2006 Research results

1. Gene Silencing by RNA Interference

Author: O. Yamada, R .Ikeda, Y. Ohkita, R. Hayashi, K. Sakamoto, and O. Akita
Abstract: We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This systI utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.
Source: Bioschi. Biotechnol. Biochem., 71, 138-144 (2007)

2. Effects of milling ratio on properties of endosperm starches and rice flours from milled Japanese rice grains

Author: M. Okuda, K. Kobayashi, T. Itani, I. Aramaki and K. Hashizume
Abstract: Flour and endosperm starch prepared from rice grains of three Japanese rice cultivars milled from 90 to 30% of their original weight were subjected to physicochIical/structural analysis in order to examine the relationship between milling ratio and physicochIical/starch structural properties. The peak viscosity of the rice flours was found to increase with decreasing milling ratio, while that of the purified starches did not change significantly. The gelatinization peak tIperature of the rice flours was found to decrease while enthalpy changes increased with decreasing milling ratio. The gelatinization peak tIperature of the purifiedstarches was not found to change significantly with different milling ratios. Differences in structural properties of purified starches were examined by gel-filtration chromatography of isoamylase debranched starch. The FI (percentage of amylose) and FIIb/FIIa (ratio of short-to-long chain amylopectin) were found tobe constant for all three cultivars for milling ratios ranging from brown rice to 30%.
Source: J. Appl. Glycosci., 54, 1-5 (2007)

3. The Driselase-treated fraction of rice bran is a more effective dietary factor to improve hypertension, glucose and lipid metabolism in stroke-prone spontaneously hypertensive rats compared to ferulic acid

Author: Ardiansyah, H. Shirakawa, T. Koseki, K. Hashizume, and M. Komai
Abstract: The aim of this study is to investigate the effects of dietary supplIentation with the Dreselase-treated fraction (DF) of rice bran and ferulic acid (FA) on hypertension and glucose and lipid metabolism in stroke-prone spontaneously hypertensive rats (SHRSP). Male SHRSP at 4 weeks of age were divided into three groups, and for 8 weeks were fed (1) a control diet based on AIN-93M, (2) a DF of rice bran-supplIented diet at 60g/kg and (3) an FA-supplIented diet at 0.01g/kg. Means and standard errors were calculated and the data were tested by one-way ANOVA followed tolerance, plasma nitric oxide (NOx), urinary 8-hydroxy-2’-deoxyguanosine and other parameters. In particular, compared to the FA diet, the DF diet produced a significant improvIent in urinary NOx, hepatic triacylglycerol and several mRNA expressions of metabolic parameters involved in glucose and lipid metabolism. The results of the metabolic syndrome-related parameters obtained from this study suggest that the DF diet is more effective than the FA diet.
Source: British Journal of Nutrition, 97, 67 -76, (2007)

4. Genome-Wide Expression Profile of Sake Brewing Yeast under Shaking and Static Conditions

Author: M. Shobayashi, E. Ukena, T. Fijii and H. Iefuji
Abstract: To identify the genes responsible for characteristics, that are different as betweensake brewing yeasts and laboratory yeast strains, we used a DNA microarray to compare the genome-wide gene expression profiles of a sake yeast, Saccharomyces cerevisiae K-9 (kyokai 9), and a laboratory yeast, S. cerevisiae X2180-1A, under shaking and static conditions.The genes overexpressed in K-9 more than in X2180-1A were related to C-metabolism, including the HXT, ATP, and COX genes, ergosterol biosynthesis, ERG genes, and thiamine metabolism, THI genes. These genes may contribute to higher growth rates and fermentation ability and the ethanol tolerance of sake yeast.The genes underexpressed in K-9 more than in X2180-1A were CUP1-1 and CUP1-2, PHO genes, which may explain the low copper tolerance and low acid phosphatase activity of sake yeast. These underexpressed genes agree with the features and the alteration of the genome structure of sake yeast.
Source: Biosci., Biotechnol., Biochem., 71, 323-335、(2007)

5. S-adenosylmethionine (SAM)-accumulating Sake Yeast Suppresses Acute Alcohol-induced Liver Injury in Mice

Author: H. Izu, M. Shobayashi, Y. Manabe, K. Goto, H. Iefuji
Abstract: Suppressive effects on acute alcoholic liver injury by S-adenosylmethionine (SAM) and sake yeast, Saccharomyces cerevisiae Kyokai No.9, were shown previously. To enhance the suppression of acute alcoholic liver injury by sake yeast, we prepared SAM-accumulating sake yeast (SAM yeast). Male C57BL/6 mice that had been fed a diet containing 0.25% SAM yeast or sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed SAM yeast, ethanol-induced increases in both triglyceride (TG) and alanine aminotransferase (ALT) were significantly repressed. In addition, SAM yeast-fed mice did not show an ethanol-induced decrease in hepatic SAM level, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by SAM yeast. Theses results suggested that SAM yeast has much effect on the suppression of acute alcoholic liver injury in mice compared with sake yeast.
Source: Biosci. Biotechnol. Biochem., 70, 2982-9 (2006)

