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.

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.

Preparation of calcium- and magnesium-fortified potato starches with altered pasting properties.

Calcium- and magnesium-fortified potato starches were prepared by immersion in various concentrations of CaCl2 and MgCl2 aqueous solutions, respectively. The pasting properties, i.e., peak viscosity and breakdown, of all the starches obtained above were analyzed using a Rapid Visco Analyzer. Furthermore, the gelatinization properties and in vitro digestibility of the representative calcium- and magnesium-fortified starches were tested. The maximum calcium content of the fortified potato starches was as high as 686 ppm with the addition of a high-concentration CaCl2 solution, while the calcium content of the control potato starch was 99 ppm. The magnesium content increased from 89 to 421 ppm by treatment of the potato starch with an MgCl2 solution. Markedly lower values of peak viscosity and breakdown were observed in calcium- and magnesium-fortified potato starches than in the control potato starch. However, the gelatinization temperature and enthalpy as well as resistant starch content of calcium- and magnesium-fortified potato starches were similar to those of the control potato starch. It is concluded that potato starches with altered pasting properties can be easily manufactured by the use of solutions containing high levels of calcium and magnesium.

Traits of shattering resistant buckwheat 'W/SK86GF'.

Seed shattering is a significant problem with buckwheat, especially at harvesting time. Several reports have shown that a green-flower mutant of buckwheat, such as W/SK86GF, has a strong pedicel. Although a strong pedicel may provide some resistance to shattering in the field, no study has thoroughly examined this issue. In this paper, we demonstrate that a W/SK86GF has shattering resistance by comparing the degrees of shattering of W/SK86GF and Kitawasesoba (leading variety of Hokkaido with non-green-flower traits) through a test for four years, including a typhoon hit year in the field. In a non-typhoon year, the shattering seed ratio (shattering seed weight/(yield + shattering seed weight) × 100) of W/SK86GF at maturing time +15 days (+15D) was lower than that of Kitawasesoba. In a typhoon hit year, the shattering seed ratios of Kitawasesoba at maturing time and +15D were surprisingly high, 14.4 and 21.1%, respectively. On the other hand, those of W/SK86GF were only 3.08% and 2.57%, respectively; indicating W/SK86GF is promising as a shattering resistant line even in a typhoon hit year. From these results, shattering resistance of W/SK86GF can be evaluated after maturing time such as +15D and pedicel strength would confer W/SK86GF a shattering resistant trait.

Development of Novel Detection Method for Rutinosidase in Tartary Buckwheat (Fagopyrum tataricum Gaertn.).

Contamination of rutinosidase is a major problem for rutin-rich food due to the hydrolysis of the functional compound rutin and generation of strong bitterness caused by the hydrolyzed moiety quercetin. This problem sometimes occurs between the trace and normal rutinosidase Tartary buckwheat varieties. Here, we developed a simple and rapid method for rutinosidase detection in 'ripening seeds using UV light' and in 'dough using alum-flavonoid complexation' from Tartary buckwheat (Fagopyrum tataricum Gaertn.). Normal rutinosidase seeds can be distinguished from trace-rutinosidase mature seeds and ripening young seeds using a rutin solution by comparing the muddiness, which corresponds to quercetin generation. In dough, we detected a threefold relative increase in rutinosidase activity corresponding to 1% contamination of normal rutinosidase flour with trace-rutinosidase flour within 65 min. These methods do not require expensive apparatuses and toxic chemicals and are therefore promising for detecting and preventing contamination by rutinosidase, e.g., in food processing factories.

Effects of lipase, lipoxygenase, peroxidase and free fatty acids on volatile compound found in boiled buckwheat noodles.

BACKGROUND: Relationships between buckwheat (Fagopyrum esculentum Moench) flour lipase, lipoxygenase and peroxidase activity, along with levels of individual free fatty acids (FFAs) and levels of headspace volatile compounds of boiled buckwheat noodles, were investigated for 12 different buckwheat varieties. Enzyme activities and FFA levels in flour were correlated with their respective varietal arrays of boiled noodle headspace volatile compounds, measured by gas chromatography-mass spectrometry. RESULTS: The volatiles hexanal, tentative butanal, tentative 3-methylbutanal and tentative 2-methylbutanal showed significant positive correlation with one another, indicating that they may be generated through similar mechanisms. These important volatile components of buckwheat flavor were also positively correlated with lipase and/or peroxidase activity, indicating that enzymatic reactions are important in flavor generation in boiled buckwheat noodles. On the other hand, pentanal, which showed no significant correlation with any enzyme activity, showed a significant positive correlation to the levels of C18:2 and C18:3 FFAs, suggesting the existence of a 'non-enzymatic' and/or 'uncertain enzymatic pathway' for flavor generation in boiled buckwheat noodles. CONCLUSION: Lipase and peroxidase in buckwheat flour are important for flavor generation of boiled buckwheat noodles. This information is important for increasing desirable flavor of buckwheat products as well as for selecting varieties with improved flavor.

