Tatsuro Suzuki

Factors affecting the digestibility of raw and gelatinized potato starches.

The enzymatic digestibilities of raw and gelatinized starches in various potato starches, as well as sweet potato, cassava, and yam starches, were estimated, along with other starch properties, such as the phosphorus content, median granule size, and rapid visco analyzer (RVA) pasting properties. Furthermore, correlation coefficients were calculated between the hydrolysis rates (HR) by amylase and other starch quality parameters. A larger granule size was closely associated with a lower HR in raw starch, while the HR in gelatinized starch did not correlate with the median granule size. An increase in phosphorus content resulted in a definitely lower HR in raw starch and tended to decrease the HR in gelatinized starch for the composite of potato and other starches. In contrast, no correlation coefficients of the phosphorus content with the HRs in raw and gelatinized starches were observed within potato starches. Starches with higher peak viscosity and breakdown showed a lower HR in raw starch, while few or no effects of these RVA parameters on the HR in gelatinized starch were observed for the composite of potato and other starches or among potato starches, respectively.

Comparison of phenolic compositions between common and tartary buckwheat (Fagopyrum) sprouts

The phenolic compositions of non-germinated/germinated seeds and seed sprouts (at 6-10 day-old) of common (Fagopyrum esculentum Möench) and tartary (Fagopyrum tataricum Gaertn.) buckwheats were investigated. Phenolic compounds, including chlorogenic acid, four C-glycosylflavones (orientin, isoorientin vitexin, isovitexin), rutin and quercetin, were determined in the seed sprouts by high-performance liquid chromatography (HPLC). In the edible parts of common buckwheat sprouts, individual phenolics significantly increased during sprout growth from 6 to 10 days after sowing (DAS), whereas in tartary buckwheat sprouts they did not. While the sum contents of phenolic compounds in the edible part (mean 24.4mg/g DW at 6-10 DAS) of tartary buckwheat sprouts were similar to those of common buckwheat sprouts, rutin contents in the non-germinated/germinated seeds (mean 14.7mg/g DW) and edible parts (mean 21.8mg/g DW) of tartary buckwheat were 49- and 5-fold, respectively, higher than those of common buckwheat. Extracts of the edible parts of both species showed very similar free radical-scavenging activities (mean 1.7μmol trolox eq/g DW), suggesting that the overall antioxidative activity might be affected by the combination of identified phenolics and unidentified (minor) components. Therefore, buckwheat seed sprouts are recommended for their high antioxidative activity, as well as being an excellent dietary source of phenolic compounds, particularly tartary buckwheat sprouts, being rich in rutin.

Purification and characterization of flavonol 3-glucosidase, and its activity during ripening in tartary buckwheat seeds

To obtain basic information about rutin metabolism in buckwheat seeds, flavonol 3-glucosidase (f3g) was purified from tartary buckwheat (Fagopyrum tataricum) seeds. The f3g consisted of two isozymes, and their characteristics were very similar to the rutin-degrading enzyme (RDE) reported in [Phytochemistry 37 (1994) 133], although the molecular weight and kinetic constants were quite different. In addition, rutin and isoquercitrin concentrations and corresponding f3g activities were measured during the ripening of tartary and common buckwheat seeds. Rutin and isoquercitrin increased during ripening, and the rutin concentration remained high in the fully ripe seeds. The rutin 3-glucosidase activity and isoquercitrin 3-glucosidase activity also increased during ripening and remained high with a slight reduction in fully ripe seeds. In such seeds, the major part of the rutin was found in the embryo, and almost all the f3g activity was detected in the testa. These results support the hypothesis that the f3g activity is not the major limiting factor of the rutin concentration in fully ripe seeds.

Breeding of 'Manten-Kirari', a non-bitter and trace-rutinosidase variety of Tartary buckwheat (Fagopyrum tataricum Gaertn.).