6. PCR using primers targeting the FLO5 and YHR213W to discern sake yeast, shochu yeast and wine yeast.

Author: H. Fukuda, Y. Zhou, S. Mikami
Abstract: In order to discern sake yeast, shochu yeast and wine yeast, a specific polymerase chain reaction (PCR) was investigated. PCR products between shochu yeast strains and sake yeast was differed by using primer designed the open reading frame of FLO5. Between wine yeasts and most of other yeasts was distinguished by primer designed the open reading frame of YHR213W. But PCR products of shochu yeast KF1 are resIble that of some wine yeasts, we could not discern thI. As a method based on the PCR to discern brewing yeasts was simpler than classical physiological methods, it was useful to discern brewing yeast with different purpose of application.
Source: J. Brew. Soc. Japan, 102, 139-145 (2007)

7. Effect of high temperature on anthocyanin composition and transcription of flavonoid hydroxylase genes in ‘Pinot noir’ grapes (Vitis vinifera)

Author: K. Mori, N. Goto-Yamamoto, M. Kitayama and K. Hashizume
Abstract: Anthocyanin accumulation in grape berry skins is influenced by temperature during the maturation period. Although it has been reported that high temperatures result in lower anthocyanin concentrations in berry skins, their effects on anthocyanin composition remain unclear. To study the effect of high temperature on anthocyanin composition, the accumulation of individual anthocyanins in the skin of ‘Pinot noir’ berries was investigated in two experiments: Experiment 1, high morning and evening temperature treatment; Experiment 2, high day-time temperature treatment. High temperatures (30°C) in the morning and evening did not the affect the total anthocyanin content in the skin, but decreased the levels of delphinidin 3-glucosides, petunidin 3-glucosides and malvidin 3-glucosides, which are 3’,5’- hydroxylated, or methylated following 3’,5’-hydroxylation. A high day-time temperature (35°C) also decreased the levels of delphinidin, petunidin and malvidin 3-glucosides. In both Experiments, grape berries grown under high temperature had a low abundance of mRNA for flavonoid 3’, 5’-hydroxylase (F3’5’H) in the skin. These results suggest that changes in the accumulation of individual anthocyanins in the skins of ‘Pinot noir’ grape berries, due to high temperature, are regulated at the level of transcription of the F3’5’H gene.
Source: J. Hort. Sci. Biotechnol., 82, 199-206 (2007)

8. Rice Protein Digestion by Sake Koji Enzymes: Comparison between Steamed Rice Grains and Isolated Protein Bodies from Rice Endosperm

Author: K.Hashizume, M.Okuda, S.Sakurao, M.Numata, T.Koseki, I.Aramaki, T.Kumamaru and H.Sato
Abstract: The digestion of proteins in steamed rice grains by sake koji enzymes under simulated sake mash conditions was analyzed by comparing the hydrolysis of steamed rice grains and heat-treated protein bodies (PBs) isolated from seven rice samples including four endosperm-storage protein mutants. The disappearance of peptides in the digest of isolated PBs was faster than that of steamed rice grains; however, more insoluble proteins formed in the case of isolated PBs. Not all of the hydrolyzed PB proteins were immediately solubilized in the digestion tests. High-molecular-weight peptides were more abundant in the solubilized digest of steamed rice grains than in that of isolated PBs. Variance in Ile, Ser, Glu, and Gly levels in the digest of steamed rice grains was relatively high among the seven samples, but was not found to be high in digests of isolated PBs. These results indicate that factors that may be derived from the steamed rice grains profoundly affect the digestion of proteins in steamed rice grains by sake koji enzymes.
Source: J.Biosci.Bioeng., 102、340-345（2006）

9. N-Linked Oligosaccharides of Aspergillus awamori Feruloyl Esterase Are Important for Thermostability and Catalysis

Author: T.Koseki, K.Takahashi, T.Handa, Y.Yamane, S.Fushinobu and K.Hashizume
Abstract: A unique N-linked glycosylation motif (Asn79-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from Aspergillus spp. To clarify the function of the flap, the role of N-linked oligosaccharides located in the flap region on the biochIical properties of feruloyl esterase (AwFAEA) from Aspergillus awamori expressed in Pichia pastoris was analyzed by rIoving the N-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. N-glycosylation-free N79A and N79Q mutant enzymes had lower activity than that of the glycosylated recombinant AwFAEA wild-type enzyme toward α-naphthylbutyrate (C4), α-naphthylcaprylate (C8), and phenolic acid methyl esters. Kinetic analysis of the mutant enzymes indicated that the lower catalytic efficiency was due to a combination of increased Km and decreased kcat for N79A, and to a considerably decreased kcat for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.
Source: Biosci.Biotechnol.Biochem., 70, 2476-2480（2006）

