FAGOPYRUM Volume 37(2), September 2020 ISSN 0352 – 3020 Scientific Journal on Buckwheat Research International Buckwheat Research Association Slovenska akademija znanosti in umetnosti Slovenian Academy of Sciences and Arts FAGOPYRUM volume 37(2), September 2020 An international journal on buckwheat research published by The Slovenian Academy of Sciences and Arts, Ljubljana, Slovenia, under the auspices of The International Buckwheat Research Association (IBRA). Confirmed by the Class of Natural Sciences of the The Slovenian Academy of Sciences and Arts on June 6, 2019 and the Presidency of the Academy on November 12, 2019. Managing Editorial Board Ivan Kreft (Editor-in-Chief) (Slovenia) Christian Zewen (Associate Editor) (Luxembourg) Blanka Vombergar (Associate Editor) (Slovenia) Mateja Germ (Associate Editor) (Slovenia) Kiyokazu Ikeda (Associate Editor) (Japan) Clayton Campbell (Language Editor) (Canada) Advisory Board Y. Asami, Ryukoko University, Ohtsu, Japan T. Bjorkman, Cornell Univerity, Geneva, USA C. Campbell, Chairperson of the 7thISB, Canada N. K. Chrungoo, North Eastern University, Shillong, India N. N. Fesenko, All-Russia Research Institute of Legumes and Groat Crops, Orel, Russia M. Germ, University of Ljubljana, Ljubljana, Slovenia H. Hayashi, Tsukuba University, Tsukuba, Japan Y. Honda, National Agriculture and Food Research Organization, Tsukuba, Japan S. Ikeda, Kobe Gakuin University, Kobe, Japan N. Inoue, Shinshu University, Minami-Minowa, Japan D. Janovska, Crop Research Institute, Praha, Czech R. Lin, Shanxi Academy of Agricultural Science, Taiyuan, China R. L. Obendorf, Cornell University, Ithaca, USA O. Ohnishi, Kyoto University, Kyoto, Japan R. Ohsawa, Tsukuba University, Tsukuba, Japan C. H. Park, Kangwon National University, Chunchon, Korea J. C. Rana, ICAR-National Bureau of Plant Genetic Resources, New Delhi, India G. N. Suvorova, All-Russia Research Istitute on Legumes and Groat Crops, Orel, Russia G. Wieslander, Department of Medical Sciences, Uppsala University and University Hospital, Uppsala, Sweden S.-H. Woo, Chungbuk National University, Cheongju, Korea Y. Yasui, Kyoto University, Kyoto, Japan Editor Emeritus: Toshiko Matano, Ohmi Ohnishi, Kiyokazu Ikeda Subscription Information: One volume per year: subscription price for printed issues 2018 is US $80.00 for individuals and scientific institutions. Authors, reviewers and editors of the issue receive free printed copies. Electronic versions are untill further freely available for academic and non-commercial use at http://www.sazu.si/publikacije-sazu FAGOPYRUM is open to everyone who is interested in buckwheat and will cover all aspects of buckwheat research: genetics, cytology, breeding, agronomy, nutrition, utilization, biochemistry, ethnobotany and others. FAGOPYRUM will accept manuscripts in English only, which meet the scientific requirements set by the Editorial Board and which have not been published or submitted for publication elsewhere. Announcements concerning the promotion of research on buckwheat (workshops, symposium and so on), bibliographies and other information related to buckwheat will also be published. Deadline for receiving manuscripts for volume 38: September 30, 2020, to e-mail address: ivan.kreft@guest.arnes.si Page setup: Medija grafično oblikovanje d.o.o., Prečna ulica 6, 1000 Ljubljana. Front page photo: A prototype of commercial product of buckwheat noodles with added blue-green alga, ishi-kurage (Nostoc commune Vauch.) (See paper of Asami et al.). FAGOPYRUM volume 37(2) (2020) CONTENTS ORIGINAL PAPERS Mechanical characteristics of buckwheat noodles made with blue-green alga, ishi-kurage (Nostoc commune Vauch.) Yuya ASAMI, Mitsuki ZENNO, Keina MIKAMI, Hikaru OSUGA, Rui SETOYAMA, Kenta SAKANASHI, Tesshu TAMAI, Tsuyoshi FURUMOTO and Kiyokazu IKEDA ............................................................................... 29 Extraction of rutin and quercetin from Tartary buckwheat grains, hydrothermally treated at different temperatures Filip BRAJČIČ, Patricia LUŠTEK, Tinara ŠUŠTARIČ, Janja PUST ....................................................................... 