 (2009)

5. Outcome of Methamidophos, Acetamiprid, and Aflatoxin B1 in brewing

Author: Sachie HORII, Yukari IGI, Kuniyasu GOTO
Abstract: The effects of brewing on the levels of Methamidophos, Acetamiprid, and Aflatoxin B1 in sake samples were investigated. In the fermentation process, the concentration of the pesticides and Aflatoxin B1 decreased with time when we added these substances at 10 or 20 ppb. These values differed depending on the kind of pesticide. When we compared the residual rate in the liquid and the solid part at 20 days after a 3rd adding, a greater amount of Metamidophos remained in the liquid part. In the case of Aflatoxin B1, the solid part contained more. On the other hand, Acetamiprid remained at the same rate in both the liquid and solid part. Activated carbon (100mg/L or 300mg/L) absorbed more than 50% Acetamiprid and Aflatoxin B1 for 30 minutes. Methamidophos, however, was not influenced by the activated carbon process. Ninety percent or more of the pesticides and Aflatoxin B1 remained after heating (75℃). In other words, the pesticides and Aflatoxin B1 could not be effectively degraded or eliminated in the heating processes. The storing time did not influence the concentration of Methamidophos and Acetamiprid. After 5 months, Aflatoxin B1 in the solid part was degraded, but it did not show a significant difference.
Source: J. Brew. Soc. Japan, 104, 900-907 (2009)

6. Aspergillus oryzae atfA controls conidial germination and stress tolerance

Author: Kazutoshi Sakamoto, Kazuhiro Iwashita, Osamu Yamada, Ken Kobayashi, Akihiro Mizuno, Osamu Akita, Shigeaki Mikami, Hitoshi Shimoi and Katsuya Gomi
Abstract: We compared atfA and atfB, the genes encoding the respective ATF/CREB-type transcription factors in Aspergillus oryzae. The germination ratio of △atfA conidia was low without any stress, unlike that of △atfB conidia. The △atfA conidia were more sensitive to oxidative stress than the △atfB conidia, which are also sensitive to oxidative stress. We compared the gene expressions of these strains by using a DNA microarray, GeneChip. Almost all the genes regulated by atfB were also regulated by atfA, but atfA also regulated many genes that were not regulated by atfB, including some genes putatively involved in oxidative stress resistance. The level of glutamate, the major amino acid in A. oryzae conidia, was significantly low only in the △atfA conidia, and the glycerol accumulation during germination was not observed only in the △atfA strain. We therefore concluded that atfA is involved in germination via carbon and nitrogen source metabolism.
Source: Fungal Genetics and Biology、46、887-897、(2009)

7. Influence of Sulfur and Nitrogen Content of Rice Grains on Flavor in Stored Sake

Author: M. Okuda, A. Isogai, M. Joyo, N. Goto-Yamamoto and S. Mikami
Abstract: The influence of nitrogen and sulfur compounds in rice grains on changes in flavor in stored sake was investigated using Japanese rice cultivars for sake making. Nitrogen content exhibited a significant positive correlation with sulfur content. Based on the molar ratio of nitrogen to sulfur in the rice grain, the sulfur compounds appeared to be derived from protein-associated sulfur-containing amino acids, as reported previously. The higher the protein content of the rice, the greater the amount of nitrogen and sulfur compounds found in both the digest of steamed rice grains and in the sake. Physicochemical changes were investigated in the stored sake to confirm the influence of total sulfur content. Polysulfides in the stored sake appeared to be higher when made from rice grains of high total sulfur content. Staling of stored sake was affected by levels of protein-associated sulfur-containing amino acids in the rice.
Source: Cereal Chemistry, 86(5):534-541, (2009)

8. Influence of starch characteristics on digestibility of steamed rice grains under sake-making conditions, and rapid estimation methods of digestibility by physical analysis