10. Mutational analysis of N-glycosylation recognition sites on the biochIical properties of Aspergillus kawachii α-l-arabinofuranosidase 54

Author: T.Koseki, Y.Miwa, Y.Mese, A.Miyanaga, S.Fushinobu, T.Wakagic, H.Shoun, H.Matsuzawa and K.Hashizume
Abstract: A role for N-linked oligosaccharides on the biochIical properties of recombinant α-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn83- Thr- Thr and Asn202- Ser- Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn83, Asn202, and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochIical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn202 may contribute to thermostability and catalysis.
Source: Biochimica et Biophysica Acta, 1760, 1458-1464（2006）

11. Effect of gene disruption and overexpression of acetate metabolic enzymes of yeast on acetate production during alcohol fermentation

Author: N.Goto-Yamamoto and Dang Hong Anh
Abstract: To study the mechanism of acetate production by yeast during alcohol fermentation, effect of gene disruption of ACS2, ACS3, ACS6, and ACH1, as well as overexpression of ACS1 and ACS2, on acetate production was examined using laboratory strains. Disruption of ACH1 and double disruption of ALD2 and ALD3 did not affect the acetate production. Although the overexpression of ACS2 reduced the acetate production, it also inhibited alcohol fermentation. As was reported, disruption of ALD6 reduced the acetate production. However, it caused overproduction of pyruvate, which was probably caused by shortage of reducing force.
Source: J. Brew. Soc. Japan, 101, 949-956 (2006)

12. Expression of VvmybA1 Gene and Anthocyanin Accumulation in Various Grape Organs

Author: S.T. Jeong, N.Goto-Yamamoto, K.Hashizume, S.Kobayashi and M.Esaka
Abstract: VvmybA1 is known to encode a transcriptional factor that induces the anthocyanin biosynthesis in grape skins. However, no VvmybA1 mRNA was detected in the young leaves of Cabernet Sauvignon, Dornfelder, or Muscat Hamburg, nor in the tendrils or stI epidermis of Muscat Bailey A, although these organs accumulated a little anthocyanin. These results indicate that VvmybA1 in these cultivars was transcribed specifically in the berry skin. On the other hand, VvmybA1 of Bailey Alicant A, a teinturier cultivar, was transcribed in all the organs that accumulate anthocyanin, which shows that the organ specificity of its expression was probably reduced.
Source: Am. J. Enol. Vitic., 57, 507-510 (2006)

13. Global Gene Expression Analysis of Yeast Cells during Sake Brewing

Author: H.Wu, X.Zheng, Y.Araki, H.Sahara, H.Takagi and H.Shimoi
Abstract: During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing.
Source: Appl. Environ. Microbiol., 72, 7353-7358 (2006)

14. TIperature-dependence of enantioselectivity and desymmetrization in the acetylation of 2-mono- and 2,2-di-substituted 1,3-propanediols by a novel lipase isolated from the yeast Cryptococcus spp. S-2

Author: C.Lin, Y.Hiraga, K.Masaki, H.Iefuji and K.Ohkata
Abstract: The effect of tIperature on enantioselectivity and desymmetrization in the acetylation of 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c) and 2-benzyl-2-methyl-1,3-propanediol (1d)by a novel lipase (CSL) isolated from the yeast Cryptococcus spp. S-2 was studied. Desymmetrization of 1a, 1c and 1d by CSL-catalyzed acetylation was observed in the tIperature range of -20oC to 40oC, while diacetylation of 1b occurred considerably even at 0oC. The kinetic parameters of the selectivity indicated that the acetylation of 1a is an entropy controlled process whereas the reaction of 1c and 1d is mainly controlled by the enthalpy term. In the monoacetylation of the diol 1d, the preferred configuration in the enantiomeric induction by CSL was opposite to that of immobilized porcine pancreatic lipase (PPL).
Source: Biocatalysis and Biotransformation, 24, 390-395 (2006)

