Heat-treatment of Tartary Buckwheat (Fagopyrum tataricum Gaertn.) Provides Dehulled and Gelatinized Product with Denatured Rutinosidase

Abstract

Despite the increasing attention garnered by Tartary buckwheat (Fagopyrum tataricum Gaertn.) for its health benefits, its use has been limited by the difficulty in dehulling and a rutin hydrolysis-induced bitter taste caused by endogenous rutinosidase activity. Using circulating fluidized bed at 200 °C, the buckwheat was observed to produce a popcorn-like gelatinized product. Gelatinization was confirmed by differential scanning calorimetry, and rutinosidase activities were examined using activity staining on native-PAGE, and the substrate (rutin) and product (quercetin) were quantified by LC-MS. Owing to denaturation of rutinosidase in the heat-treated products, the rutin content was retained without the formation of the bitter product quercetin. Nutritional analysis of pre- and post-treated products showed retention of the macro and micro nutrients even after heat-treatment. The popped Tartary buckwheat could, therefore, be obtained by simple treatment, and might reveal new opportunities to utilize pre-gelatinized and rutin-rich properties by rutinosidase denaturation.

Introduction

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is a pseudo-cereal that probably originated in Eastern Tibet, China (Tsuji and Ohnishi, 2001), and is currently cultivated and utilized in many other countries. Historically, Tartary buckwheat has been cultivated in areas with severe environmental conditions, since it is a relatively low-demanding crop and has the ability to tolerate environmental stresses such as water deficiency, diseases, insects, and ultraviolet radiation (Bonafaccia et al., 2003; Fabjan et al., 2003). Recently, Tartary buckwheat has started drawing attention for its nutritional or physiological benefits, owing to essential nutrients such as proteins, vitamins, minerals, and the extremely high polyphenol content (Bonafacca et al., 2003; Huang et al., 2014; Ikeda et al., 2004). Numerous reports have mentioned the approximately 100-fold higher rutin (quercetin 3-rutinoside) content of Tartary buckwheat compared to common buckwheat (Fagopyrum esculentum), which is also well known for its functionality owing to the rutin content (Jian et al., 2007; Morishita et al., 2007).

However, utilization of Tartary buckwheat is limited, owing to the difficulty in dehulling its hard pericarp and the strong bitterness of products (Kawakami et al., 1995), attributed to quercetin formation by rutinosidase-induced rutin hydrolysis in Tartary buckwheat seed (Suzuki et al., 2002; Yasuda et al., 1992; Yasuda and Nakagawa, 1994). The enzyme activity is sufficiently strong to hydrolyze its rutin to quercetin in a very short time, and is estimated to be almost 680-fold that of common buckwheat (Yasuda et al., 1992). Thus, products using Tartary buckwheat as an ingredient are strongly bitter in taste, even if mixed with common buckwheat, which is otherwise not bitter. Therefore, massive breeding efforts have been invested to grow the novel non-bitter Tartary buckwheat “Manten-Kirari”, containing trace rutinosidase activity, using a genetic resource from Nepal (Suzuki et al., 2014). This variety could represent an innovative approach to providing high rutin content to a product.

Heat-treatments of > 70 °C have been reported to de-activate rutinosidase in Tartary buckwheat (Kawakami et al., 1995; Yasuda et al., 1992). Due to the simplicity of the procedure, heat-treatment has been thoroughly investigated to obtain a higher rutin content without the formation of bitter quercetin (Kočevar Glavač et al., 2017; Lukšič et al., 2016; Sun et al., 2018).

However, heat-treatment or breeding efforts may not circumvent all of the limitations mentioned above, especially dehulling difficulties. We had observed that Tartary buckwheat, upon certain heat-treatment conditions, “pops” to result in a popcorn-like product, which easily removes the pericarp. This study therefore aimed to develop an effective method to overcome both the limitations of dehulling and rutinosidase activity in Tartary buckwheat utilization by heat-treatment with circulating fluidized bed.