Here, we developed a new Tartary buckwheat cultivar ‘Manten-Kirari’, whose flour contains only trace amounts of rutinosidase and lacked bitterness. The trace-rutinosidase breeding line ‘f3g-162’ (seed parent), which was obtained from a Nepalese genetic resource, was crossed with ‘Hokkai T8’ (pollen parent), the leading variety in Japan, to improve its agronomic characteristics. The obtained progeny were subjected to performance test. ‘Manten-Kirari’ had no detectable rutinosidase isozymes in an in-gel detection assay and only 1/266 of the rutinosidase activity of ‘Hokkai T8’. Dough prepared from ‘Manten-Kirari’ flour contained almost no hydrolyzed rutin, even 6 h after the addition of water, whereas the rutin in ‘Hokkai T8’ dough was completely hydrolyzed within 10 min. In a sensory evaluation of the flour from the two varieties, nearly all panelists detected strong bitterness in ‘Hokkai T8’, whereas no panelists reported bitterness in ‘Manten-Kirari’. This is the first report to describe the breeding of a Tartary buckwheat cultivar with reduced rutin hydrolysis and no bitterness in the prepared flour. Notably, the agronomic characteristics of ‘Manten-Kirari’ were similar to those of ‘Hokkai T8’, which is the leading variety in Japan. Based on these characteristics, ‘Manten-Kirari’ is a promising for preparing non-bitter, rutin-rich foods.

Changes in rutin concentration and flavonol-3-glucosidase activity during seedling growth in tartary buckwheat (Fagopyrum tataricum Gaertn.)

To investigate the physiological roles of rutin (quercetin 3-O-rutinoside) and flavonol-3-glucosidase (f3g, a rutin-degrading enzyme) during cotyledon growth in tartary buckwheat, changes in rutin and quercetin (aglycone of rutin) concentrations, and f3g activities were measured. The rutin concentration gradually increased (about 50 mg g-1 DW cotyledons) up to 12 d after germination (DAG). The quercetin concentration in the tartary buckwheat cotyledon increased immediately after germination, reached a maximum at 4 DAG, and then remained around 1.5 mg g-1 DW until 12 DAG. The f3g activity in the cotyledon and testa decreased gradually after germination. However, it retained 80% activity at 4 DAG compared with dry seed. In addition, the f3g activity was high on the surface of the cotyledon. We also investigated spatial distribution of rutin and f3g activity in 4 DAG seedlings. While rutin was mostly present in the cotyledon, f3g activity was observed in the testa, the surface of the testa, and root. These r...

Purification and characterization of lipase in buckwheat seed.

To obtain basic information about enzymatic deterioration of buckwheat flour, triacylglycerol lipase (LIP; EC 3.1.1.3) was purified from buckwheat seed. The LIP consisted of two isozymes, LIP I and LIP II, and they were purified with purification folds of 60 and 143 with final specific activities of 0.108 and 0.727 mumol of fatty acid released per minute per milligram of protein at 30 degrees C using triolein as a substrate. Molecular weights were estimated to be 150 (LIP I) and 28.4 kDa (LIP I) by gel filtration and 171 (LIP I) and 26.5 kDa (LIP II) by SDS-PAGE. Optimal pHs of LIP activities were 3.0 (LIP I) and 6.0 (Lip II) using triolein as a substrate. Both LIP I and II reacted in the acidic pH range. Optimal temperatures were 30 (LIP I) and 40 degrees C (LIP II), and both LIP I and II were stable below 30 degrees C when p-nitrophenyl-laurate was used as a substrate. However, they were inactivated above 60 degrees C. On the other hand, when triolein was used as a substrate, optimal temperatures were 30 degrees C for both LIP I and II, and they retained 40% of their activity after a 4 h incubation of enzymes at 70 degrees C. LIP I and II had higher activity against triolein than monoolein or tri/monopalmitin. Most of the LIP activity was distributed in the embryo.

Discovery and genetic analysis of non-bitter Tartary buckwheat (Fagopyrum tataricum Gaertn.) with trace-rutinosidase activity