15. GC-Olfactometry analysis of the aroma components in sake koji

Author: M.Takahashi, A.Isogai, H.Utsunomiya, S.Nakano, T.Koizumi and A.Totsuka
Abstract: In order to clarify the aroma compounds responsible for the aroma of sake koji, we identified these and examined the contribution of each aroma compound to the whole aroma. First of all, the intensity of koji aroma was evaluated by sensory analysis. Iploying GC-Olfactometry(GC-O) and GC-MS analysis using a head space sorptive extraction (HSSE) method, we found novel 5 compounds:1-octen-3-one (mushroom-like) , 2-methyl-2-hepten-6-one (nut-like) , methional (potato-like) , phenylacetaldehyde (rose-like) and (Z)-1,5-octadien-3-one (geranium-like) besides isobutyraldehyde, isovaleraldehyde, and 1-octen-3-ol in sake koji. The concentrations of 1-octen-3-one, 1-octen-3-ol, and phenylacetaldehyde were higher in strong-smelling koji than in weak ones. The flavor dilution factors (FD) of 1-octen-3-one and methional were highest (FD=125), and those of 2-methyl-2-hepten-6-one, Phenylacetaldehyde, and (Z) -1,5-octadien-3-one were second (FD=25). It was possible to reproduce the aroma of koji by blending 2-methyl-2-hepten-6-one, methional, 1-octen-3-one, 1-octen-3-ol, and phenylacetaldehyde. It was concluded that methional, 1-octen-3-one, and phenylacetaldehyde were important for the aroma of koji.
Source: J. Brew. Soc. Japan, 101, 957-963 (2006)

16. Sake yeast suppresses acute alcohol-induced liver injury in mice

Author: H.Izu, M.Shobayashi, Y.Manabe, K.Goto and H.Iefuji
Abstract: Brewer’s and baker’s yeasts appear to have components that protect the liver injury. Whether Sake yeast, Saccharomyces cerevisiae Kyokai No.9, also has a hepatoprotective effect has not been examined. Here we show that Sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% Sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed Sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, Sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methonine metabolism in the liver caused by ethanol was relieved by Sake yeast. These results indicate that Sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.
Source: Biosci. Biotechnol. Biochem., 70, 2488-2493 (2006)

17. Effect of Sake Cakes on D-Galactosamine-induced Liver Injury in Mice

Author: H.Izu, K.Goto and H.Iefuji
Abstract: We showed that Sake cakes suppresse D- galactosamine (GalN) -induced liver injury in mice. Male BALB/c mice received D-GalN (1200 mg/kg) after feeding a diet containing 10% Sake cakes for 14 days. In the mice fed Sake cakes, D-GalN-induced increases in glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) were significantly suppressed. D-GalN-induced increase in DNA fragmentation in liver was also suppressed by feeding Sake cakes, indicating that D-GalN-induced apoptosis was decreased by Sake cakes. These results suggest that Sake cakes protect against D-GalN-induced liver injury in mice. In addition, ingestion of Sake cakes increased hepatic S-adenosylmethionine (SAM) that is an intermediate in methionine metabolism and is a precursor of glutathione. Sake cakes may enforce methionine metabolism and contribute to suppression of liver injury.
Source: J. Brew. Soc. Japan, 101, 893-899 (2006)

18. Rsp5 regulates expression of stress proteins via post-translational modification of Hsf1 and Msn4 in Saccharomyces cerevisiae

Author: Y. Haitania, H. Shimoi, H. Takagi
Abstract: Rsp5 is an essential E3 ubiquitin ligase in Saccharomyces cerevisiae and is known to ubiquitinate plasma membrane permeases followed by endocytosis and vacuolar degradation. We previously isolated the rsp5 mutant that is hypersensitive to various stresses, suggesting that Rsp5 is involved in degradation of stress-induced abnormal proteins. Here, we analyzed the ability to refold the proteins by stress proteins in the rsp5 mutant. The transcription of stress protein genes in the rsp5 mutant was significantly lower than that in the wild-type strain when exposed to temperature up-shift, ethanol or sorbitol. Interestingly, the amounts of transcription factors Hsf1 and Msn4 were remarkably defective in the rsp5 mutant. These results suggest that expression of stress proteins are mediated by Rsp5 and that Rsp5 primarily regulates post-translational modification of Hsf1 and Msn4.
Source: FEBS Lett., 580, 3433-3438(2006)

19. Aspergillus oryzae strains with a large deletion of the aflatoxin biosynthetic homologous gene cluster differentiated by chromosomal breakage

