2020 Volume 26 Issue 5 Pages 597-603
Although Manten-Kirari is a new Tartary buckwheat variety with trace rutinosidase activity, the trace levels of rutinosidase gradually hydrolyze rutin in dough. In addition, contamination from normal rutinosidase varieties can sometimes hydrolyze rutin during dough making. To reduce the hydrolysis, we evaluated the effects of sodium bicarbonate (NaHCO3). The following variables were adjusted: water content, blending ratio of Tartary buckwheat flour, and dough storage temperature. The rutin residual ratio increased with decreasing dough storage temperature, water content, and time after addition of water, and with increasing NaHCO3 concentration. Addition of 0.5% NaHCO3 can prevent rutin hydrolysis almost completely in dough made from Manten-Kirari. When the contamination rates from normal rutinosidase varieties was less than 1%, addition of NaHCO3 to dough made from Manten-Kirari under low-temperature storage of dough (5 °C) can retain a rutin residual ratio of more than 80% for a water content of 30% for up to 2 hours after addition of water.
Rutin, a type of flavonoid, is widely found in plants (Sando and Lloyd 1924, Couch et al., 1946, Haley and Bassin 1951, Fabjan et al., 2003). Rutin-containing food has many biological functions, such as strengthening of blood capillaries (Shanno, 1946; Griffith et al., 1944), alpha-glucosidase inhibitory activity (Li et al., 2009), anti-oxidative activity (Jiang et al., 2007; Awatsuhara et al., 2010; Ishiguro et al., 2016), and anti-hypertensive activity (Matsubara et al., 1985). In addition, Wieslander et al. have reported the clinical effects of rutin in double-blind crossover studies on serum myeloperoxidase and cholesterol levels (Wieslander et al., 2011), and occurrence of headaches, mucosal symptoms, and tiredness (Wieslander et al., 2012). Buckwheat is the only known cereal to contain rutin in its seeds. Therefore, buckwheat has been identified as a rutin-rich food (Kreft et al., 2006; Ikeda et al., 2012). Among cultivated buckwheat species, Tartary buckwheat (Fagopyrum tataricum Gaertn.) is known to contain about 100-fold greater rutin content in the seeds than common buckwheat (Fagopyrum esculentum Moench.). The rutin content in Tartary buckwheat flour is sometimes more than 2% w/w. However, Tartary buckwheat is also known to have high rutinosidase activity, which is sufficient to hydrolyze seed rutin within a few minutes after the addition of water (Yasuda et al., 1992; Yasuda and Nakagawa 1994; Suzuki et al., 2002) (Fig. 1). Rutin is a functional compound, and therefore hydrolysis of rutin is undesirable in Tartary buckwheat. In addition, rutin hydrolysis causes a strong bitter taste in processed foods. The hydrolyzed moiety of rutin, quercetin (the aglycone of rutin), is an important compound that causes the strong bitter taste (Kawakami et al., 1995; Suzuki et al., 2002; 2014a, 2014b). Some food-processing techniques such as heat treatment of the seeds or flour can prevent rutin hydrolysis. However, this leads to a serious deterioration of texture (Yoo et al., 2012), flavor, and color, as well as adding to the cost of preparation. It is in this context that a new Tartary buckwheat variety ‘Manten-Kirari’ (MK) with trace-rutinosidase activity was developed; the variety has desirable characteristics with respect to production of foods with high rutin content (Suzuki et al., 2014a, 2014b). The rutinosidase activity in MK seed is two to three orders of magnitude less than that of other traditional varieties such as ‘Hokkai T8’ (T8) which is one of the parents of MK (Suzuki et al., 2014b). Processed foods such as bread (Suzuki et al., 2015) and noodles (Suzuki et al., 2019) made from MK have high rutin concentrations and reduced bitterness. However, there remains some trace rutinosidase activity in MK. This gradually hydrolyzes rutin during dough storage. In addition, contamination of MK seeds or flour by varieties with normal rutinosidase activity such as T8 sometimes occurs, e.g., during sowing, harvesting, and food processing. Consequently, there is a need for information on the effects of different variables (such as water content, dough storage temperature, and blending ratio for Tartary buckwheat flour) on the rutin residual ratio. In addition, techniques to retain a high rutin residual ratio, even in contaminated dough, are also required. In this study, we tested the effects of sodium bicarbonate (NaHCO3) addition on rutinosidase activity in buckwheat dough. NaHCO3 is added to food as a raising agent and to control pH, for example, in bread and cookies, and it is used to prevent degeneration of proteins such as milk in coffee as a pH control agent. In water, bicarbonate is re-equilibrated as in Eq. 1.
