2019 Volume 25 Issue 6 Pages 915-920
To investigate rutin hydrolysis in dough made with ‘Manten-Kirari’, a new Tartary buckwheat variety with trace rutinosidase activity that lacks bitterness, we performed a time course study of the residual rutin ratio in dough at different water contents and blending ratios of Tartary buckwheat flour. In the common rutinosidase variety, ‘Hokkai T8’, a large portion of the rutin was hydrolyzed within 30 min after the addition of water, whereas about 80–90% remained in the dough made with ‘Manten-Kirari’. We also investigated the residual rutin ratio in noodles such as soba-noodle and pasta containing Tartary buckwheat flour. With ‘Hokkai T8’, rutin was hydrolyzed almost completely in all noodles tested. In contrast, about 90% of rutin remained in ‘Manten-Kirari’-containing noodles. In addition, while ‘Hokkai T8’ noodles exhibited strong bitterness, ‘Manten-Kirari’ noodles lacked or had only slight bitterness. These results indicate that ‘Manten- Kirari’ holds promise as a material for rutin-rich noodles with minimal bitterness.
Rutin and rutin-containing food have many biological functions, such as antioxidative (Jiang et al. 2007, Awatsuhara et al. 2010, Ishiguro et al. 2016), strengthening of blood capillaries (Shanno 1946, Griffith et al. 1997), antihypertensive (Matsubara et al. 1985) and alpha-glucosidase inhibitory activities (Li et al. 2009). In addition, the clinical effects of rutin in a double-blind crossover study were investigated, and reductions in serum myeloperoxidase and cholesterol levels (Wieslander et al. 2011), mucosal symptoms, headache, and tiredness were observed (Wieslander et al. 2012).
Although rutin is widely distributed in plants (Sando et al. 1924, Couch et al. 1946, Haley et al. 1951, Fabjan et al. 2003), buckwheat is the only known cereal to contain rutin in its seeds. For this reason, buckwheat has been identified as a rutin-rich material for food products (Kreft et al. 2006, Ikeda et al. 2012). Among cultivated buckwheat species, Tartary buckwheat (Fagopyrum tataricum Gaertn.) contains approximately 100-fold greater rutin content in its seeds than common buckwheat. Rutin contents in Tartary buckwheat flour are about 1%–2% w w-1. However, Tartary buckwheat also has extremely high rutinosidase activity, which is sufficient to hydrolyze the rutin contained in buckwheat flour within a few min after the addition of water (Yasuda et al. 1992, Yasuda et al. 1994, Suzuki et al. 2002) (Figure 1). Although food processing technology such as heat treatment of the seeds or flour can prevent rutin hydrolysis, this leads to the deterioration of texture (Yoo et al. 2012), color and flavor and consequent added costs. Therefore, a Tartary buckwheat variety with low rutinosidase activity is desirable for production of foods with high rutin content. Tartary buckwheat seeds contain at least two rutinosidase isozymes with close similarities in substrate specificity (Km), optimum temperature and optimum pH (Yasuda et al. 1992, Yasuda et al. 1994, Suzuki et al. 2002). Therefore, to develop a variety with low rutinosidase activity, it was proposed that the expression of both isozymes be suppressed. On the other hand, the use of Tartary buckwheat (also known as ‘bitter buckwheat’) in food products is currently limited due to the presence of at least three bitter compounds (Kawakami et al. 1955): quercetin and two unidentified compounds. Among them, quercetin is generated by rutinosidase activity. Therefore, the development of a Tartary buckwheat variety with low rutinosidase activity and minimal bitterness has been highly anticipated.
Rutin hydrolysis in Tartary buckwheat seeds
Recently, our research team developed a novel Tartary buckwheat variety, “Manten-Kirari” (Suzuki et al. 2014a, Suzuki et al. 2014b), with trace rutinosidase activity; the rutinosidase activity in the flour is about two or three orders of magnitude less than in the common variety. This variety is suitable to cultivate in the northern dimension area due to its short growth period of about 80 days. Additionally, the flour of this variety lacks bitterness. Therefore, we investigated the possibility of making rutin-rich and non-bitter foods using “Manten-Kirari” buckwheat flour.