Author: Yun-Hae LEE, M. Tominaga, R. Hayashi, K. Sakamoto, O. Yamada, O. Akita
Abstract: Recently we divided Aspergillus oryzae RIB strains into group 1, having seven aflatoxin biosynthesis homologous genes (aflT, nor-1, aflR, norA, avnA, verB, and vbs), and group 2, having three homologues (avnA, verB, and vbs). Here, partial aflatoxin homologous gene cluster of RIB62 from group 2 was sequenced and compared with that of RIB40 from group 1. RIB62 showed a large deletion upstream of ver-1 with more than half of the aflatoxin homologous gene cluster missing including aflR, a positive transcriptional regulatory gene. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a unique sequence of about 8 kb and a telomere. Southern analysis of A. oryzae RIB strains with four kinds of probe derived from the unique sequence of RIB62 showed that all group 2 strains have identical hybridizing signals. Polymerase chain reaction with specific primer set designed to amplify the junction between ver-1 and the unique sequence of RIB62 resulted in the same size of DNA fragment only from group 2 strains. Based on these results, we developed a useful genetic tool that distinguishes A. oryzae group 2 strains from the other groups’strains and propose that it might have differentiated from the ancestral strains due to chromosomal breakage.
Source: Appl. Microbiol. Biotechnol., 72, 339-345 (2006)

20. Simple Method for the Determination of Ethyl Carbamate in Alcoholic Beverages

Author: T.Hashiguchi, Y. Igi, K. Goto
Abstract: We developed a simple method for the determination of ethyl carbamate in alcoholic beverages. Butyl carbamate was added to sake sample as an internal standard and then added to Extrelut column. Ethyl carbamate and butyl carbamate were eluted with hexane-ethyl acetate (1:1). The eluate was concentrated in a rotary evaporator or TurboVap concentrator. The concentrate was analyzed by GC/MS in a selected ion monitoring mode. The detection limit and quantification limit of ethyl carbamate were 2.5 and 7.5μg/l. This method was suitable for the analysis of many sake, Shaoxing rice wine (shokoshu), and plum liquor (umeshu) samples. Applying this extraction and concentration method, we could analyze ethyl carbamate in sake by FID-GC. Twenty times concentrated extract of sake obteined the approximate ethyl carbamate concentration.
Source: J. Brew. Soc. Japan、101、519-525、(2006)

21. Furan content in Alcoholic Beverages

Author: T. Hashiguchi, K. Goto
Source: Report of the Research Institute of Brewing、178、41-44、(2006)

22. Analysis of Sake Component Presented to the Sake Contest in 2004

Author: S. Nakano, H. Utsunomiya, A. Isogai, J. Hiramatsu
Abstract: We made sum of the inquiry cards and analyzed Sake components presented to the Sake Contest in 2004 in order to study and investigate the current state and the trends of their quality. The average values of alcohol, nihonshudo(gravity) and acidity were 17.7%, 4.0 and 1.3. In case of gold prize winners, the average values of isoamylalcohol, isoamylacetate and ethylcaproate were 123ppm, 2.2ppm and 6.6ppm, respectively.
Source: Report of the Research Institute of Brewing、178, 1-12 (2006)

23. Square-plate culture method allows detection of differential gene expression and screening of novel, region-specific genes in Aspergillus oryzae

Author: K. Masai, J. Maruyama, K. Sakamoto, H. Nakajima ,O. Akita, K. Kitamoto
Abstract: When grown on solid agar medium, the mycelium of a filamentous fungus, Aspergillus oryzae, forms three morphologically distinct regions: the tip (T), white (W), and basal (B) regions. In this study, we developed the square-plate culture method, a novel culture method that enabled the extraction of mRNA samples from the three regions and analyzed the differential gene expression of the A. oryzae mycelium in concert with the microarray technique. Expression of genes involved in protein synthesis was predominant in the T region; relative expression was, at most, six times higher in the T region compared to the other regions. Genes encoding hypothetical proteins were expressed at high levels in the W and B regions. In addition, genes coding transporters/permeases were predominantly transcribed in the B region. By analyzing the expression patterns of genes in the three regions, we dIonstrated the dynamic changes in the regulation of gene expression that occur along the mycelium of filamentous fungi. Consequently, our study established a method to analyze and screen for region-specific genes whose function may be essential for morphogenesis and differentiation in filamentous fungi and whose traits may be beneficial to the biotechnology industry.
Source: Appl Microbiol Biotechnol. 2006 Aug;71(6):881-891

24. Yeast genes involved in response to lactic acid and acetic acid: Acidic conditions caused by the organic acids in Saccharomyces cerevisiae cultures induces expression of intracellular metal metabolism genes regulated by Aft1p