37 INFORMATION Information for authors ........................................................................................................................................ 41 Research paper Mechanical characteristics of buckwheat noodles made with blue-green alga, ishi-kurage (Nostoc commune Vauch.) Yuya ASAMI1, Mitsuki ZENNO1, Keina MIKAMI1, Hikaru OSUGA1, Rui SETOYAMA1, Kenta SAKANASHI1, Tesshu TAMAI1, Tsuyoshi FURUMOTO1 and Kiyokazu IKEDA*2 1 Faculty of Agriculture, Ryukoku University, Seta oe-cho, Otsu, 520-2194, Japan, yuya_asami@agr.ryukoku.ac.jp 2 Faculty of Nutrition, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan, nk102009@nutr.kobegakuin.ac.jp * Corresponding author: nk102009@nutr.kobegakuin.ac.jp, Fax +81 78 974 5689 DOI https://doi.org/10.3986/fag0016 Received: July 27, 2020; accepted September 7, 2020. Keywords: common buckwheat, mechanical characteristics, noodles, Nostoc commune ABSTRACT The present study was conducted to clarify the effect of a kind of blue-green algae, i.e., ishi-kurage, on the mechanical characteristics of buckwheat noodles. Mechanical analysis of buckwheat noodles with ishi-kurage showed that incor- poration of ishi-kurage into buckwheat noodles enhanced breaking stress and energy. Sensory evaluation with human panels showed that buckwheat noodles with ishi-kurage were more preferred when compared with noodles without ishi- kurage. On the other hand, incorporation of ishi-kurage into buckwheat noodles enhanced decreased solubility of the albumin plus globulin fraction. The present study finding suggests that the endogenous protein may be an important factor responsible for the mechanical characteristic of buckwheat noodles with ishi-kurage. Fagopyrum 37(2):29-36 (2020) 29 INTRODUCTION Buckwheat (Fagopyrum spp.) is an important crop in some regions of the world (Kreft et al., 2003; Ikeda, 2002). Buckwheat flour contains high levels of essential nutrients such as protein (Ikeda et al., 1991) and miner- als (Ikeda and Yamashita, 1994). Thus buckwheat flour is an important dietary source of these essential nutrients. On the other hand, components in buckwheat flour have still not been well characterized from the viewpoints of both nutrition and food-functionality. Careful characteri- zation of the components is needed to better understand their nutritional and food-functional properties. There is a variety of buckwheat foods, such as bread, pancake, crepe, galettes, pasta, blini, kasha etc., around the world (Ikeda, 2002). In view of their processing and cooking, increasing attention has been paid to the palata- bility and acceptability of buckwheat products. Clarifying the mechanical characteristics of buckwheat products, including noodles and pasta, is a subject of great interest. Noodles made from buckwheat flour-water dough are popular in some regions including Japan (Ikeda, 2002). In Japan, buckwheat noodles are a popular, traditional food. Traditional processing methods for buckwheat noodles have been long recorded in Japanese history for approximately four hundred years or more (Zen-men- kyo, 2014). As buckwheat flour has low cohesiveness, dough-binders, such as wheat flour, egg, seaweed, Japa- nese yam flour, are often added in preparing buckwheat noodles (Zen-men-kyo, 2014). A variety of buckwheat noodles with various dough-binders has been tradition- ally available in Japan. We reported mechanical effects by addition of various dough-binders to buckwheat noodles (Ikeda et al., 2005; Asami et al., 2019). However, further systematic analysis is needed to understand the exact mechanical effects of various dough-binders to buck- wheat products. Japanese people prefer to eat edible algae including marine algae. As Japan is surrounded by the sea on all sides, there is a variety of edible marine algae in Japan. In view of their color, algae are classified into four groups, i.e., brown algae, red algae, blue green algae, and indigo algae. In relation to buckwheat processing, various edible marine algae are traditionally utilized as dough-binders (Zen-men-kyo, 2014). They include buckwheat noodles with red algae, called hegi-soba in Niigata, the central re- gion of Japan, and soba with agar-agar in several regions (Zen-men-kyo, 2014). These buckwheat noodles with red