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Rutin hydrolysis in Tartary buckwheat seeds.
Plant materials and preparation of flour Tartary buckwheat of two varieties (the trace-rutinosidase, MK and T8, were sown in early June in an experimental field in Memuro, Hokkaido, Japan (42°52′54″N, 143°03′18″E, altitude 131 m). MK is one of the most widely used varieties of Tartary buckwheat in Japan. We harvested plants in late August and dried them at 35 °C for about one week. Harvested plants were threshed, and seeds were purified using grain fan. After removal of stones, seeds were stored at 10 °C. Tartary buckwheat seeds were milled using a test mill (Quadrumat® Junior, Brabender® GmbH & Co., Duisburg, Germany). Subsequently, the flour was passed through a 2-mm sieve; the flour milling percentage was 63%. A commercially available wheat flour (‘Yuki,’ Nisshin Seifun Group Inc., Tokyo, Japan) was used for blending with buckwheat flour in the preparation of the dough.
Evaluation of prevention of rutin hydrolysis in dough by addition of NaHCO3 We added 0%, 0.125%, 0.25%, 0.5% and 1.0% solutions of NaHCO3 to the flour. To make the dough, we added the following ratios of water to the flour: 30% for dried noodles, 45% for ‘fresh’ noodles, 100% for bread, and 400% for galette. The water and flour were mixed immediately for one minute to obtain a uniform dough. After that, the dough was placed at 25 °C for 60, 120, 240, 360, and 480 minutes.
Evaluation of the effects of contamination of trace rutinosidase variety dough by normal rutinosidase variety flour on rutin hydrolysis In order to prepare buckwheat noodles, Tartary buckwheat flour was mixed with wheat flour. The final proportion of Tartary buckwheat flour ranged from 30% to 80%; 30% is generally used as the lower limit for the blending ratio for preparation of buckwheat noodles in Japan, and 80% is the upper limit for hand-made Tartary buckwheat noodles because of the fragility of Tartary buckwheat dough. MK flour (trace rutinosidase variety) was deliberately contaminated with T8 flour (normal rutinosidase variety) in the following proportions: 29.91% MK, 0.09% T8; 29.73% MK, 0.27% T8; 79.7% MK, 0.3% T8; and 79.2% MK, 0.8% T8. For each flour mixture, we added a 0.125%, 0.25% or 0.5% solution of NaHCO3. To make the noodle dough, we added 30% or 45% water to the flour. After mixing the flour and water, the dough was held at 5 °C or 25 °C for 120 and 480 minutes.
Evaluation of pH in dough We added 10 mL of deionized water to 0.5 g of each dough and mixed it well to obtain a uniform solution. The pH of the solution was measured using a pH meter.
Analysis of rutin residual ratio of dough After storage of the dough, we added 9 mL of methanol containing 20% volume of 0.1% (v/v) phosphoric acid and mixed in 1.0 g of the dough. Samples were held in an oven at 80 °C for 3 hours. After removal, the centrifuged supernatant was subjected to high-performance liquid chromatography (HPLC) according to the method of Suzuki et al., (2005). Rutin and quercetin concentrations were determined by the retention time of commercially available standards. The rutin residual ratio was calculated as the percentage of rutin content in the dough relative to the rutin content in the flour before addition of water.
Evaluation of prevention of rutin hydrolysis in dough by addition of NaHCO3 In T8, rutin was completely hydrolyzed in dough for every water content in which 0%, 0.125% and 0.25% NaHCO3 was added (Fig. 2A–C). In the dough where 0.5% NaHCO3 was added, rutin was mostly hydrolyzed after addition of water (Fig. 2D). This trend is clearer in dough with lower water content (such as 30% for the dry noodles) than in dough with higher water content. On the other hand, rutin in MK was gradually hydrolyzed after addition of water (Fig. 2F–I). This trend is also clearer in the MK dough with lower water content (30%) than that with higher water content, as in the case for 0.5% NaHCO3 in T8. In the dough in which a 1.0% NaHCO3 solution was added, no rutin was hydrolyzed after addition of water for any of the doughs (30%, 45%, 100%, 400% water) (Fig. 2J).
Effects of NaHCO3 concentration on rutin residual ratio in dough.