Author: M. Kawahata, K. Masaki, T. Fujii and H. Iefuji
Abstract: We found that the acidic condition affects metal metabolism in this study using two types of genome wide analysis to investigate yeast genes involved in response to lactic acid and acetic acid. One is an expression analysis using DNA microarray to investigate yeast acid shock response as the first step to adapt to an acidic condition and yeast acid adaptation response as the result of maintaining integrity in the acidic condition. The other is a functional screening using the non-essential genes deletion collection of S. cerevisiae. The expression analysis showed that genes involved in stress response such as YGP1, TPS1, and HSP150 were induced under the acid shock response conditions. Genes such as FIT2, ARN1, and ARN2 involved in metal metabolism regulated by Aft1p were induced under the acid adaptation conditions. AFT1 was found to be induced under the acid shock response conditions and under the acid adaptation condition by lactic acid. Moreover, GFP-fused Aft1p was localized to the nucleus in cells grown in media containing lactic acid, acetic acid, or hydrochloric acid. The expression analysis and the functional screening both suggested that the acidic condition affects cell wall architecture. The depletion of cell wall components encoded by SED1, DSE2, CTS1, EGT2, SCW11, SUN4, and YNL300W and histone acetyltransferase complex proteins encoded by YID21, EAF3, EAF5, EAF6, and YAF9 increased resistance to lactic acid media. Depletion of the cell wall mannoprotein Sed1p gave resistance to lactic acid, although the expression of SED1 was induced by exposure to lactic acid. Depletion of V-ATPase and HOG MAPK proteins caused acid sensitivity. Moreover, our quantitative PCR analysis showed that expression of PDR12 increased under the acid shock response condition by lactic acid and decreased under the acid adaptation condition by hydrochloric acid.
Source: FIS Yeast Res. 6, 924-936 (2006)

25. Nitrogen balance of the sake mash

Author: K. Hashizume, M. Okuda, M. Numata, M. Tajiri, and T. Koseki
Abstract: The behavior of nitrogen was analyzed in conditions of small scale sake brewing and subsequent pressing under actual manufacturing like conditions. About 55% of the nitrogen in the material rice was liquefied in the sake mash, 30-33% was transferred to the sake obtained, and 22-25% was transferred to the yeast. About 44-47% of the nitrogen in the sake was free amino acid, and almost all the residue of the nitrogen was believed to be peptides. We could not classify about 17-20% of nitrogen in the sake cake and lee.
Source: J. Brew. Soc. Japan、101、723-726（2006）

26. Investigation of PCR using primers targeted the minisatellite sequence to discern sake yeast strains

Author: H. Fukuda, Y. Zhou, S. Mikami
Abstract: In order to discern sake yeast strains, a specific polymerase chain reactions (PCR) which targeted 22 ORFs containing the minisatellite sequences were tested. We found the difference in DNA fragments amplified by using five pair primers designed YOR009W(TIR4), YDR150W(NUM1), YKR102W(FLO10), YDL037C(BSC1), and YNL190W of sake yeast strains. Since shochu yeast strains were difference in PCR products by using primers that sake yeast strains were no difference in the length of PCR products, the lower polymorphic of the amplified DNA fragments of sake yeast strains were speculated to caused that the genetic difference of these strains was lower in compare with other brewing yeast strains.
Source: J. Brew. Soc. Japan、101、601-613（2006）

27. Analysis of Traditional Shochu Presented to the 28th Sake Contest in 2005

Author: S. Mikami, H. Fukuda, Y. Satoh and J. Hiramatsu
Source: Report of the Research Institute of Brewing、178, 31-40 (2006)

28. Results of Sensory Evaluation and Analysis of the Western Type Alcoholic Beverages Presented to the 43th Contests

Author: K. Hashizume, N. Goto, K. Koyama and J. Hiramatsu
Source: Report of the Research Institute of Brewing、178, 13-30 (2006)

29. Flavor Terminology and Reference Standards for Sensory Analysis of Sake

Author: H. Utsunomiya, A. Isogai, H. Iwata, S. Nakano
Source: Report of the Research Institute of Brewing, 178, 45-52, (2006)

30. Chemo-enzymatic production of fuel ethanol from cellulosic materials utilizing yeast expressing β-glucosidases

Author: T. Uryu, M. Sugie, S. Ishida, S. Konoma, H. Kato, K. Katsuraya, K. Okuyama, G. Borjihan, K. Iwashita, H. Iefuji
Abstract: Ethanol was produced in a considerably high yield by fermenting hydrolyzates from cellulosic materials by means of a recombinant laboratory yeast expressingβ-glucosidases. Tissue paper, cotton, and sawdust were hydrolyzed by two-step sulfuric acid hydrolysis to give mixtures containing glucose, cellobiose, and high cello-oligosaccharides. After the cellulosic material was partially hydrolyzed with 80% sulfuric acid, the hydrolysis was continued with 5% sulfuric acid. Except for non-carbohydrate components, all constituents in the hydrolyzates were fermented by the yeast that was preincubated in the medium that the plasmid encoded by the β-glucosidases gene was kept in the multiplicated yeast. A solution containing 4% hydrolyzates from paper was fermented to give as high as 1.9% maximum ethanol concentration and 70% ethanol conversion. Cotton also gave a similar result. Sawdust was converted into ethanol in approx 22% conversion. Accordingly, it was revealed that the β-glucosidases-expressing yeast can ferment the cello-oligosaccharided obtained by hydrolysis of cellulosic materials into ethanol. In addition, a hydrolyzate cotaining a high glucose proportion gave a high ethanol concentration in a short time.
Source: Applied Biochem. Biotechnol., 135, 15-31, 2006

31. Effects of Candida utilis treatment on the nutrient value of rice bran and the effect of Candida utilis on the degradation of forages in vitro.

