Ken'ichi Ohtsubo

Detection of changes in taste of japonica and indica brown and milled rice (Oryza sativa L.) during storage using physicochemical analyses and a taste sensing system.

Changes in the taste of japonica, hybrid, and indica brown and milled rice, stored for 10 months at low (5 degrees C, 65-70% relative humidity) and room temperatures were observed by physicochemical analyses and a novel method using a taste sensing system. During storage, some properties increased or decreased while others were fairly constant. The main taste components of cooked rice such as sweetness (sucrose) and umami tastes (glutamic acid and aspartic acid) were reduced during storage, whereas glucose and fructose increased. The increase of fat acidity and consequent decrease of the pH value of the cooking solution may contribute to the off-taste of cooked stored rice. A taste sensing system with 10 lipid membrane sensors was also used to classify new and old rice samples using principal component analysis. Fresh and room temperature stored japonica and indica rice could be clearly distinguished; however, it was not possible to differentiate the samples stored at low temperature.

Discovery of β-1,4-d-Mannosyl-N-acetyl-d-glucosamine Phosphorylase Involved in the Metabolism of N-Glycans

Background: N-Glycans are metabolized by sequential glycoside hydrolase-catalyzed reactions. Results: A phosphorylase encoded in a gene cluster involved in N-glycan metabolism in the genome of Bacteroides thetaiotaomicron catalyzed reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine. Conclusion: An N-glycan metabolic pathway containing a unique phosphorylase was discovered. Significance: B. thetaiotaomicron efficiently utilizes the energy of ATP via a phosphorylase-dependent metabolic pathway. A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase as the short name for BT1033.

Rheological Properties and Lipid Oxidation of Rice Decontaminated with Low-Energy Electrons

Microbial load and quality of rice grains which were exposed to electrons at different acceleration voltages of 170 to 200 kV were examined to evaluate the efficacy of decontaminating rice with low-energy electrons. Electrons at any acceleration voltage between 170 and 200 kV reduced microbial loads of brown rice grains to levels lower than 10 colony-forming units (CFU)/g. Higher acceleration voltages resulted in a lower viscosity and a higher thiobarbituric acid value (TBA). Milling at a yield of 90 or 88% after electron treatment made the viscosity and TBA of rice treated with electrons at 170 kV almost the same as untreated rice. Low and high compression analyses of rice grains which were exposed to electrons at 170 to 180 kV and milled at a yield of 90% followed by cooking showed almost the same hardness and stickiness as untreated grains which were milled at 90%. The results indicate that milling at a yield of 90% or lower removes the portion of rice exposed to electrons at 170 kV and that treatment of brown rice with low-energy electrons enables the preparation of milled rice with extremely low levels of microbial load and little quality deterioration.

Characteristics of Bread Prepared from Wheat Flours Blended with Various Kinds of Newly Developed Rice Flours

Characteristics of the bread prepared from wheat flour blended with the flour of various kinds of newly developed rice cultivars were investigated. Qualities of the bread made from wheat flour blended with rice flour have been reported to be inferior to those from 100% wheat flour bread. To improve its qualities, we searched for the new-characteristic rice flours among the various kinds of newly developed rice cultivars to blend with the wheat flour for the bread preparation. The most suitable new characteristic rices are combination of purple waxy rice, high-amylose rice, and sugary rice. Specific volume of the bread from the combination of wheat and these 3 kinds of rice flours showed higher specific volume (3.93) compared with the traditional wheat/rice bread (3.58). We adopted the novel method, continuous progressive compression test, to measure the physical properties of the dough and the bread in addition to the sensory evaluation. As a result of the selection of the most suitable rice cultivars and blending ratio with the wheat flour, we could develop the novel wheat/rice bread, of which loaf volume, physical properties, and tastes are acceptable and resistant to firming on even 4 d after the bread preparation. To increase the ratio of rice to wheat, we tried to add a part of rice as cooked rice grains. The specific volume and qualities of the bread were maintained well although the rice content of total flour increased from 30% to 40%.

Molecular Cloning and Functional Expression of cDNA Encoding a Cysteine Proteinase Inhibitor, Cystatin, from Job's Tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A λZAP II cDNA library was constructed from mRNA in immature seeds of the grass Jobs tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Jobs-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.