Materials and Methods

Tartary buckwheat sample preparation    Two kinds of Tartary buckwheat grain, commercial seeds from Hokkaido, Japan (Kissui, Sapporo, Japan) and China (Tsurushin-syubyou, Matsumoto, Japan), were purchased from the Japanese market in 2017.

Milled Tartary buckwheat groats-flour was obtained from the above-mentioned buckwheat grains. Pericarps of the grains (200 g) were removed using a milling machine (IFM-800DG; Iwatani, Osaka, Japan) and test sieves (pore size: 500 µm, 1 mm, 2 mm, and 4 mm; Iida manufacturing, Japan), following the methods reported earlier (Fujita et al., 2003). All the portions of crushed groats with testa were milled to flour using an ultra-centrifugal rotor-mill with a 0.5-mm screen mesh (ZM-100; Retsch, Haan, Germany).

Popped Tartary buckwheat was prepared from the purchased buckwheat grains (200 g) as shown in Figure 1. The grains with pericarps (30 g per batch) were heated at 200 °C by circulating fluidized bed using a small popcorn maker (KK-000285, D-STYLIST; Peanuts club, Osaka, Japan); the grains popped in 4 min, and the hulls were removed using the test sieves. The separated popped product was milled using the ultra-centrifugal rotor-mill with 0.5-mm screen mesh.

Fig. 1.

Tartary buckwheat and its popped product

Four Tartary buckwheat samples, different in origin and treatment, were prepared for further analysis as follows: milled Tartary buckwheat from Hokkaido (TBH-M), popped and milled Tartary buckwheat from Hokkaido (TBH-P), milled Tartary buckwheat from China (TBC-M), popped and milled Tartary buckwheat from China (TBC-P).

Chemicals    Acetonitrile, methanol, and formic acid (LC-MS grade) were obtained from Fujifilm Wako Pure Chemical (Osaka, Japan). Rutin, quercetin, and methyl 2,4-dihydroxybenzoate (used as an internal standard for LC-MS analysis) were purchased from Tokyo Chemical Industry (Tokyo, Japan).

Determination of starch gelatinization by DSC    Starch gelatinization of the samples was evaluated using DSC6100 (Hitachi Hi-Tech Science, Tokyo, Japan). In total, 10 mg (TBH-M and TBC-M) and 5 mg (TBH-P and TBC-P) of samples were weighed accurately into a 70-µL silver sample pan (AG70-CAPSULE; Hitach Hi-Tech Science, Tokyo, Japan) with 50 µL or 55 µL of water, respectively. The pans were sealed and heated from 30 to 120 °C at a heating rate of 0.5 °C/min to obtain a thermogram. A thermogram recorded with 60 µL water in a silver pan was taken as the reference. Parameters such as onset (T₀), peak (T_p), and conclusion (T_c) temperatures, and enthalpy (ΔH) of gelatinization were determined using the supplied software. Enthalpy (ΔH) was calculated by integrating the area enclosed by the thermogram over the base line.

LC-MS quantification of rutin and quercetin    Rutin and quercetin were extracted from the samples (50 mg) using methanol (1 mL), containing methyl 2,4-dihydroxybenzoate as the internal standard (100 µg/mL), and heating the sample vial at 80 °C for 2 h. The extract was centrifuged, and the supernatant filtered through a 0.45-µm membrane filter (Toyo Roshi Kaisha, Ltd., Tokyo, Japan) prior to analysis. A 1-µL aliquot was injected for LC-MS.