To make food products with a high rutin content, a rutin residual ratio above 80% is an important practical threshold for the following reasons. Although rutin hydrolysis causes a strong bitter taste (Suzuki et al., 2002), a rutin residual ratio around 80% is acceptable in terms of bitterness for Tartary buckwheat noodles (Suzuki et al., 2019). In addition, a rutin residual ratio of about 80% corresponds to the rutin amount at which clinical trials have revealed positive effects on humans (Nishimura et al., 2016). In MK, addition of 0.125% NaHCO3 meant the rutin content for all doughs remained roughly at or above 80% 60 minutes after addition of water (Fig. 2G). This would be sufficient time to make and cook noodles in a home or factory environment. However, hand-made noodle makers sometimes store raw noodles for more than 480 minutes. In addition, bread-making requires a fermentation procedure which can take several hours. In such cases, addition of a 0.125% to 0.5% solution of NaHCO3 should be effective (Fig. 2G–I). In bread dough (100% water to flour ratio), the rutin residual ratio is also affected by the blending ratio of Tartary buckwheat flour to wheat flour; a low blending ratio tends to give rise to a high rutin residual ratio in noodles (Suzuki et al., 2019). In galette dough (containing a 400% water to flour ratio), only the 1.0% NaHCO3 solution retained a rutin residual ratio of 80% for over 120 minutes after addition of water (Fig. 2J).
In the T8 doughs, the 1.0% solution of NaHCO3 maintained the rutin residual ratio above 80% for the dried noodle dough (30% water to flour ratio) for 60 minutes after addition of water (Fig. 2E). These results indicate that the rutin residual ratio could be higher if larger amounts of NaHCO3 were added. However, NaHCO3 has a bitter taste, and doughs containing higher NaHCO3 concentrations also have a bitter taste even if the rutin residual ratio is higher than 80%. In addition, the color of the dough turns darker (light yellow to dark brown) as the concentration of NaHCO3 increases (data not shown). Tartary buckwheat noodle makers in Japan generally prefer a light-yellow color, and therefore higher concentrations of NaHCO3 solutions are not suitable.
Evaluation of effects of contamination of trace rutinosidase variety dough with normal rutinosidase variety flour on rutin hydrolysis The contamination tests simulated contamination of MK flour with T8 or other normal rutinosidase varieties. The effects of addition of NaHCO3 to prevent or reduce rutin hydrolysis in contaminated doughs were evaluated. The rutin residual ratio was negatively correlated with dough storage temperature, dough water content, and time after addition of water, and positively correlated with NaHCO3 concentration. In MK:T8 (30:0) and 29.91:0.09, the rutin residual ratio was over 80%, such as in the case of a 30% water content at 5 °C (Fig. 3A,B). This trend is also observed for MK:T8 = 80:0 and 79.7:0.3 (Fig. 3 D, E). For MK:T8 = 29.73:0.27, the rutin residual ratio was over 80% for the dried noodle dough (30% water to flour ratio) 6 hours after addition of water for a 30% water content at 5 °C and 2 hours after addition of water (Fig 2. C). Even for MK:T8 = 79.2:0.8, the rutin residual ratio was over 80% for the dried noodle dough (30% water to flour ratio) at 2 and 6 hours after addition of water at 5 °C (Fig. 3F).
Effects of NaHCO3 concentration on rutin residual ratio in dough.
The rutinosidase activity in Tartary buckwheat seeds consists of at least two isozymes with very similar enzymatic characteristics (Suzuki et al., 2002). The optimal pH for rutinosidase activity is around 4.0 and the optimal temperature is around 40 °C. The pH of the doughs after addition of 0% 0.125%, 0.25%, 0.5%, or 1.0% NaHCO3 solutions was 6.5, 8.9, 9.2, 10.0, and 10.5, respectively. Therefore, addition of NaHCO3 would reduce rutinosidase activity in terms of divergence from the optimal pH (Yasuda et al., 1992). In addition, the inhibitory effects of sodium ions on rutinosidase activity should be investigated in a future study. The difference in the rutin residual ratio between the 5 °C and 25 °C treatments is also related to divergence from the optimal temperature.
When the contamination rate due to the normal rutinosidase variety was over 1%, it was difficult to reach a rutin residual ratio above 80% in the fresh noodle dough (45% water to flour ratio) at 25 °C. In a recent study, Katsu et al., (2019) identified a DNA marker that discriminates MK from other Tartary buckwheat varieties. This technique should be effective for the evaluation of contamination rates for MK buckwheat. Using this method, it should be possible to efficiently produce foods (such as fresh noodles, dried noodles, breads, and galettes) that are rutin rich and that do not have a bitter taste.
Acknowledgments We would like to thank Dr. Mukasa for his useful advice on planning the experiment. We also thank Kobayashi-shokuhin Co. Ltd. for useful advice on 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, 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 supported in part by a grant from the Research Project on Development of Agricultural Products and Foods with Health-promoting benefits (NARO), Japan.