Flour preparation and time course analysis of rutin hydrolysis in dough Tartary buckwheat seeds of ‘Manten- Kirari’ (trace-rutinosidase variety) and ‘Hokkai T8’ (normal rutinosidase variety) were milled using a test mill (Quadrumat® Junior, Brabender® GmbH and Co., Duisburg, Germany) at the flour milling percentage of 63%. Tartary buckwheat flour and wheat flour were mixed at the final Tartary buckwheat ratios of 5%, 10%, 30%, 60% and 100%. Portions of the mixed flour (150 mg) were individually placed in test tubes and pre-incubated at 25 °C for 3 hours. Aliquots of water (45 and 67.5 µL) at 25 °C were added to each tube to make dough (final water contents of the doughs were 30% and 45%, respectively). These dough water contents were selected to reflect that the water contents of soba-noodle and pasta are generally about 30% to 45%. Immediately after adding water to the tube, the flour and water were mixed for one min to obtain a uniform dough. After sealing the tube, the dough was placed at 25 °C for 30, 60, 120, 240 and 480 min. Next, to extract rutin and quercetin (aglycone of rutin), 9 mL of methanol containing 20% volume of 0.1% (v v-1) phosphoric acid was added and mixed, and the samples were subjected to flavonoid extraction at 80 °C for 3 hours (Suzuki et al. 2002). After extraction, the centrifuged supernatant was analyzed using HPLC (Suzuki et al. 2002), and the rutin and quercetin concentrations were determined. The residual rutin ratio was calculated as the percentage of rutin content in the dough relative to the weight of the flour component of the dough.
Raw Soba-noodle For raw soba-noodle, 100 g of wheat flour and 100 g of Tartary buckwheat flour (the control contained 200 g of wheat flour) were combined, and 94 mL of water was added using a mixer for 10 min. After mixing, the crumbly dough mixture was transferred to a noodle-sheeting machine, passed through rollers 3.0 mm apart, folded, and then sheeted using the same space between rollers. The same folding and sheeting were performed again. The sheet was passed between the rollers three times with the clearance progressively reduced to 2.2 mm. The final thickness of the sheeted dough was adjusted to 1.4 mm. The sheet was cut into noodle strips 25 cm in length using No. 20 cutting rolls. For boiled noodles, the cut noodles were placed in 4 L of boiling water for 2.5 min and then rinsed with cold water. Boiled noodles were stored at −30 °C until used for extraction of rutin and quercetin. Boiled noodles were also subjected to sensory evaluation of the parameters ‘color’, ‘flavor’, ‘taste’, ‘bitterness’, ‘hardness’, and ‘buckwheat noodleness’. For each parameter, 13 panels rated the samples as ‘very bad’, ‘a little bad’, ‘slightly bad’, ‘ordinary’, ‘slightly good’, ‘a little good’, and ‘very good’. For evaluation of each parameter, the common variety, ‘Hokkai T8’, was taken as ‘ordinary’, and panelists evaluated the novel variety, ‘Manten-Kirari’, in comparison to ‘Hokkai T8’. In ‘color’, ‘flavor’, ‘hardness’, and ‘buckwheat noodleness’, the evaluation scores were defined as 8, 10, 12, 14, 16, 18 and 20 points for ‘very bad’, ‘a little bad’, ‘slightly bad’, ‘ordinary’, ‘slightly good’, ‘a little good’, and ‘very good’, respectively. In ‘taste’ and ‘bitterness’, the evaluation scores were defined as 4, 5, 6, 7, 8, 9 and 10 points for ‘very bad’, ‘a little bad’, ‘slightly bad’, ‘ordinary’, ‘slightly good’, ‘a little good’, and ‘very good’, respectively. The sensory evaluation was performed according to the method of Honda et al. (2010).
Dried Soba-noodle For dried soba-noodle, 140 g of wheat flour, 60 g of Tartary buckwheat flour (control contained 200 g of wheat flour) and 4.0 g of salt were combined, and 80 mL of water was added using a mixer for 10 min. After mixing, the crumbly dough mixture was transferred to a noodle-sheeting machine as described above to make cut noodles. The cut noodles were dried at 50 °C until a water content of 13.0% was reached. Dried noodles were boiled in 4 L of water for 6 min and then rinsed with cold water. Boiled noodles were stored at −30 °C until used for extraction of rutin and quercetin.