algae or agar-agar have a unique preferred texture. In Japan, there is a kind of blue-green algae, i.e., Nostoc commune Vauch., called ishi-kurage in Japanese (Fig.1). Ishi-kurage belongs to a cyanobacterium phyrum and a Nostoc genus (Itoh, 2015). Ishi-kurage grows natu- rally on some conditions such as a surface of soil, but is fragile to drying (Fig. 1-(A)) and wetting, i.e., this algae becomes swollen (Fig. 1-(B)) when wetting (Itoh, 2015). In Japan, ishi-kurage is traditionally utilized as an edible algae in some limited areas such as Ane-gawa River in Shiga Prefecture, Western region of Japan, and Miyako- jima Island in Okinawa Prefecture. Ishi-kurage contains functional components such as effect of reducing serum and liver cholesterol concentrations that may exhibit beneficial effects to humans (Hori at al., 1990; Ishibashi et al., 1994; Itoh, 2015). Therefore, the nutritional value as a functional food of ishi-kurage has been increasing in recent years. From the nutritional importance of buck- wheat and ishi-kurage, it is an interesting subject to utilize ishi-kurage as their dough-binder to buckwheat noodles. There are up to now no buckwheat noodles prepared with ishi-kurage. If buckwheat noodles with high palatability can be prepared with ishi-kurage, much attention to such buckwheat noodles will be attracted. This study aimed to prepare buckwheat noodles pre- pared with ishi-kurage and to clarify mechanical charac- teristics of buckwheat noodles with added ishi-kurage. MATERIALS AND METHODS Materials Mechanical characteristics of buckwheat noodles were analyzed in the present study. Two mechanical anal- ysis, I and II, were conducted in this study. Mechanical analysis I was conducted to clarify the ef- fects of the ishi-kurage on the buckwheat noodles. Buck- wheat flour (Fagopyrum esculentum Moench, var. Kita- wase-soba), which was harvested in Hokkaido (in 2018), was used in this research. Buckwheat flour was kindly provided prepared from Terao Milling Co. (Hyogo, Japan) and stored at -80oC until use. Ground blue-green algae, i.e., ishi-kurage in Japanese (Nostoc commune Vauch.) (Fig. 1-(C)) used in this study was a commercial product (Mi- cro Algae Co., Gifu, Japan). Mechanical analysis II, buckwheat noodles with ishi- kurage as a commercial dried noodle product as were pro- totyped and their mechanical characteristics were meas- ured. Production of buckwheat noodles with ishi-kurage Asami et al. (2020): Buckwheat noodles with Nostoc commune 30 was outsourced to the Tanaka Seimenjyo Co. Ltd., Japan. Buckwheat flour used was that harvested in Japan, and wheat flour used was that harvested in the USA, Cana- da, and Australia. The same ishi-kurage sample, as used in mechanical analysis I, was used in mechanical analysis II. Mechanical measurements Mechanical analysis I: Mechanical characteristics of buckwheat noodles were evaluated by breaking analysis. Prior to mechani- cal analysis, buckwheat flour, which had been stored at -80oC, was placed in a desiccator at room temperature until the flour exhibited a constant moisture content. The moisture of the flour was measured with a moisture analyzer (ML-50, A&D Co. Ltd., Japan). Ishi-kurage was boiled, and then sticky gel of ishi-kurage obtained by boil- ing was added to buckwheat flour. The buckwheat dough was prepared just prior to mechanical analysis to have a moisture content of about 42% (w/w) by adding an appropriate amount of distilled water. Then buckwheat noodles were made from the buckwheat dough using a hand-made pasta machine (SP-150, Imperta Co., Torino, Fig. 1 Ishi-kurage. (A), ishi-kurage in the dry state; (B), ishi-kurage in the wet state; and (C), ground ishi-kurage. (A) (B) (C) Fagopyrum 37(2):29-36 (2020) 31 Italy). The buckwheat noodles obtained were subjected to mechanical analysis. Before the mechanical analysis, buckwheat noodles prepared were heated in boiling water for a period of 150 sec and subsequently were cooled for a period of 150 sec at 4oC. Immediately after cooling, me- chanical measurements of the noodles were performed. Breaking analysis of buckwheat noodles was performed with Rheoner RE2-3305C (Yamaden Co. Ltd., Japan). Measurements of breaking analysis