Depth-selective x-ray diffraction using energy-dispersive x-ray detector and straight capillary optics.

Depth-selective x-ray diffraction (XRD) technique was developed. In this technique, XRD spectra were measured using an energy dispersive (ED) x-ray detector at fixed angles. A straight capillary optic was used to define the incident x-ray beam, and a second straight capillary defined the beam path from the sample to detector. Thereby, only the XRD spectrum at the small intersection of two capillary optics could be obtained. A depth-selective XRD is possible by changing the sample position in depth. Many XRD peaks appear in a high-energy range more than 10 keV in the ED spectrum. The detection of these peaks will be advantageous for depth analysis because of low absorption in the sample. Depth-selective measurement would be advantageous over general XRD. In this study, depth-selective and ED-XRD spectra are demonstrated for the layered sample, which consisted of film-like Si powder and a muscovite film.

Spatial Distribution and Characteristics of Protein Content and Composition in Japonica Rice Grains: Implications for Sake Quality.

The quantity and composition of rice proteins play a crucial role in determining taste quality of sake, Japanese rice wine. However, the spatial distribution of proteins within rice grains, especially in endosperm tissue, and the differences between rice varieties remain unclear. Here, we analyzed the crude protein contents and composition ratios of table (Nipponbare and Koshihikari) and genuine sake rice varieties (Yamadanishiki, Gohyakumangoku, Dewasansan, Dewanosato, and Yumenokaori) to elucidate their spatial distribution within the Japonica rice grain endosperm. Seven sake rice varieties were polished over five harvest years using a brewer's rice-polishing machine. We obtained fractions at 90-70% (the outermost endosperm fraction), 70-50%, 50-30%, and 30-0% (the central region of the endosperm fraction). Yamadanishiki and Dewanosato exhibited considerably lower crude protein contents than the other cultivars. After applying SDS-PAGE, the protein composition, comprising glutelin/total protein (G/TP), prolamin/TP (P/TP), and G/P ratios of these fractions was determined. In white rice (at a 90% rice-polishing ratio), the average ratio of the major protein composition was G/TP 41%, P/TP 21%, and G/P ratios of 1.97. Gohyakumangoku and Yamadanishiki had higher G/TP ratio, while Dewanosato had a lower value. Despite having lower crude protein contents, Yamadanishiki and Dewanosato exhibited significantly varying G/TP ratios. The G/TP ratio markedly varied among rice varieties, particularly in the rice grains' central region. The 50-30% fraction had the highest P/TP ratio among all tested rice varieties, suggesting spatial differences in P/TP within rice grains. Koshihikari had the lowest P/TP ratio. In addition, the 50-30% fraction had the lowest G/P ratio among all tested rice varieties, with Gohyakumangoku having the highest G/P ratio. Dewanosato had the lowest G/P value, and this value significantly differed from that of Yamadanishiki in the 30-0% fraction. We found substantial differences in protein composition within distinct spatial regions of rice grains, and larger differences among rice varieties were observed in the rice grain's central region.

Carbon stable isotopic compositions of glucose and ethanol in sake after simultaneous saccharification and fermentation processes.

The carbon stable isotopic composition of glucose is transferred to that of ethanol during the simultaneous saccharification and fermentation processes of sake production. However, there is limited information regarding carbon isotope discrimination between the ingredient rice and the sake components. Our fermentation experiments show that the carbon stable isotopic composition of rice is intermediate between those of glucose and ethanol in sake and do not differ significantly from those of rice koji and sake lees. Carbon isotope discrimination from rice to ethanol and from glucose to ethanol is 0.9 ± 0.1‰ (mean ± standard deviation, n = 18) and 1.9 ± 0.2‰, respectively. This is approximately half of the isotope discrimination observed in grape wine due to the saccharification process during sake manufacture. Carbon isotope discrimination from ingredient rice to sake components provides valuable insights for the manufacturing process and the authentication of sake.

Modeling the sake brewing characteristics of rice from brown rice metabolites.