Discovery of Two β-1,2-Mannoside Phosphorylases Showing Different Chain-Length Specificities from Thermoanaerobacter sp. X-514

We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789.

Purification and characterization of a heat stable inulin fructotransferase (DFA I-producing) from Arthrobacter pascens a62-1

An inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter pascens a62-1 was purified and the properties of the enzyme were investigated. The enzyme was purified from culture supernatant of the microorganism 58.5 fold with a yield of 8.32% using Super Q Toyopearl chromatography and butyl Toyopearl chromatography. It showed maximum activity at pH 5.5 and 45 °C and was stable up to 75 °C. This heat stability was highest in the inulin fructotransferases (DFA I-producing) reported until now. The molecular mass of the enzyme was estimated to be 37,000 by SDS-PAGE and 60,000 by gel filtration, and was considered to be a dimer. The N-terminal amino acid sequence (20 amino acid residues) was determined as Ala-Asn-Thr-Val-Tyr-Asp-Val-Thr-Thr-Trp-Ser-Gly-Ala-Thr-Ile-Ser-Pro-Tyr-Val-Asp.

Quality Prediction of Rice Flour by Multiple Regression Model with Instrumental Texture Parameters of Single Cooked Milled Rice Grains

ABSTRACT The purpose of this study was to develop highly accurate regression models with texture parameters of cooked milled rice grains for predicting pasting properties in terms of quality index of rice flour. Two methods were adopted as the texture measurement to acquire predictors for the models. In the calibration set, all the multiple regression models by a single-grain method exhibited a higher R2 than those by a three-grain method. Each of the former models also showed a lower SEP and a higher RPD in the validation set. The prediction performance was best for consistency (RPD = 2.4). The single-grain method was more advantageous for the pasting prediction. These results suggest that the models based on grain texture could predict rice flour quality.

Search for Cell-Wall-Degrading Enzymes of World-Wide Rice Grains by PCR and Their Effects on the Palatability of Rice

Such rice cultivars as Japonica, Japonica-Indica hybrid, Javanica and Indica, were evaluated for their main chemical components (amylose content and protein content), pasting property of rice flour (consistency), physical property of the cooked rice grains (adhesion, L3), and enzyme activities (cellulase and xylanase). The amylose content, cellulase activity and xylanase activity showed significant positive or negative correlation with the pasting property (consistency) of rice flour (r = 0.89, r = 0.58, r = 0.70, respectively) and with the physical property of the cooked rice grains (adhesion, L3: r = -0.51, r = -0.61, r = -0.71, respectively) at the level of 1%. Endogenous xylanase and cellulase played important roles to determine the texture of the cooked rice grains similarly to the amylose content. Part of the DNA sequences of the α-glucosidase gene differed among the Japonica, Japonica-Indica hybrid and Indica subspecies. We found discriminative DNA bands appearing by PCR, corresponding to 1,4-β-xylanase and endo-1,4-β-glucanase 13 in the case of Indica rice, Indica-Japonica hybrid rice, and Javanica rice (non-Japonica subspecies). The equation for estimating the physical property (adhesion) of cooked rice grains by PCR was improved by adding novel primers related to the cell-wall-degrading enzymes.

Molecular Cloning and Structural Characterization of the Hagfish Proteinase Inhibitor of the Alpha-2-Macroglobulin Family

The “most primitive” living vertebrate the hagfish has a dimeric proteinase inhibitor, a protein homologous to human α2-macroglobulin, in its plasma at high concentration. Although the hagfish proteinase inhibitor has been isolated and its function and quaternary structure studied, its primary structure, subunit composition and fragmentation process remain unclear. In this study, hagfish proteinase inhibitor cDNA was cloned, sequenced and cDNA-deduced amino acid sequence was analyzed. A large fraction of homosubunits in the dimeric structure of the protein has undergone a cleavage at a specific arginyl residue (Arg833) while the rest retained their chain integrity without being processed. Thus random combinations of processed and nonprocessed subunits in the dimeric structure of this protein result in different molecular conformers and generate a complicated multiband pattern in SDS-PAGE. It was further demonstrated by proteolytic analysis that the hagfish inhibitor has no susceptible arginyl residues within its bait region and thus incapable of trapping arginine specific proteinases. This implies that the specific subunit cleavage at Arg833 was caused by an unknown arginine specific proteinase which escaped from the entrapment by the hagfish inhibitor.