were performed with a load cell of 200N and measurement speed of 0.50 mm/ sec. A wedge-style plunger (No.49: W 13mm, D 30mm, H 25mm) was used in measurements with the Rheoner RE2-3305C. Mechanical measurements were replicated twenty times for each sample. Mechanical analysis II: Noodles were prepared with a buckwheat flour-to- wheat flour ratio of 1:4. Two types of buckwheat noodles were prepared. Two types of buckwheat noodles were prepared with or without addition of ishi-kurage. In the case of buckwheat noodles with added ishi-kurage, the amount of ishi-kurage added was 2% of the flour weight. Mechanical analysis of the buckwheat noodles was meas- ured in the same as in mechanical analysis I, except that the noodle boiling time was 5 minutes. Figure 2 shows buckwheat noodles with and without addition of ishi- kurage. Sensory evaluation Sensory evaluation was conducted by a scoring-scale method (Toda, 1994) with volunteer panels (n=26). The evaluation criteria which was selected consisted of six items, i.e., overall evaluation, hardness, springiness, eas- iness to bite through, smoothness and color. The scoring scales consisted of seven points: +3, the most prefer; +2, moderately prefer; +1, slightly prefer; 0, medium prefer; -1, slightly less prefer; -2, moderately less prefer; and -3, the least prefer. The buckwheat noodle samples in soy- sauce soup were presented to the panels immediately af- ter cooking and were immediately evaluated. This study was implemented after the permission from the Ryuko- ku University Ethics Committee. The panels in this study gave their consent regarding the purpose of the study, study methodology and publication of the study results. Protein determination For chemical analysis of the combined fractions of buckwheat albumin plus globulin in the heated noodle samples which had been subjected to the mechanical measurements, the noodle samples were lyophilized and (A) (B) Fig. 2 A prototype of commercial product of buckwheat noodles with added ishi-kurage. (A), non added ishi-kurage and (B), added ishi-kurage. Asami et al. (2020): Buckwheat noodles with Nostoc commune 32 then ground into flour. The flours obtained were extract- ed with a ten-fold (v/w) volume of 0.2M NaCl for 1hr at 4oC. After the extraction, the suspensions were centri- fuged at 17,000 X g for 20 min. Protein concentration was determined using the Bradford method (Bradford, 1976) with bovine serum albumin as a standard protein. Statistical analysis Statistical analysis was conducted using a personal computer with the program Excel (Microsoft Co., USA), Ekuseru-Toukei (Social Survey Research Information Co., Japan) and SPSS Ver.23.0 (IBM, USA). RESULTS AND DISCUSSION Mechanical analysis I: mechanical characteristics of buckwheat noodles made with ishi-kurage Figure 3 shows breaking characteristics of buckwheat noodles prepared without or with ishi-kurage. As amounts of ishi-kurage added into buckwheat noodles increased, breaking stress and energy of the buckwheat noodles concomitantly increased (Fig. 3 (A and B)). A significant high breaking stress and breaking energy was found with buckwheat noodles with a concentration of ishi-kurage with 1.8% or over as compared with buckwheat noodles without ishi-kurage (P<0.05) (Fig. 3 (A and B)). These find- ings showed unique mastication buckwheat noodles pre- pared with ishi-kurage noodles. Mecanical analysis II: mechanical characteristics of prototype of buckwheat product with ishi-kurage Figure 4 shows the comparison of breaking character- istics between prototype noodles made without and with ishi-kurage. There was a significant (P<0.05) difference in breaking stress and breaking energy between the two dif- ferent buckwheat noodles examined (Fig. 4). The effect of addition of ishi-kurage could be shown as in the results of mechanical analysis I (Fig. 3). 