Sake, soy sauce, miso (Japanese bean paste), and beer are made from grains. The characteristics of the grain significantly affect the quality of the final product. Many studies have been performed to evaluate the sake-brewing characteristics of rice. However, current rice analysis methods are time and labor intensive and require large samples. We developed a novel method for predicting the brewing characteristics of sake rice using <1 g of sample. Brown rice extracts were analyzed by ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry, and mass chromatogram data were used as explanatory variables. The objective variables were the physical and chemical properties of the rice, the enzymatic activity of the rice-koji, the fermentation properties of the sake mash, the standard analytical values of the sake, and the flavor component concentrations in the sake. Prediction models were developed using the orthogonal projections to latent structures method. The prediction performances of the models were verified, and 32 out of the 54 objective variables were used in well-performing models. In conclusion, we developed a method for predicting the rice properties and brewing characteristics from results acquired by analyzing <1 g of brown rice. The method is a powerful tool for breeding new sake rice cultivars for good brewing characteristics in early generations and will improve our understanding of fluctuations in the brewing characteristics of sake rice before each sake brewing season starts.

Aspergillus oryzae acetamidase catalyzes degradation of ethyl carbamate.

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.

Theoretical Modeling of Electronic Structures of Polyiodide Species Included in α-Cyclodextrin.

The molecular mechanism of blue color formation in an iodine-starch reaction is studied by employing the iodine-α-cyclodextrin (α-CD) complex as a practical model system that resembles the structural properties of the blue amylose-iodine complex. To this end, we construct, using the quantum chemistry method, a molecular model of the complex (I5-/Li+/2α-CD) that consists of one I5-, two molecules of α-CD, and a lithium cation, and this model is employed as a basic unit in constructing the structural models of polyiodide ions (I5-)n. The initial structure in the geometry optimization is adopted from the α-CD-iodine complex structure obtained from the X-ray crystallography study. The structural models of (I5-)n are built by adding the basic unit n times along the crystal axis and by optimizing the structure using quantum mechanics/molecular mechanics (QM (iodine)/MM (α-CD)) calculations. The electronic absorption spectra of the resulting model structures are calculated by time-dependent density functional theory (TD-DFT). We find that I5- acts as a basic unit of coloration in the visible region. The visible color originates from the electronic transition within the I5- molecule, and any charge transfer between the I5- ion and either of α-CD or a coexisting counter cation is not involved. We also reveal that the electronic transitions of (I5-)n are delocalized, which accounts for the well-known observation that the color of the iodine-starch reaction becomes bluish with an increase in the chain length of amylose. Furthermore, the preresonance Raman spectra calculated from the model suggest that the vibrational motions are localized in the I5- subunit dominantly. A comparison between an experimental absorption spectrum feature of the α-CD-iodine complex and the calculated ones of (I5-)n ions with various n values suggests that (I5-)4 polyiodide ions tend to be populated dominantly in the α-CD-iodine complex under aqueous conditions.

Long-term responses to climate change of the carbon and oxygen stable isotopic compositions and gelatinization temperature of rice.

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°C to 70.0 °C) 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.

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

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 (-0.612**) to their formation whereas an ACP to Cbz-Glu-Tyr was not (r = 0.220). It was suggested that (pGlu) oligo-peptide ethyl esters were formed during the decomposition of bitter-tasting peptides to which the ACP to (pGlu)LFGPNVNPWH might contribute.

Wash- and Amplification-Free Digital Immunoassay Based on Single-Particle Motion Analysis.

Digital enzyme-linked immunosorbent assay (ELISA) is a powerful analytical method for highly sensitive protein biomarker detection. The current protocol of digital ELISA requires multiple washing steps and signal amplification using an enzyme, which could be the potential drawback in in vitro diagnosis. In this study, we propose a digital immunoassay method, which we call "Digital HoNon-ELISA" (digital homogeneous non-enzymatic immunosorbent assay) for highly sensitive detection without washing and signal amplification. Target antigen molecules react with antibody-coated magnetic nanoparticles, which are then magnetically pulled into femtoliter-sized reactors. The antigens on the particles are captured by antibodies anchored on the bottom surface of the reactor via molecular tethers. Magnetic force enhances the efficiency of particle encapsulation in the reactors. Subsequent physical compartmentalization of the particles enhances the binding efficiency of antigen-carrying particles to the antibodies. The tethered particles show characteristic Brownian motion within a limited space by the molecular tethering, which is distinct from free diffusion or nonspecific binding of antigen-free particles. The number of tethered particles directly correlates with the concentration of the target antigen. Digital HoNon-ELISA was used with a prostate-specific antigen to achieve a detection of 0.093 pg/mL, which is over 9.0-fold the sensitivity of commercialized highly sensitive ELISA (0.84 pg/mL) and comparable to digital ELISA (0.055 pg/mL). This digital immunoassay strategy has sensitivity similar to digital ELISA with simplicity similar to homogeneous assay. Such similarity allows for potential application in rapid and simple digital diagnostic tests without the need for washing and enzymatic amplification.