Colored potato extracts induce superoxide dismutase-2 mRNA via ERK1/2 pathway in HepG2 cells.

Rats fed a diet containing Shadow Queen (SQ), an anthocyanin-rich potato cultivar, previously showed an increase in the hepatic superoxide dismutase (SOD)-2 mRNA level. We investigated whether an extract of SQ would directly increase the hepatic SOD-2 mRNA level in HepG2 cells. Furthermore, we estimated the intracellular signaling pathway for the induction of SOD-2 mRNA expression. HepG2 cells were stimulated using extracts of four crops, including SQ, for 12 h; only extracts of colored potatoes induced SOD-2 mRNA expression significantly. This induction of SOD-2 mRNA expression was blocked by an inhibitor of the extracellular signal-related kinase (ERK) 1/2 pathway. Furthermore, an extract of SQ increased the phosphorylation of ERK1/2 after 15 or 30 min of stimulation. These data indicate that an extract of SQ directly induces hepatic SOD-2 mRNA expression via activation of ERK1/2 pathway in HepG2 cells.

Yam contributes to improvement of glucose metabolism in rats.

To investigate whether yam improves glucose metabolism, yam-containing diets were given to Wistar rats. In a short-term experiment, fasted-rats were given 1.0 g of a control and 20% yam-containing diets. At 60 min after start of the feeding, glucose level in the yam diet group was lower or tended to be lower than that in the control diet. Insulin levels at 30 min and 60 min were significantly lower than those in the control group. In a long-term experiment, a normal diet (N) or 25% high fat diets with (Y) or without 15% yam powder (HF) were given to rats for 4 weeks. At 4 weeks, in an oral glucose tolerance test, the area under the curve (AUC) of plasma glucose level was higher in the HF group than that in the N group, whereas those in the Y groups did not differ from that in the N group. Glycosylated hemoglobin levels had similar tendency to the AUCs. Plasma leptin levels in the Y groups were significantly higher than that in the N group. In conclusion, yam may contribute to improvement of glucose metabolism. Additionally, we speculated that leptin level is possibly involved in the insulin-response to yam diets.

Anti-inflammatory effect of buckwheat sprouts in lipopolysaccharide-activated human colon cancer cells and mice.

In conducting an in vitro screening of ethanol extracts from various natural foods using a human colon cancer cell line (CoLoTC cells), an extract of buckwheat sprouts (ExtBS) was found to express significant anti-inflammatory activity. The anti-inflammatory activity of ExtBS was confirmed by oral administration of lipopolysaccharide (LPS) to mice. Inflammatory cytokines (interleukin 6 and tumor necrosis factor alpha) were markedly up-regulated in the spleen and liver from LPS-administrated mice, and combinatory treatment with LPS and ExtBS decreased up-regulation of them in both cytokines. Both serum cytokine levels corresponded to their gene expressions in tissues, but no anti-inflammatry effect in mice was observed when ExtBS was treated intraperitoneally. ExtBS oral administration also showed protective activity as to hepatic injury induced by galactosamine/LPS treatment. Based on these data, we suggest that ExtBS contains anti-inflammatory compounds.

Structural identification of anthocyanins and analysis of concentrations during growth and flowering in buckwheat (Fagopyrum esculentum Moench) petals.

The anthocyanin profiles and variety/breeding-line differences of anthocyanin concentrations in petals of common buckwheat flowers have been studied. Four anthocyanins, cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-rhamnoside, and cyanidin 3-O-galactosyl-rhamnoside were isolated from the petals of common buckwheat (Fagopyrum esculentum Moench), separated using high performance liquid chromatography and identified using reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry techniques. In every variety/breeding line tested, cyanidin 3-O-rutinoside was detected as the major anthocyanin and the next is cyanidin 3-O-glucoside whereas cyanidin 3-O-rhamnoside and cyanidin 3-O-galactosyl-rhamnoside were trace or not detectable in white and pink flowered buckwheat. Of all the varieties/breeding lines tested, Gan-Chao, a Chinese variety, contained the highest amount of anthocyanins. The largest part of cyanidin moiety was presented as a proanthocyanidin form (PAs-Cy). Anthocyanins and PAs-Cy in petals were increased along with increase of flower development stages. Therefore, fully developed petals of red flowered buckwheat, especially Gan-Chao, are promising as a new anthocyanin-rich material for food processing.

Identification of anthocyanins in the sprouts of buckwheat.

The anthocyanin profiles and varieties/breeding line differences of anthocyanin concentrations in common/tartary buckwheat sprouts have been studied. Four anthocyanins, cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-galactoside, and cyanidin 3-O-galactopyranosyl-rhamnoside, were isolated from the sprouts of common buckwheat, were separated using high-performance liquid chromatography (HPLC), and were identified using reversed-phase liquid chromatography (LC)/electrospray ionization-mass spectrometry (ESI-MS)/MS techniques. In tartary buckwheat sprouts, two anthocyanins (cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside) were identified. Among 19 common/tartary buckwheat varieties/breeding lines, Hokkai T10 contained the highest amounts of anthocyanins. Cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside concentrations in 6-10 days after seeding sprouts of Hokkai T10 ranged from 0.16 to 0.20 mg/g dry wt and from 5.55 to 6.57 mg/g dry wt, respectively. In addition, dark-grown sprouts of Hokkai T10 accumulated 0.091 and 2.77 mg/g dry wt of cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside whereas other varieties/breeding lines accumulated trace amounts of anthocyanins. Given its anthocyanin-rich red cotyledons, Hokkai T10 is a promising line for use as "Moyashi" type sprouts and is strongly recommended as a new functional food, rich in dietary anthocyanins.