Pasta For pasta preparation, 170 g of wheat flour, 30 g of Tartary buckwheat flour (the control sample contained 200 g of wheat flour), and 4.0 g of salt were mixed, and then 100 g of egg was added using a mixer for 10 min. After mixing, the crumbly dough mixture was transferred to a noodle-sheeting machine, passed twice through rollers 3.0 mm apart, folded, and then sheeted using the same space between rollers. The same folding and sheeting procedure was performed again. The sheet was passed between the rollers three times with the clearance progressively reduced to 2.2 mm. The final thickness of the sheeted dough was adjusted to 1.8 mm. The sheet was cut into noodle strips 25 cm in length using No. 15 cutting rolls. For boiled pasta, cut noodles were boiled in 4 L of water for 9 min. Boiled noodles were stored at −30 °C until used for extraction of rutin and quercetin. For dried pasta, cut noodles were dried at 50 °C until a water content of 13.0% was reached. Dried noodles were boiled in 4 L of water for 11 min and then rinsed with cold water. Boiled noodles were stored at −30 °C until used for extraction of rutin and quercetin.
Determination of rutin and quercetin concentrations in noodles Each type of noodle was lyophilized and milled to a fine powder. Rutin and quercetin were extracted from 0.1 g samples with 10 mL of extraction solution containing a 20% volume of 0.1% (v v-1) phosphoric acid at 80 °C for three hours. After extraction, the centrifuged supernatant was separated using HPLC, and the rutin and quercetin contents were determined (Suzuki et al. 2005)
In the variety ‘Hokkai T8’, the residual rutin ratio decreased rapidly immediately after the addition of water (Figure 2). At 30 min after water addition, the residual rutin ratio was approximately 30% in the sample with 30% water content and 5% buckwheat flour (Figure 2). In other samples, the residual rutin ratio was < 5%, and approached 0% at 480 min after water addition in the sample with 45% water content. The residual rutin ratio was higher in the 30% than in the 45% water content sample. This result is consistent with the results of cookie and its dough containing Tartary buckwheat flour (Brunori et al. 2010, Suzuki et al. 2015a). On the other hand, the residual rutin ratio of ‘Manten-Kirari’ was very high compared to ‘Hokkai T8’. Specifically, in the 30% water content sample, about 50% residual rutin was observed at 480 min after water addition. The residual rutin ratio showed an inverse relationship with ‘water content’ and ‘blending ratio of Tartary buckwheat’.
Rutin residual ratio of dough on different blending ratio of Tartary buckwheat flour to wheat flour
In ‘Hokkai T8’, rutin was almost completely hydrolyzed in soba-noodles and showed minimal levels under both raw and dried conditions, whereas rutin levels of > 90% were present in ‘Manten-Kirari’ noodles (Table 1). In this experiment, about 60 min was required to make noodles starting from the addition of water. In the time course analysis of the residual rutin ratio in doughs at 60 min after addition of water, about 90% rutin was present in the samples where the ‘water content’ and ‘blending ratio of Tartary buckwheat’ were similar to raw noodle: 45% water and 60% Tartary buckwheat flour (raw-soba noodle), and 45% water and 15% Tartary buckwheat flour (raw pasta) (Figure 2). These results are consistent with the residual rutin ratio in raw noodles. On the other hand, about 8 hours and 12 hours were required to dry soba-noodles and pasta, respectively, after noodle preparation.