Author: M. Okuda, K. Hashizume, I. Aramaki, M. Numata, M. Joyo, N. Goto-Yamamoto and S. Mikami
Abstract: Retrogradation of starch in steamed rice grains under sake-making conditions was examined. The enthalpy change derived from retrogradation of amylopectin increased during the koji and sake mash processes. Ethanol in the sake mash accelerated retrogradation of amylopectin. Enzyme digestibility and properties of endosperm starches of 136 Japanese rice samples used for sake production were examined. A digestion test of steamed rice grains was conducted after 24 hr storage at 15℃ to estimate more accurately digestibility during the sake-making process. The ratio of short-to-long chain amylopectin exhibited a significant correlation with enzyme digestibility, suggesting that the structure of amylopectin may be used as a predictor of digestibility. The gelatinization temperature of brown/milled rice and purified starch measured by DSC (Differential Scanning Calorimetry) correlated well with the ratio of short-to-long chain amylopectin and with enzyme digestibility. Furthermore, the pasting temperature of milled rice as measured by RVA (Rapid Visco Analyser) correlated well with the ratio of short-to-long chain amylopectin and with enzyme digestibility. Therefore, DSC and RVA appear to be useful tools for estimating enzyme digestibility of steamed rice grains under sake-processing conditions because the measurements are done rapidly and require only small weights of brown/milled rice flour samples.
Source: J. Appl. Glycosci. 56, 185-192 (2009)

9. Relationship between sake making properties of rice grains and meteorological data

Author: M. Okuda, K. Hashizume, M. Numata, M. Joyo, N. Goto-Yamamoto and S. Mikami
Abstract: The relationship between meteorological conditions and sake making properties of rice grains was investigated. Mean air temperatures during the month after heading showed a high positive correlation with the ratio of short/long chain amylopectin and a negative correlation with amylose contents. Both gelatinization temperatures measured by differential scanning calorimetry (DSC) and pasting temperature measured by Rapid Visco Analyser (RVA) also showed high correlations with mean air temperatures during the month after heading. Enzyme digestibility of steamed rice grains was examined by a method mimicking sake making. The enzyme digestibility of steamed rice grains was lower when the mean air temperature during the month after heading was higher. The regression formula was constructed by meteorological data for the years 2003 - 2007. The predicted values showed a high correlation with actual values using the 2008 samples. This indicates the enzyme digestibility of steamed rice grains can be predicted by meteorological conditions.
Source: J. Brew. Soc. Japan, 104, 9, 699-711 (2009)

10. Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris

Author: Watanabe T, Ikeda H, Masaki K, Fujii T, Iefuji H.
Abstract: Phosphohydrolysis of organic phosphorus compounds by acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2) is an important method for efficient removal of phosphorus from high concentration organic wastewater. Another important method is supplementation of animal feed with phytase (EC 3.1.3.8 and EC 3.1.3.26), which improves the availability of phytate-phosphates (phosphate that are hydrolyzed by phytases), making it possible to add less phosphate to animal feed and resulting in the excretion of less phosphorus by the animals. In the present study, we purified a novel phytase from the wastewater treatment yeast Hansenula fabianii J640 (Hfphytase), cloned the 1456 bp open reading frame (ORF) encoding Hfphytase, and characterized Hfphytase. The molecular weight of Hfphytase after deglycosylation by PNGaseF was 49 kDa. The optimal pH and temperature for enzyme activity were 4.5 and 50 °C, respectively. Hfphytase exhibits 40% identity with Debaryomyces castellii phytase, 37% identity with Aspergillus niger PhyB, and 34% identity with Saccharomyces cerevisiae Pho5p. Recombinant Hfphytase was transformed and expressed in Pichia pastoris. The yield was 23 g/l by jar fermenter cultivation. The marked phosphohydrolysis activity exhibited by Hfphytase on six substrates (pNP-P, sodium phytate, glucose-1 phosphate, glucose-6 phosphate, α-glycerophosphate and β-glycerophosphate) indicated that it is a non-specific acid phosphatase.
Source: J. Biosci. Bioeng., 108, 225-235, 2009