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

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

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

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

Rice used for Japanese sake making.

Sake is a Japanese traditional alcoholic beverage made from rice and water. Recently, its export and its production in countries other than Japan have increased. In accordance, both the breeding and the cultivation of sake rice varieties have been extended to wide areas of Japan. To breed new sake rice cultivars and to cultivate rice with high suitability for sake production, an understanding of the properties of the ingredient rice grains and the factors affecting sake making is important. The influence of various rice properties, such as starch structure, protein, water absorption, and grain polishing, on sake making are being revealed in detail. In this review, the properties of rice used for sake making are summarized in terms of fermentation science and rice cultivation.

Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation.

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

Rotational Dynamics of Solutes with Multiple Single Bond Axes Studied by Infrared Pump-Probe Spectroscopy.

To investigate the relationship between the structural degrees of freedom around a vibrational probe and the rotational relaxation process of a solute in solution, we studied the anisotropy decays of three different N3-derivatized amino acids in primary alcohol solutions. By performing polarization-controlled IR pump-probe measurements, we reveal that the anisotropy decays of the vibrational probe molecules in 1-alcohol solutions possess two decay components, at subpicosecond and picosecond time scales. On the basis of results showing that the fast relaxation component is insensitive to the vibrational probe molecule, we suggest that the anisotropy decay of the N3 group on a subpicosecond time scale results from a local, small-amplitude fluctuation of the flexible vibrational probe, which does not depend on the details of its molecular structure. However, the slow relaxation component depends on the solute: with longer alkyl chains attached to the N3 group, the anisotropy decay of the slow component is faster. Consequently, we conclude that the slow relaxation component corresponds to the reorientational motion of the N3 group correlated with other intramolecular rotational motions (e.g., rotational motions of the neighboring alkyl chain). Our experimental results provide important insight into understanding the rotational dynamics of solutes with multiple single bond axes in solution.

Discrimination of wine from grape cultivated in Japan, imported wine, and others by multi-elemental analysis.

Differences in mineral concentrations were examined among three types of wine in the Japanese market place: Japan wine, imported wine, and domestically produced wine mainly from foreign ingredients (DWF), where Japan wine has been recently defined by the National Tax Agency as domestically produced wine from grapes cultivated in Japan. The main objective of this study was to examine the possibility of controlling the authenticity of Japan wine. The concentrations of 18 minerals (Li, B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba, and Pb) in 214 wine samples were determined by inductively coupled-plasma mass spectrometry (ICP-MS) and ICP-atomic emission spectrometry (ICP-AES). In general, Japan wine had a higher concentration of potassium and lower concentrations of eight elements (Li, B, Na, Si, S, Co, Sr, and Pb) as compared with the other two groups of wine. Linear discriminant analysis (LDA) models based on concentrations of the 18 minerals facilitated the identification of three wine groups: Japan wine, imported wine, and DWF with a 91.1% classification score and 87.9% prediction score. In addition, an LDA model for discrimination of wine from four domestic geographic origins (Yamanashi, Nagano, Hokkaido, and Yamagata Prefectures) using 18 elements gave a classification score of 93.1% and a prediction score of 76.4%. In summary, we have shown that an LDA model based on mineral concentrations is useful for distinguishing Japan wine from other wine groups, and can contribute to classification of the four main domestic wine-producing regions of Japan.

The relationship between rice protein composition and nitrogen compounds in sake.

The relationship between the protein composition of rice and nitrogen compounds (amino acids and oligo-peptides) in the produced sake were investigated using endosperm protein mutant rice (LGC-1, LGC-Jun, Kx433, QA28), sake rice (Yamadanishiki) and cooking rice (Nipponbare, Nihonmasari, Koshihikari). The total nitrogen concentration, amino acid concentration and most peptide peak areas determined by RP-HPLC and gel filtration chromatography of the produced sake were lower when sake was made from a low glutelin content rice mutant compared with other rice varieties. The concentration of nitrogen compounds in the sake positively correlated with the glutelin content of the highly milled rice grains used for sake production. Sake produced using a low glutelin content rice mutant is generally evaluated as having a light taste. Our findings suggest that nitrogen compounds (oligo-peptides and amino acids) derived from rice glutelin significantly contribute to the taste of sake.