| Variety | Blending ratio of Tartary buckwheat flour | Rutin | Quercetin | Rutin residual ratio | |
|---|---|---|---|---|---|
| % | mg/100gDW | mg/100gDW | % | ||
| Raw Soba-noodle | Manten-Kirari | 50 | 1,210 ± 8.55 | 53.9 ± 9.97 | 91.7 |
| Hokkai T8 | 50 | 1.54 ± 1.22 | 521 ± 74.1 | 0.15 | |
| (Control) | 0 | 0.00 ± 0.00 | 0.00 ± 0.00 | - | |
| Raw pasta | Manten-Kir | 15 | 365 ± 44.0 | 14.9 ± 0.51 | 92.3 |
| Hokkai T8 | 15 | 10.2 ± 0.00 | 170 ± 20.7 | 2.87 | |
| (Control) | 0 | 0.00 ± 0.00 | 0.00 ± 0.00 | - | |
| Dried Soba-noodle | Manten-Kirari | 30 | 836 ± 43.4 | 37.0 ± 12.6 | 91.8 |
| Hokkai T8 | 30 | 3.30 ± 0.00 | 308 ± 49.9 | 0.53 | |
| (Control) | 0 | 0.00 ± 0.00 | 0.00 ± 0.00 | - | |
| Dried pasta | Manten-Kirari | 15 | 391 ± 39.7 | 12.1 ± 4.62 | 94.1 |
| Hokkai T8 | 15 | 2.70 ± 0.00 | 269 ± 57.1 | 0.49 | |
| (Control) | 0 | 0.00 ± 0.00 | 0.00 ± 0.00 | - |
Data are means ± SD of three independent experiment
Generally, soba-noodle producers and customers prefer to consume soba-noodles as soon as possible after noodle preparation. Typically, 120 min is sufficient from the start of soba-noodle production to noodle consumption in a Japanese soba noodle shop. In contrast, for pasta, after mixing the flour and dough, the dough is sometimes stored for 240 min to allow for the uniform distribution of water in the dough. In this experiment, at least 80% and 50% rutin were present in every sample of ‘Manten-Kirari’ at 120 min and 240 min after water addition, respectively. During the drying process of the noodles, we hypothesized that rutinosidase would hydrolyze rutin. However, residual rutin ratios in dried noodles were almost the same as in the raw noodles. This should be associated with the rapid decrease in water content of noodles during the drying process.
In the sensory analysis of soba-noodles, each test score for ‘Manten-Kirari’ was significantly higher (desirable) than that for ‘Hokkai T8’. The non-bitterness of the flour is an important characteristic of ‘Manten-Kirari’. In soba-noodles, most panelists evaluated the bitterness of ‘Manten-Kirari’ as ‘good’, because bitterness was absent or very weak compared to ‘Hokkai-T8’ (Table 2). Generally, the strong bitterness of ‘Tartary buckwheat’ is a significant drawback to its use. Therefore, the reduced bitterness of ‘Manten-Kirari’ is a very favorable characteristic for noodles.
| Variety | Color | Flavor | Taste | Bittemess | Texture | Total | |
|---|---|---|---|---|---|---|---|
| Hardness | Buckwheat noodleness | ||||||
| Manten-Kirari | 15.8** | 14.7* | 8.2** | 8.3** | 15.0** | 15.6** | 77.8 ** |
| Hokkai T8 | 14.0 | 14.0 | 7.0 | 7.0 | 14.0 | 14.0 | 70.0 |
Some papers described that intestinal bacteroides hydrolyze rutin in rats (Bokkenheuser et al. 1987). On the other hand, some flavonoid glycosides from foods have been identified in human plasma (Paganga and Rice-Evans 1997). Although most rutin in Tartary buckwheat noodles may be hydrolyzed after eating, a little amount of rutin may be absorbed without hydrolysis. Further studies are required to clarify this.
Recently, a DNA marker that discriminates ‘Manten- Kirari’ from other common and Tartary buckwheat flour and dried noodles has been identified (Katsu et al. 2019). In addition, Nishimura et al. (2016) reported the clinical effects of ‘Manten-Kirari’-containing noodles. The dough of ‘Manten- Kirari’ at a dose of 5,000 mg flour kg-1 is at a non-effect level in acute and subacute tests on experimental animals (Suzuki et al. 2015b). These results indicate that ‘Manten-Kirari’-containing dough is promising for the production of rutin-rich foods made in Japan.
Acknowledgments We thank Dr. Mukasa for his useful advice for planning the experiments. We also thank Kobayashi- Shokuhin Co. Ltd. for useful advice for the noodle preparation technique. We thank Mr. A. Morizumi, Mr. T. Hirao, Mr. S. Nakamura, and Mr. K. Suzuki for technical assistance. We thank Mr. K. Abe and Mr. T. Fukaya for their assistance in the field and noodle preparation. We also thank Ms. K. Shimizu, Ms. K. Fujii, Ms. M. Hayashida, and Ms. T. Ando for technical assistance. This work was partly supported by a grant from the Research Project on Development of Agricultural Products and Foods with Health-promoting Benefits (NARO), Japan.