11. Crystal structure and enhanced activity of a cutinase-like enzyme from Cryptococcus sp. strain S-2.

Author: Kodama Y, Masaki K, Kondo H, Suzuki M, Tsuda S, Nagura T, Shimba N, Suzuki EI, Iefuji H
Abstract: The structural and enzymatic characteristics of a cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 A resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long-chain substrates, as well as the preferred specificity of FS cutinase for short-chain substrates. In addition, site-directed mutagenesis was performed to increase the hydrolysis activity on long-chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long-chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity.
Source: Proteins: Structure, Function, and Bioinformatics, Volume 77 Issue 3, Pages 710 - 717)

12. QTL mapping of sake brewing characteristics of yeast

Author: Taku Katou, Masahiro Namise, Hiroshi Kitagaki, Takeshi Akao and Hitoshi Shimoi
Abstract: A haploid sake yeast strain, derived from a commercial diploid sake yeast strain Kyokai no. 7, showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in specification of sake brewing characteristics including ethanol fermentation and the production of aromatic components.
Source: Journal of Bioscience and Bioengineering, 107, 383-393 (2009)

13. Overexpression of MSN2 in a sake yeast strain promotes ethanol tolerance and increases ethanol production in sake brewing

Author: Mamoru Watanabe, Daisuke Watanabe, Takeshi Akao and Hitoshi Shimoi
Abstract: To improve the ethanol tolerance of sake yeast we constructed a sake yeast strain that overexpresses MSN2, a transcription factor that is activated by several environmental stresses, including ethanol. We showed that this strain is more ethanol tolerant and produced more ethanol in a sake mash than a control strain.
Source: Journal of Bioscience and Bioengineering, 107, 516-518(2009)

14. Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Author: Hong Wu, Tomoko Watanabe, Yoshio Araki, Hiroshi Kitagaki, Takeshi Akao, Hiroshi Takagi and Hitoshi Shimoi
Abstract: Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.
Source: Journal of Bioscience and Bioengineering, 107, 636-640(2009)

15. Characterization of Oriental cultivars of grapevine using a reference allele system of microsatellite data and assignment test

Author: Nami GOTO-YAMAMOTO, Mineyo NUMATA, Guang-Hua WAN, Toshi SHIMAMOTO, Katsumi HASHIZUME
Abstract: Six Oriental cultivars of Vitis vinifera, which are native to Japan or China, as well as 2 cultivars of V. labrusca as references, were characterized by 6 microsatellite markers with coded reference alleles. One allele in Oriental cultivars and 3 in V. labrusca have not been reported for Occidental cultivars. Assignment analysis using these data and reported data with grouping of V. labrusca, Oriental cultivars, and vine-growing regions of Occidental cultivars showed that 4 out of 8 Oriental cultivars, but only 3 out of 162 Occidental cultivars, were assigned to Oriental cultivars. These assignments are in accordance with the genetic distances calculated from microsatellite data. These results show that Oriental cultivars have a certain degree of genetic difference from Occidental cultivars within the species V. vinifera.
Source: J. Japan. Soc. Hort. Sci., 78, 175-179 (2009)

16. Color recovery in berries of grape (Vitis vinifera L.) 'Benitaka', a bud sport of 'Italia', is caused by a novel allele at the VvmybA1 locus

Author: Akifumi AZUMA, Shozo KOBAYASHI, Nami GOTO-YAMAMOTO, Mikio SHIRAISHI, Nobuhito MITANI, Hiroshi YAKUSHIJI and Yoshiko KOSHITA
Abstract: Color mutations in grape berry skin are relatively frequent events, and can be easily seen in the vineyard. Both light-red-skinned 'Ruby Okuyama' and more intense and uniform rosy-skinned 'Benitaka' (Vitis vinifera L.) are bud sports of white-skinned 'Italia'. Previously, we reported that 'Ruby Okuyama' was caused by the recovery of VvmybA1 expression, which may have occurred as a result of intra-LTR (long terminal repeat) recombination within a retrotransposon, Gret1. However, the molecular basis of the color recovery in 'Benitaka' has not been elucidated so far. Here, we found that the VvmybA1 locus of 'Benitaka' is heterozygous for the VvmybA1a allele (non-functional) and a novel VvmybA1^BEN allele, and that VvmybA1^BEN restored VvmybA1 transcripts. We hypothesized that VvmybA1^BEN allele was caused by homologous recombination between VvmybA1a and VvmybA3. In addition, the content and composition of anthocyanins in berry skins differed greatly between 'Ruby Okuyama' and 'Benitaka'. The levels of expression of the genes for flavonoid 3',5'-hydroxylase (F3'5'H), O-methyltransferase (OMT), and glutathione-S-transferase (GST) were associated with differences in the anthocyanin content and composition between the two cultivars.
Source: Plant Sci., 176, 470-478, 2009