A Shimadzu LC-MS system (Kyoto, Japan) equipped with a pump (LC-20AD), a column oven (CTO-20A), an auto sampler (SIL-20A) along with a mass spectrometric detector, LCMS2020, and equipped with an electrospray ion source (ESI) was used. Chromatographic separations were performed with a TSKgel Super-ODS column (4.6 × 50 mm, Tosoh, Tokyo, Japan). The mobile phase consisted of 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B) at a total flow rate of 0.4 mL/min. Chromatographic separation was achieved using a 14-min gradient elution. The initial mobile phase composition was 1% B, which was gradually increased to 80% B in 10 min, and then maintained at 100% B for 2 min, and back to the initial condition of 1% B in 2 min for re-equilibration. All analytes were detected using ESI with the following settings: nebulizer gas 1.5 L/min, drying gas 15 L/min, desolvation line temperature 250 °C, and heat block temperature 200 °C. Mass spectrometric detection was carried out in negative mode, scanning m/z 160–700 in 1-sec event time, covering molecular ions of the internal standard (methyl 2,4-dihydroxybenzoate), quercetin, and rutin. Under these conditions, the internal standard, quercetin, and rutin were eluted at 8.88, 8.66, and 7.23 min as molecular ions with m/z 167, 301, and 609, respectively. Quantification was achieved using the peak area ratio of the internal standard versus standard quercetin and rutin.

Rutinosidase assay with substrate and product quantification    Crude rutinosidase was prepared from the samples (50.0 mg) with extraction buffer (1.0 mL) consisting of 20 mM sodium acetate buffer (pH 5.0). The supernatant was precipitated with 80% saturated (NH₄)₂SO₄, followed by re-suspension of the precipitate in extraction buffer. The obtained crude rutinosidase was desalted by a 100-kDa ultrafiltration membrane (Ultrafree-C3HK; MerckMillipore, Darmstadt, Germany) for the rutinosidase assay.

The rutinosidase assay was performed using 0.1% rutin in 20% methanol (0.3 mL) with crude rutinosidase, following desalting (0.1 mL). The assay mixture was incubated at 40 °C for 10 min. Methanol (1.2 mL), containing the internal standard, was added to stop the hydrolysis, and thereafter, the mixture was filtered through a 0.45-µm membrane filter (Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The filtered assay mixture was applied for rutin and quercetin analysis as mentioned earlier.

Activity staining of rutinosidase on native-PAGE    Activity staining of rutinosidase on native-PAGE was carried out for each sample following the method mentioned by Suzuki et al., 2004. Crude rutinosidase, prepared as above, was used, and glycerol (10% as final concentration) was added. A 10-µL aliquot was applied onto a 5–20% polyacrylamide gel (c-PAGEL C520L; ATTO, Tokyo, Japan) following the electrophoresis conditions suggested by Davis (1964). After electrophoresis, the gel was rinsed with water and equilibrated for 10 min with sodium-acetate buffer (pH 5.0) containing 20% (v/v) methanol. The equilibrated gel was then stained in sodium-acetate buffer (pH 5.0) containing 20% (v/v) methanol, 0.6% (w/v) rutin, and 5 mM copper (II) sulfate for 20 min. Yellow-brown bands of the quercetin-copper complex were visualized wherever rutinosidase activity was available; the stained gel was rinsed with water for use in the flat-bed scanner (GT-X970; EPSON, Suwa, Japan).

Nutritional values    Macro and micro nutrients were evaluated by Japan Food Research Laboratories according to the Standard Tables of Food Composition in Japan (STFCJ) (2015), based on methods recommended by the Association of Official Agricultural Chemists. Macro nutrients are essential food ingredients, and include moisture, protein, fat, carbohydrate, and ash contents, while micro nutrients include minerals such as Fe, K, Mg, Zn, and vitamins such as folic acid and vitamin E. Each measurement was carried out in duplicate. Protein content was analyzed using the Kjeldahl method and quantified as 6.25 times the N content. Carbohydrate content was calculated by subtracting measured moisture, protein, fat and ash from the total weight. Energy was calculated using Atwater's coefficient as a conversion factor: 4 for protein and carbohydrate and 9 for fat.