Time-course study and effects of drying method on concentrations of gamma-aminobutyric acid, flavonoids, anthocyanin, and 2''-hydroxynicotianamine in leaves of buckwheats.

Concentrations of gamma-aminobutyric acid (GABA), rutin, minor flavonoids (such as orientin), anthocyanin, and 2''-hydroxynicotianamine (2HN) were quantified in the leaves of common and tartary buckwheat (Fagopyrum esculentum Moench and Fagopyrum tataricum Gaertn., respectively), at 14, 28, and 42 days after sowing (DAS). GABA and rutin concentrations peaked at 42 DAS, whereas anthocyain, 2HN, and minor flavonoid concentrations declined with the age of the plants. However, at 42 DAS, anthocyanin concentrations in the leaves of tartary buckwheat Hokkai T10 leaves were at least 10-fold greater than in the other buckwheats tested. In addition, the effects on target compound concentrations and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of three different drying methods (20 h at 40 degrees C, 7 h at 70 degrees C, or lyophilization) were investigated. In general, the drying method had no significant effect on the parameters tested. These results indicate that, in terms of GABA, rutin, and anthocyanin concentrations, leaf powder from 42 day old Hokkai T10 has the potential to be a useful food ingredient, such as Ao-jiru juice.

Effects of storage temperature on the contents of sugars and free amino acids in tubers from different potato cultivars and acrylamide in chips.

To clarify the effects of storage temperature on potato components and acrylamide in chips, tubers from five cultivars were stored at various temperatures (2, 6, 8, 10, and 18 degrees C) for 18 weeks, and the contents of sugars, free amino acids in tubers, and acrylamide in chips after frying were analyzed. At temperatures lower than 8 degrees C, the contents of reducing sugars increased markedly in all cultivars, with similar increases in the acrylamide level and dark brown chip color. Free amino acids showed little change at the storage temperatures tested and varied within certain ranges characteristic of each cultivar. The contents of reducing sugars correlated well with the acrylamide level when the fructose/asparagine molar ratio in the tubers was <2. When the fructose/asparagine ratio was >2 by low-temperature storage, the asparagine content, rather than the reducing sugar content, was found to be the limiting factor for acrylamide formation.

Change in content of sugars and free amino acids in potato tubers under short-term storage at low temperature and the effect on acrylamide level after frying.

Changes in the sugar and amino acid contents of potato tubers during short-term storage and the effect on the acrylamide level in chips after frying were investigated. The acrylamide content in chips began to increase after 3 days of storage at 2 degrees C in response to the increase of glucose and fructose contents in the tubers. There was strong correlation between the reducing sugar content and acrylamide level, R(2)=0.873 for fructose and R(2)=0.836 for glucose. The sucrose content had less correlation with the acrylamide content because of its decrease after 4 weeks of storage at 2 degrees C, while the reducing sugar in potato tubers and the acrylamide in chips continued to increase. The contents of the four amino acids, i.e., asparatic acid, asparagine, glutamic acid and glutamine, showed no significant correlation with the acrylamide level. These results suggest that the content of reducing sugars in potato tubers determined the degree of acrylamide formation in chips. The chip color, as evaluated by L* (lightness), was correlated well with the acrylamide content.

Changes in sugar content and activity of vacuolar acid invertase during low-temperature storage of potato tubers from six Japanese cultivars.

The storage of potato tubers at low temperatures leads to the accumulation of sugars in a process called "low-temperature sweetening." To understand this phenomenon, we measured the sugar contents and the activity of acid invertase over several months in tubers of six Japanese cultivars stored at 4 degrees C or 20 degrees C. At 20 degrees C, few changes in sugar contents took place in any of the tubers. On the other hand, when stored at 4 degrees C, three types of changes were observed among the cultivars: (1) increased levels of reducing sugars during storage; (2) a pattern similar to that of type 1, but with 4- to 6-fold lower levels of reducing sugars throughout storage; and (3) increased sucrose, but not reducing sugars. The activity of vacuolar acid invertase increased in the type-1 cultivars, whereas, in the type-2 and type-3 cultivars, the activities were very low during storage at 4 degrees C. Reverse transcription-polymerase chain reaction analysis of acid invertase showed that the transcripts of the enzyme accumulated in the tubers stored at 4 degrees C in the type-1 cultivars but not in type-3. These results suggest that the activity of vacuolar acid invertase is related to the types of changes that occurred in sugar content during low-temperature storage among the potato cultivars.