0 20000 40000 60000 80000 100000 120000 0% 0.6% 1.2% 1.8% 2.4% 3.0% 0 5000 10000 15000 20000 25000 0% 0.6% 1.2% 1.8% 2.4% 3.0% 1.2 1.0 .8 .6 0.4 .2 0 2.5 2.0 1.5 1.0 0.5 0 c bc bc ab ab a c bc bc ab ab a Br ea ki ng e ne rg y (J/ m 3 X1 04 ) Br ea ki ng st re ss (P a X1 05 ) Ishi-kurage blending ratio of buckwheat flour to total Ishi-kurage blending ratio of buckwheat flour to total Fig. 3 Breaking characteristics of buckwheat noodles made with ishi-kurage. (A), breaking stress; and (B), breaking energy. Vertical bars in the figure show the standard deviations. Values within the same row that are not followed by the same letter are significantly different at P<0.05. (A) (B) Fagopyrum 37(2):29-36 (2020) 33 0 50000 100000 150000 200000 250000 300000 350000 400000 450000 500000 無添加 添加 5.0 4.5 4. 3.5 3. 2.5 2. 1.5 0 1. 0.5 0 20000 40000 60000 80000 100000 120000 無添加 添加 12.0 10.0 8.0 6.0 4.0 2.0 0 Br ea ki ng st re ss (P a X1 05 ) Br ea ki ng e ne rg y (J/ m 3 X1 04 ) Non added ishi-kurage Added ishi-kurage Non added ishi-kurage Added ishi-kurage ** * Sensory evaluation of buckwheat noodles with ishi- kurage Figure 5 shows the comparison of sensory evaluation between noodles made without and with ishi-kurage. Sig- nificant differences (P<0.05) between two types of buck- wheat noodles were found for springiness, smoothness and color (Fig. 5), respectively. Springiness and color of buckwheat noodles with ishi-kurage were significantly higher than without ishi-kurage noodles (Fig. 5). On the other hand, smoothness of buckwheat noodles with ishi- kurage was significantly lower than without ishi-kurage noodles (Fig. 5). The present findings (Figs. 3, 4 and 5) suggest that incorporating ishi-kurage as a dough-improv- er into buckwheat noodles can produce buckwheat noo- dles with stable masticatory characteristics together with high palatability and acceptability. Protein compositions of buckwheat noodles made with ishi-kurage Figure 6 shows the NaCl-soluble protein content of buckwheat noodles made with ishi-kurage, i.e., noodles 0 50000 100000 150000 200000 250000 300000 350000 400000 450000 500000 無添加 添加 5.0 4.5 4. 3.5 3. 2.5 2. 1.5 0 1. 0.5 0 20000 40000 60000 80000 100000 120000 無添加 添加 12.0 10.0 8.0 6.0 4.0 2. 0 Br ea ki ng st re ss (P a X1 05 ) Br ea ki ng e ne rg y (J/ m 3 X1 04 ) Non added ishi-kurage Added ishi-kurage Non added ishi-kurage Added ishi-kurage ** * (A) (B) Fig. 4 Comparison of breaking characteristics between prototype noodles made without and with ishi-kurage. (A), breaking stress; and (B), breaking energy. Vertical bars in the figure show the standard deviations. Significant difference between the two buckwheat noodles: *P<0.05, **P<0.01. 1.23 1.00 1.00 1.00 1.54 0.50 1.73 1.42 1.58 0.81 0.92 1.81 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Overall evaluation Hardness Springiness Easiness to bite through Smoothness Color Non addied ishi-kurage Added ishi-kurage ** *** Ishi-kurage I i kurage Fig. 5 Sensory evaluation of prototype noodles made without and with ishi-kurage. Significant difference between the two buckwheat noodles: *P<0.05, ***P<0.001. Asami et al. (2020): Buckwheat noodles with Nostoc commune 34 0.00 0.50 1.00 1.50 2.00 2.50 3.00 0% 0.6% 1.2% 1.8% 2.4% 3.0% Na Cl so lu bu le pr ot ei n co nt en t (g /1 00 g flo ur ) Ishi-kurage blending ratio of buckwheat flour to total et al., 1999; Asami et al., 2008). Actually, statistical anal- ysis showed that the AG fraction content (Fig. 6) nega- tively correlated to their observed breaking stress (Fig. 3 (A)) with r = −0.869 (P<0.05), breaking energy (Fig. 3 (B)) with r = −0.865 (P<0.05). These statistical findings suggest that the proteins of AG fraction (Fig. 6) may be associated with the observed mechanical characteristics (Fig. 3) of buckwheat noodles made with ishi-kurage. Finally, the present study shows clear alterations in me- chanical characteristics of buckwheat noodles made with ishi-kurage. The present study suggests that changes in the protein of AG fraction in buckwheat noodles with ishi-kurage may be an important factor affecting the mechanical char- acteristics of buckwheat noodles, although the exact mech- anism remains uncertain. In this study, ishi-kurage, which was reported to have high functionality (Hori at al., 1990; Ishibashi et al., 1994; Itoh, 2015), was added to buckwheat noodles. The findings of the present study will hopefully stimulate further development of new buckwheat products. Fig. 6 NaCl-soluble protein content of buckwheat noodles made with ishi-kurage. Vertical bars in the figure