Results and Discussion

Starch gelatinization of each sample    The thermograms of Tartary buckwheat samples from Hokkaido differed according to treatment (TBH-M and TBH-P), as shown in Figure 2. The endothermic peak, correlated to starch gelatinization, was observed clearly with T₀ at 58 °C, T_c at 79 °C, and T_p at 68.8 °C in the untreated sample (TBH-M, Fig. 2A). Enthalpy (ΔH) was calculated to be 1.488 mJ/mg. On the other hand, a clear endothermic peak was not observed in the popped sample (TBH-P), although a small peak was obvious (Fig. 2B). Therefore, enthalpy of the popped sample could not be calculated. As the popped sample had almost no endothermic peak (Tp), it was concluded that the starch in the popped sample was completely gelatinized. The thermograms also showed a possible thermal history over their gelatinization temperature (Tp), 68.8 °C.

Fig. 2.

DSC thermograms of non-treated (TBH-M) and heat-treated (popped; TBH-P) Tartary buckwheat

Rutin and quercetin contents of Tartary buckwheat samples    Two kinds of Tartary buckwheat (TBH and TBC) subjected to different treatments (-M and -P) showed different rutin and quercetin contents, as shown in Table 1. The contents were consistent with those reported previously (Morishita et al., 2007; Lukšič et al., 2016; Kočevar Glavač et al., 2017; Sun et al., 2018). However, the rutin content was slightly decreased in the popped samples (TBH-P and TBC-P), thus the quercetin contents were increased in response to rutin degradation. Heat-treatment using circulating fluidized bed at 200 °C for 4 min may induce rutinosidase activity until heat-denaturation of the enzyme; therefore, popped samples had a higher quercetin content than the untreated samples. As reported by Buchner et al. (2006), thermal processing induced degradation of both rutin and quercetin, and quercetin showed faster degradation in the quercetin and rutin model system. The increase in quercetin was smaller than the equivalent decrease in rutin. The bitterness of milled and popped Tartary buckwheat samples was checked and confirmed to be strong and without bitterness in the milled and popped samples, respectively.

Table 1. Rutin and quercetin content (mg/100 g, dry matter basis) of the samples

Sample name	Rutin	Quercetin
TBH-M	1637 ± 118.2	35 ± 2.3
TBH-P	1469 ± 49.5	52 ± 6.2
TBC-M	1548 ± 77.8	10 ± 1.3
TBC-P	1202 ± 71.2	111 ± 13.2

Analysis were triplicated and expressed as average ± SD.

Rutinosidase assay with substrate and product quantification    In order to estimate rutinosidase activity, the mixture of rutin solution and crude enzyme extract was subjected to HPLC after incubation. The chromatograms obtained are shown in Figure 3. The rutin peak in the assay completely disappeared only after the addition of the crude enzyme extract from both the untreated Tartary buckwheat samples (TBH-M and TBC-M; Fig. 3A and C); instead, the quercetin peak was intensely detected as the result of rutinosidase activity. In contrast, the rutin peak remained in both popped Tartary buckwheat sample crude enzyme extracts (TBH-P and TBC-P; Fig. 3B and D). This suggests the heat-denaturation of rutinosidase in popped Tartary buckwheat by use of circulating fluidized bed.

Fig. 3.

LC-MS chromatograms of rutinosidase assay mixtues using crude enzymes.

The assay were performed with crude enzyme from Tartary buckwheat samples with 0.1% rutin (w/v), then substrate (rutin) and product (quercetin) were quantified.

Activity staining of rutinosidase in native-PAGE    Rutinosidase activity was directly displayed in native-PAGE using activity staining based on copper (II) complex formation (Fig. 4). In un-treated Tartary buckwheat samples (TBH-M and TBC-M), the active bands included several isozymes that were clearly stained as yellow-brown bands in the gel (Fig. 4A and C). On the other hand, popped Tartary buckwheat samples (TBH-P and TBC-P) showed no active bands (Fig. 4B and D). As reported earlier by Kawakami et al. (1995) and Yasuda et al. (1992), endogenous rutinosidase activity should be denatured at > 70 °C. Popped Tartary buckwheat samples (TBH-P and TBC-P) could be heated to greater than 70 °C by using circulating fluidized bed, thereby resulting in denatured rutinosidase.