Vibrational dynamics of azide-derivatized amino acids studied by nonlinear infrared spectroscopy.

Recently, biomolecules which are labeled by azide or thiocyanate groups in solutions and proteins have been studied to examine microscopic environment around a solute by nonlinear infrared (IR) spectroscopy. In this study, we have performed two-dimensional infrared (2D-IR) spectroscopy to investigate the vibrational frequency fluctuations of two different azide-derivatized amino acids, Ala (N3-Ala) and Pro (N3-Pro), and N3(-) in water. From the 2D-IR experiments, it was found that the frequency-frequency time correlation function (FFTCF) of solute can be modeled by a delta function plus an exponential function and constant. FFTCF for each probe molecule has a decay component of about 1 ps, and this result suggests that the stretching mode of the covalently bonded azide group is sensitive to the fluctuations of hydrogen bond network system, as found in previous studies of N3(-) in water. In contrast to FFTCF of N3(-), FFTCF of the azide-derivatized amino acids contains static component. This static component may reflect dynamics of water affected by the solutes or the structural fluctuations of the solute itself. We also performed the IR pump-probe measurements for the probe molecules in water in order to investigate vibrational energy relaxation (VER) and reorientational relaxation. It was revealed that the charge fluctuations in the azide group are significant for the VER of this mode in water, reflecting that the VER rate of N3(-) is faster than those of the azide-derivatized amino acids. While the behaviors of the anisotropy decay of N3-Ala and N3(-) are similar to each other, the anisotropy decay of N3-Pro contains much slower decaying component. By considering the structural difference around the vibrational probe between N3-Ala and N3-Pro, it is suggested that the structural freedom of the probe molecules can affect the reorientational processes.

The transfer of radioactive cesium and potassium from rice to sake.

Using rice grains contaminated with radioactive cesium ((134)Cs and (137)Cs) that was released by the Fukushima Daiichi Nuclear Power Plant Accident in March of 2011, we investigated the behaviors of the radioactive cesium and potassium (total K and (40)K) during sake brewing. Cesiumis a congener of K, and yeast cells have the ability to take up Cs using known K transporters. During rice polishing, the concentrations of radioactive Cs and K in the polished rice grains decreased gradually until a milling ratio (polished rice weight/brown rice weight) of 70% was reached. No significant changes were observed below this milling ratio. Sake was brewed on a small scale using the 70% polished rice. The transfer ratio of radioactive Cs to sake and to the sake cake was significantly different than the ratio of K. Approximately 36% and 23% of radioactive Cs in the polished rice was transferred to the sake and sake cake, respectively; however, 40% was removed by washing and steeping the rice grains. On the other hand, 25% and 40% of K in the polished rice was recovered in the sake and sake cake, respectively, and 35% was removed by washing and steeping the rice grains. From the present results, the concentration of radioactive Cs in sake would be 4 Bq/kg fresh weight, which is well below the regulation values (100 Bq/kg), even using brown rice containing 100 Bq/kg of radioactive Cs.

The transfer of stable ¹³³Cs from rice to Japanese sake.

Possible contamination by radioactive cesium (Cs), released by the Fukushima Daiichi Nuclear Plant Accident in Japan on March 2011, has been a matter of concern with respect to Japanese sake made from rice grains cultivated in affected fields. In this study, the behavior of stable (133)Cs, which is a useful analogue for predicting the behavior of radioactive Cs, was investigated in the production of sake using rice grains harvested in Japan in 2010. The concentration of stable (133)Cs in the polished rice grains decreased gradually with decreasing milling ratios until a ratio of 70% was reached, and below that point, it did not change significantly. The (133)Cs concentration in the 70% polished rice was approximately 20% of that found in brown rice. The sake was brewed on a small scale using 70% polished rice, and the transfer of (133)Cs from rice to sake was examined. Approximately 30-40% of (133)Cs in the 70% polished rice was removed during the washing and the steeping of the rice grains, and approximately 40% of the (133)Cs in the 70% polished rice was transferred to the sake. If the radioactive Cs species behaves similarly, these results suggest that brown rice containing 100 Bq/kg radioactivity of Cs would generate 70% polished rice grains containing 20 Bq/kg and that the sake brewed from these grains would contain 3-5 Bq/kg.