show the standard deviations. evaluated in mechanical analysis I (Fig. 3). The NaCl-sol- uble protein exhibits the combined fraction of the two major buckwheat proteins, i.e., albumin plus globulin (Ikeda, 2002), designated the AG fraction below. Chang- es by the addition of the ishi-kurage in solubility of the AG fraction were found (Fig. 6). Addition of ishi-kurage reduced the solubility of the AG fraction in buckwheat noodles as the ishi-kurage added into buckwheat noodles increased (Fig. 6). Ishi-kurage is reported to contain high levels of dietary fiber such as pectin (Hori et al., 1992). This observed phenomenon (Fig. 6) suggests a possibili- ty indicating that buckwheat protein may be precipitated arisen by addition of dietary fiber present in ishi-kurage, as we have suggested in our previous findings also sug- gested that buckwheat protein may be precipitated by ad- dition of some seaweeds (Asami et al., 2019). Our studies suggest that precipitation, if any, of buckwheat proteins in buckwheat products may lead to large alterations in the mechanical properties of buckwheat proteins (Ikeda, Fagopyrum 37(2):29-36 (2020) 35 ACKNOWLEDGMENT This work was supported by the Research Institute for Food and Agriculture of Ryukoku University, Japan. The present author is sincerely grateful to many persons of the Faculty of Agriculture, Ryukoku University for their kind cooperation in the sensory evaluation experiments. The present author is sincerely grateful to Ms. Kazumi Hashi- moto, Ryukoku University, for support in the experiment. REFERENCES Asami, Y., N. Mochida, R. Lin, C. Campbell, Y. Kuroko and K. Ikeda. 2008. Relationship of endogenous protein compo- nents to the mechanical characteristics of buckwheat doughs. Fagopyrum, 25: 49-56. Asami, Y., S. Ooto, M. Kitamura, K. Sakanashi, T. Tamai, T. Furumoto, S. Ikeda and K. Ikeda, 2019. Mechanical character- ization of buckwheat noodles mixed with seaweed (fu-nori). Fagopyrum, 36: 5-9. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254. Hori, K., T. Ueno-Mohri, T. Okita and G. Ishibashi, 1990. Chemical composition, in vitro protein digestibility and in vitro available iron of blue green alga, Nostoc commune. Plant Foods for Human Nutrition, 40: 223-229. Hori, K., T. Ueno-Mohri and T. Okita, 1992. Absorption of color additives and settling volume in water of blue-green alga, ishikurage (Nostoc commune). Plant Foods for Human Nutrition, 42: 31-36. Ikeda, K., T. 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Hypocholesterolemic effect of Blue-green algae, Ishikurage (Nostoc commune) and suizenji-nori (Aphanothece sacrum suringar) in rats fed high cholesterol diet. J. Home Economics of Japan, 45: 579-584. Itoh, T. 2015. Functional components in natural bioresources including Nostoc commune, a kind of blue-green algae. Nip- pon Suisan Gakkaishi, 81: 640-643. Kreft, I., L.J. Chang, Y.S. Choi and C.H. Park (eds), 2003. Ethnobotany of buckwheat, Jinsol Publishing Co., Seoul. Toda, J. 1994. Discussion on “rating scales” in sensory evaluation of foods. Nippon Shokuhin Kogyo Gakkaishi, 41: 228- 232. Zen-men-kyo. 2014. Kaitei Soba-Uti Kyouhon (Revision, Textbook of buckwheat noodle making). Shibata Shoten Co., Ltd, Tokyo. IZVLEČEK Namen te raziskave je bil ugotoviti vpliv modro-zelene alge ishi-kurage (Nostoc commune Vauch.) na mehanične last- nosti ajdovih testenin. Raziskava je pokazala, da je vključitev ishi-kurage v ajdove testenine povečala odpornost testenin na lomljenje. Senzorični preizkus je pokazal, da so bile ajdove testenine z algo ishi-kurage boljše v primerjavi s kontrolo brez te alge. Dodatek alge v ajdove testenine je povezan z zmanjšanjem topnosti albuminske in globulinske frakcije beljakovin testenin. Na osnovi rezultatov te raziskave lahko sklepamo, da so beljakovine ajde pomembne za mehanične lastnosti ajdovih testenin z dodatkom alge ishi-kurage. Asami et al. (2020): Buckwheat noodles with Nostoc commune 36 Short communication Extraction of rutin and quercetin from Tartary buckwheat grains, hydrothermally treated at different temperatures Filip BRAJČIČ1, Patricia LUŠTEK1, Tinara ŠUŠTARIČ1, Janja PUST1 1 Gimnazija Novo mesto, Seidlova cesta 9, SI-8000 Novo