Fig. 4.

Activity staining of rutinosidase in crude enzymes.

A: TBH-M, B: TBH-P, C: TBC-M, D: TBC-P

Macro and micro nutrients in samples    Table 2 shows the contents of macro and micro nutrients in Tartary buckwheat samples from Hokkaido after different treatments (TBH-M and TBH-P). The data shown here are presented on a dry matter basis, since the popped sample (TBH-P) showed a significantly lower moisture content (13.9 g/100 g in TBH-M and 7.5 g/100 g in TBH-P) due to heating. Macro nutrients in TBH-M and TBH-P showed no significant difference, as shown in Table 2A. However, the content of micro nutrients tended to be slightly higher in the non-treated sample (TBH-M) than in popped sample (TBH-P; Table 2B). Interestingly, vitamin E and folic acid, which are considered to be important vitamins relative to those in major cereals such as rice or wheat, were retained even after popping. Numerous reports on the nutritional value of Tartary buckwheat have indicated that the benefits of Tartary buckwheat consumption are attributable to the high protein, mineral, and vitamin contents. Our results here concur with previous reports, and demonstrate that heat-treatment (popping) does not, in general, induce significant differences in nutrient content.

Table 2. Macro and micro nutrients in Tartary buckwheat samples

A) Macro nutrient
Sample name	Protein* (g)	Fat (g)	Ash (g)	Carbohydrate (g)	Dietary fiber (g)	Energy (g)
TBH-M	13.7	4.1	2.3	79.9	5.7	411
TBH-P	12.5	4.0	2.2	81.3	4.5	411

B) Micro nutrient
Sample name	Fe (mg)	K (mg)	Mg (mg)	Zn (mg)	Folic acid (µg)	Vitamin E
						α-TCP** (mg)	β-TCP (mg)	γ-TCP (mg)	δ-TCP (mg)
TBH-M	5.41	584	239	3.33	49	0.5	–	5.6	0.2
TBH-P	3.82	499	214	3.16	32	0.3	–	4.0	0.2

The content expressed as per 100 g, dry matter basis.

*  Protein content was calculated based on the nitrogen conversion factor as 6.25.

**  TCP: Tocopherol

Heat-treatment of Tartary buckwheat with pericarps using circulating fluidized bed led to popping. Popped Tartary buckwheat had no endogenous rutinosidase activity, attributable to heat denaturation above the gelatinization temperature. Since Tartary buckwheat is difficult to dehull, heat-induced popping could be an attractive option to conveniently avoid the conventional dehulling procedure. Rutinosidase activity in Tartary buckwheat, which is another limitation in its utilization, could also be avoided by heat denaturing, as demonstrated in the current study. Therefore, this technology may reveal new opportunities to further utilize rutinosidase-active Tartary buckwheat, and to add pre-gelatinization and rutin-rich properties. Additionally, popped Tartary buckwheat is ready-to-eat and could be ground as pre-gelatinized flour, which could be used for various food products.

Further studies would be required to elucidate the heat-treatment conditions, the scale-up to industrial production, and the properties of popped, pre-gelatinized Tartary buckwheat.

Acknowledgements    This study was funded from an international collaborative research project entitled: “Establishment of food value chain through value-addition of local food resources for sustainable rural development”, supported by Japan International Research Center for Agricultural Sciences (JIRCAS).

Abbreviations

DSC

Differential scanning calorimetry

LC-MS

Liquid chromatography-mass spectrometry

PAGE

Polyacrylamide gel electrophoresis

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