Author: S. Ando, Y. Nishiguch, K Hayasaka, H. Iefuji, J. Takahashi
Abstract: Candida utilis can assimilate fatty acids, so it was hypothesized that the treatment of rice by Candida utilis would improve feed quality by reducing fat content and adding the yeast function that would stimulate rumen microbes. In this study, the oil assimilation ability of Candida utilis IFO1086, 0988, 0626 and the effect of treatment of Candida utilis IFO1086, IFO0626 on the nutrient contents of rice bran were examined. The effect of Candida utilis adding on the in vitro degradability of forage was also investigated. It was found that the oil assimilating ability of IFO1086 and IFO0626 was significantly (P＜0.01) higher than that of IFO0988. Candida utilis treatment reduced the EE content and increased the CP, ADF and NDF percentage. The absolute amount of ether extract was decreased by 35.9% in IFO0626 treatment. The absolute amount of crude protein was not changed by yeast treatment. The ADF and NDF amounts were increased. The addition of Candida utilis increased in vitro forage degradability significantly (p＜0.05), Based on these results it can be postulated that treatment of rice bran by Candida utilis may improve feed quality by reducing fat content, increasing the CP content and adding the function of yeast for stimulating rumen microbes.
Source: Asia-Aust.J.Anim.Sci. 19, 806-810, 2006

32. Components of Taru-Sake and Their Physiological Activity

Author: Y.Orihara, H.Wake, H.Utsunomiya, H.Aoshima
Abstract: Esters, higher alcohols, and sesquiterpenoids in Taru-Sake (sake stored in a cedar cask) stored in Akamidaru and Kotsukidaru for various length of time were analyzed by gas chromatography. Glucose and the absorbance at 490nm were also analyzed. The storage of sake in a cedar cask increased the sesquiterpenoids and the absorbance at 490 nm, but esters, higher alcohols, and glucose were not affected by the storage. Sake caused the response of GABAＡ receptors expressed in Xenopus oocytes, suggesting the presence of GABA in the sake. However, the extract of cask storaged sake with pentane did not potentiate the GABAＡ receptor response, though that of whiskey did. The storage of sake in a cedar cask increased the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and the total polyphenol.
Source: J. Brew. Soc. Japan, 101,349-356 (2006)

33. Analysis of Shochu and White spirits and classification by their compounds

Author: H.Utsunomiya, M.kida, N.Maki, A.Isogai, H.Iwata, T.Nishiya
Abstract: To classify the flavors of Shochu and White spirits, we analyzed 20 volatile compounds by head space solid phase micro extraction (SPME) , 7 volatile compounds by direct head space analysis, and 2 volatile compounds by enzymatic analysis among 28 Shochu-otsu, 2 Blended shochu, 3 Shochu-ko, 4 Baijiu (Chinese liquor), 3 Vodka, 3 White rum, 3 Gin, and 4 other Spirits. Acidity, TBA-value, and pH were also determined. In a cluster analysis using these 32 elIents, Shochu-otsu was distinguished from the other spirits. In a stepwise discriminant analysis procedure using acetic acid, ethyl acetate, 3-methyl-1-butanol, and dodecanol, they were classified into three groups: Shochu-otsu, Light spirits (Shochu-ko, Blended shochu, Vodka and White rum), and Baijiu. It was also possible to classify Shochu-otsu into six raw material categories by a discriminant analysis using methanol, 1-propanol, 2-methyl-1-propanol, and ethyl octanoate.
Source: J. Brew. Soc. Japan, 101,446-457 (2006)

34. A skin color mutation of grapevine, from black-skinned Pinot noir to white-skinned Pinot blanc, is caused by deletion of the functional VvmybA1 allele

Author: H. Yakushhi, S. Kobayashi, N. Goto-Yamamoto, S.T. Jeong, T. Sueta, N. Mitani, A. Azuma
Abstract: A white-wine grape, Pinot Blanc, is thought to be a white-skinned mutant of a red-wine grape, Pinot Noir. Pinot Noir was heterozygous for VvmybA1. One allele was the non-functional VvmybA1a, and the other was the functional VvmybA1c. In Pinot Blanc, however, only VvmybA1a was observed, and the amount of VvmybA1 DNA in Pinot Blanc was half that in Pinot Noir. These findings suggest that deletion of VvmybA1c from Pinot Noir resulted in Pinot Blanc.
Source: Biosci. Biotechnol. Biochem., 70 (6), 1506-1508 (2006)