17. Decolorization of Brown-sugar shochu wastewater using fungi.

Author: T. Watanabe, M. Tanaka, M. Okada, K. Masaki, T. Fujii, H. Iefuji
Abstract: As alcoholic distillery wastewater, brown-sugar shochu wastewater (BSW) contains high concentrations of brown pigments which are hardly decolorized by general biological wastewater treatment. Penicillium oxalicum d, which was newly isolated in the present study, decolorizes 47 % of the color from BSW without the addition of any nutrients. The mycelia of P. oxalicum d were stained dark brown when treated of BSW. therefore, the decolorization mechanism is related to adsorption. Treatment by P. oxalicum d reduced the time for decolorization by ozone by half. Treatment consisting of a combination of P. oxalicum d decolorization, physical decolorization by ozone, and biological wastewater treatment by activated sludge would be effective in treating BSW.
Source: J. Brew. Soc. Japan, 104, 495-501 (2009)

18. Decolorization and semi-batch continuous treatment of molasses distillery wastewater by Aspergillus tubingensis DCT6

Author: Watanabe T, Tanaka M, Masaki K, Fujii T, Iefuji H.
Abstract: Large quantities of deeply pigmented molasses distillery wastewater (MDW), are discharged during the production of bio-ethanol from molasses. Conventional biological wastewater treatment is not effective in removing the molasses pigments. In the present study, a MDW treatment system was developed with combination treatment involving biodecolorization and biotreatment by Aspergillus tubingensis DCT6, together with physical decolorization by ozonation after treatment by activated sludge. A. tubingensis DCT6, which was isolated from soil, decolorized 44% of the pigments in MDW without adding any nutrients. The combination treatment with A. tubingensis DCT6 and activated sludge method (fungi-activated sludge treatment) removed about 90% of organic compounds from MDW and appears to reduce the amount of space and water required for treatment. The fungi-activated sludge treatment reduced the time needed for decolorization by ozone by 83%. Replacing fresh seed sludge at regular intervals was useful to maintain the dominance and decolorization ability of A. tubingensis DCT6. The entire treatment obtained a decolorization ratio of 89-94% and removed more than 90% of each of DOC, DTN, and DTP.
Source: Water Sci Technol., 59, 2179-2185, 2009

19. Analysis of Sake Component Presented to the Sake Contest in 2007

Author: H. Iwata, A. Isogai, A. Fujita, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 181, 1-14 (2009)

20. Results of the Wine and Liqueur Contest in 2008

Author: S. Mikami, N. Goto, K. Koyama and J. Hiramatsu
Source: Report of the Research Institute of Brewing, 181, 15-25 (2009)

21. Analysis of Traditional Shochu Presented to the 31st Contest in 2008

Author: S. Mikami, H. Iefuji, N. Mukai, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 181, 26-36 (2009)

22. Analysis of 4-Methylimidazole, Semicarbazide, 1,3-Dichloropropanol and Microbes in

Author: K. Goto
Source: Report of the Research Institute of Brewing, 181, 37-40 (2009)

23. Application of the QuEChERS Method for Analysis of Pesticide in Alcoholic Beverages

Author: S. Horii, Y. Igi, K. Goto
Source: Report of the Research Institute of Brewing, 181, 41-47 (2009)