In a screening of about 500 lines of Tartary buckwheat, we identified lines that contained no detectable rutinosidase isozymes using an in-gel detection assay. We confirmed that seeds of these individuals had only a trace level of in-vitro rutinosidase activity. To investigate the heritability of the trace-rutinosidase characteristic, we analyzed the progeny of crosses between rutinosidase trace-lines, ‘f3g-162’, and the ‘Hokkai T8’. The F2 progeny clearly divided into two groups: those with rutinosidase activity under 1.5 nkat/g seed (trace-rutinosidase) and those with activity over 400 nkat/g seed (normal rutinosidase). The segregation pattern of this trait in F2 progeny exhibited 1 : 3 ratio (trace-rutinosidase : normal rutinosidase), suggesting that the trace-rutinosidase trait is conferred by a single recessive gene; rutinosidase-trace A (rutA). In addition, sensory panelists evaluated the bitterness of flour from trace-rutinosidase individuals and did not detect bitterness, whereas flour from normal rutinosidase individuals was found to have strong bitterness. Although at least three bitter compounds have been reported in Tartary buckwheat seeds, our present findings indicate that rutin hydrolysis is the major contributing factor to bitterness. In addition, the trace-rutinosidase line identified here, ‘f3g-162’, is a promising material for generating a non-bitter Tartary buckwheat variety.

Overexpression of a tartary buckwheat R2R3-MYB transcription factor gene, FtMYB9, enhances tolerance to drought and salt stresses in transgenic Arabidopsis.

Tartary buckwheat (Fagopyrum tataricum) is a traditional coarse cereal that exhibits strong plasticity in its adaptation to harsh and complicated environmental stresses. In an attempt to study the strong tolerance of tartary buckwheat, the FtMYB9 gene, which encodes an R2R3-MYB transcription factor protein, was functionally investigated. FtMYB9 expression was rapidly and strongly induced by ABA, cold, salt, and drought treatments in the seedling stage. A yeast one-hybrid system assay indicated that FtMYB9 is an activator of transcriptional activity, consistent with its roles as a transcription factor. Its overexpression in plants resulted in increased sensitivity to ABA at the germination and seedling stages compared to wild type. The overexpression of FtMYB9 increased tolerance to drought and salt stresses by the activation of some stress-related genes from both ABA-independent and ABA-dependent pathways in transgenic Arabidopsis. Furthermore, enhanced proline content and the activation of the P5CS1 gene implied that FtMYB9 may be involved in proline synthesis in plants. Collectively, these results suggest that FtMYB9 functions as a novel R2R3-MYB TF which plays positive roles in salt and drought tolerance by regulating different stress-responsive signaling pathways.

Proteomics Analysis of Embryo and Endosperm from Mature Common Buckwheat Seeds

We used proteomics analysis to generate the profiles of proteins in the endosperm and embryo of common buckwheat grains. These differentially expressed proteins are potentially involved in seed metabolism. Extractions were done by trichloroacetic acid (TCA) precipitation. The resulting proteins were separated using SDS-PAGE coupled to LC-ESI-Q/TOF-MS/MS. This allowed us to detect and identify 67 proteins with isoforms, making this the most inclusive protein profile. The proteins were determined to be functionally involved in the central metabolic pathway of the seed, with metabolic interest being reflected in the occurrence of a tissue-specific enzyme balance. For a case in point, we found a tissue-specific and subcellular compartment-specific isoform of granule-bound starch synthase 1 in the chloroplast/amyloplast. This provided proteomic verification of the presence of a distinct regulatory mechanism for the biosynthesis of glycan and starch, which produce amylase and amylopectin. Furthermore, several previously characterized allergenic proteins such as 11S and 13S globulin seed storage protein were acknowledged in our seed samples, thus representing the potential for proteomics techniques that survey food sources for any incidence of allergens. This protein profile of common buckwheat grain is a new avenue for understanding its seed physiology in dormant stage as well as suggesting commercial applications for the buckwheat industry as buckwheat flour.

Effect of rutin from tartary buckwheat sprout on serum glucose-lowering in animal model of type 2 diabetes

Abstract This study investigates the anti-diabetic effects of rutin from tartary buckwheat sprout in type 2 diabetes mouse model. The rutin content in tartary buckwheat sprout (TBS) is five times higher than that found in common buckwheat sprout (CBS) as evident from high-performance liquid chromatography analysis. Administration of either rutin or TBS ethanolic extract to diabetes mice decreased the serum glucose level significantly. Rutin down-regulated the expression levels of protein-tyrosine phosphatase 1B; it is negative regulator of insulin pathway, both transcriptionally and translationally in myocyte C2C12 in a dose-dependent manner. In conclusion, rutin can play a critical role in down-regulation of serum glucose level in type 2 diabetes.