35. Proteomic Analysis of Extracellular Proteins from Aspergillus oryzae Grown under Submerged and Solid-State Culture Conditions

Author: K. Oda, D. Kakizono, O. Yamada, H. Iefuji, O. Akita, and K. Iwashita
Abstract: Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to twodimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attIpted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as α-amylase (TAA) and β-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture.
Source: Appl. Environ. Microbiol., 72, 3448-3457 (2006)

36. Development of a New Glass Bottle Compatible with Transparency and Maintaining Sake Quality.

Author: Y. Nomura, K. Kaneko, K. Satoh, K. Kiso and A. Mizuno
Abstract: We researched the effects of the wave length of sunlight on the deterioration of sake quality by irradiating sunlight through sharp cut filters which block light under a specific wave length and pass light over a specific wave length. To measure deterioration of sake quality, we analyzed sake coloring, harman, antioxidant capacity, value of TBA reaction, and 3-D-G (3-Deoxygulucosone) of the sake following irradiated sunlight through a sharp cut filter. As a result, we found that sunlight under a 440 nm wave length deteriorates sake quality and that sunlight of over a 440 nm wave length does not. We then attIpted to make a new glass bottle which blocks the light under a 440 nm wave length and has high transmittance of light over a 440 nm wave length. We developed a new glass bottle which maintained sake quality in spite of being irradiated with sunlight and which allows more transparency than the amber glass in current use.)
Source: J. Brew. Soc. Japan, 101,275-282 (2006)

37. Application of a PCR using primers based on genes encoding cell wall proteins to discern commercial brewing yeast strains

Author: H. Fukuda, Y. Zhou, S. Mikami
Abstract: In order to discern commercial brewing yeast strains, a specific polymerase chain reaction (PCR) which targeted the open reading frame of AGA1, DAN4, HSP150 and SED1, was tested. Shochu yeast strains and wine yeast strains dIonstrated that PCR products are highly polymorphic in length, and we could discern all of the test wine yeast strains and eight of the test shochu yeast strains. However, as PCR products of sake yeast are lowly polymorphic, we could not discern these except for K-11 individually. As the method based on the PCR to discern brewing yeast strains was simpler than classical physiological methods, it was useful to discern brewing yeast strains with these primers.
Source: J. Brew. Soc. Japan,, 101, 357-364 (2006)

38. Treatment of Ethylendiamine and Hexamethylendiamine Using Yeast

Author: T. Watanabe, O. Suzuki, T. Fujii, N. Ozaki, K. Masaki and H. Iefuji
Abstract: The purpose of this study is to develop a new method for treating wastewater containing diamines using yeasts. We isolated yeasts that could assimilate ethylendiamine (EDA) and hexamethylendiamine (HMDA) from approximately fifty yeasts stocked in the National Research Institute of Brewing that are mainly wastewater treatment yeasts. Consequently, three yeasts, Hansenula fabianii J640, Hansenula anomala J224-1, and Candida utilis IFO0626 were revealed to be able to grow well in culture containing EDA or HMDA as sole nitrogen source. In these yeasts, C. utilis IFO0626 showed the best rIovable ability, that is, it rIoved greater than 98% of 1,000 ppm EDA dichloride and HMDA dichloride in 48 h. The assimilation of 1 g of these diamine dichlorides needed approximately 10 g of glucose as a carbon source; therefore the ratio of C:N was calculated as 100:5. These yeasts were shown to prefer ammonium sulfate and urea to EDA and HMDA as nitrogen sources. It was shown that EDA or HMDA could be more effective decomposed by increasing the initial number of yeast cells in the treatment medium.
Source: Journal of the Japan Society on Water Environment, 29, 339-342（2006）

39. Determination of Alcohol and Real Extract by Anton Paar Alcolyzer

Author: T. Kaneko, K. Ito, T. Izumi, A. Matsuyama, N. Mukai, T. Ohuchi, M. Takeuchi, S. Tatematsu, and T. Teramoto
Abstract: Determination of Alcohol and Real Extract by Anton Paar Alcolyzer was reported. No statistically significant differences were found between the Anton Paar Alcolyzer and SCABA methods for the determination of alcohol in beer, happou-shu, and nonalcoholic beer. The Anton Paar Alcolyzer method resulted in significantly higher real extract results than did the SCABA method for beer, happou-shu, and nonalcoholic beer. The repeatability and reproducibility coefficients of variation were judged acceptable.
Source: J. Am. Soc. Brew. Chem., 64, 252-254 (2006)