mesto, Slovenia, janja.pust@gimnm.org; filip.silver@gmail.com; tinara.sustaric@gmail.com; patricia.lustek@gmail.com DOI https://doi.org/10.3986/fag0017 Received: July 26, 2019; accepted September 25, 2019. Keywords: Tartary buckwheat, rutin, quercetin ABSTRACT Tartary buckwheat grains were hydrothermally treated to establish the conditions under which rutin remains in the grain. Tartary buckwheat grains were soaked in water at the temperatures 51 °C, 61 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 93 °C, 97 °C and 99 °C, and a control group at 21 °C. During 20 minutes soaking at 51 °C or 61 °C the concentration of rutin decreased. This effect was mostly pronounced by soaking at 70 °C and 75 °C, where instead of missing amount of rutin, some quercetin appeared. After soaking at 80 °C, 85 °C, 90 °C, 93 °C, 97 °C and 99 °C concentration of rutin was not significantly different in comparison to the concentration of rutin after soaking 20 minutes at 21°C. It is sug- gested that exposure to water at 21°C is similar to natural conditions, where rutin degrading enzymes remain mainly inactive and in grain separated from its potential substrate. Further is suggested that at the soaking temperatures 51 °C, 61 °C, 70 °C and 75 °C, grain structures are partly degraded and rutin degrading enzymes got contact to the substrate. By soaking at 80 °C, 85 °C, 90 °C, 93 °C, 97 °C and 99 °C, rutin degrading enzymes lose their activity. Thus wet treatment of Tartary buckwheat grains for 20 minutes at temperature at 80 °C or above, this threshold is enough to preserve the content of rutin in the samples. This is of importance for nutritional quality of Tartary buckwheat food products. Fagopyrum 37(2):37-40 (2020) 37 INTRODUCTION Flavonoid rutin and rutin degrading enzymes are in different structures of buckwheat grain. Rutin is in em- bryo, in the middle of the grain, while rutin degrading enzymes are located in the peripheral part of the grain. After crushing and milling of the grain, in the wet envi- ronment, rutin degrading enzymes are in the direct con- tact with their substrat, and the concentration of rutin may begin to decrease (MetaCyc, 2011). Harvested buck- wheat grain contain among flavonoids mainly rutin, and just a low concentration of quercetin. During dough and bread making, due to transforming of rutin to quercetin, the concentration of rutin decreases and quercetin con- centration increases (MetaCyc, 2011; Lukšič et al., 2016). Quercetin is a flavonoid, frequently present in foods and drinks, with bitter taste (Anand et al., 2016). Tartary buckwheat contains in its leaves and grain higher concentrations of rutin in comparison to common buckwheat. Bitter taste of Tartary buckwheat is due to the high concentration of rutin and quercetin (Bonafaccia et al., 2003). Buckwheat cultivation decreased for years, but recently it is coming back because of knowledge of health promoting properties (Li et al., 2001). The aim of this investigation was to establish the temperatures, at which the activity of rutin degrading en- zymes are hindered. MATERIAL AND METHODS Preparation of samples Tartary buckwheat (Fagopyrum tataricum, cv. Zla- ta) grains were obtained from Mill Rangus, Šentjernej, Slovenia. Grains were soaked in water for 20 minutes at temperatures 21 °C, 51 °C, 61 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 93 °C, 97 °C and 99 °C. After hydrothermal treatment samples were dried for 24 hours at 40 °C. Dry samples were milled in a coffee grinder (Gorenje, SMK 202, Velenje). Samples (1 g per 25 mL) were extracted for 20 min in a 80 % (v/v) methanol in horizontal shaker (Phoenix In- strument, RS-OS 5, Garbsen, Germany). After the extrac- tion solutions were filtered (Agilent Econofilters, PTFE 25 mm 0.2 µm, Santa Clara, USA). Three independent sam- ples were analyzed for each hydrothermal treatment. Determination of rutin and quercetin by HPLC Standard chemicals (rutin and quercetin), methanol (Chromasolv for HPLC), acetonitrile (LC-MS Chroma- solv) and phosphoric acid (ACS grade) were purchased by Sigma-Aldrich (Sigma Aldrich Chemie GmbH, Steiheim, Germany). Deionized water (dH2O) was treated in a deionization system DI 425 TK-0.10425 (Thermo Scien- tific, Waltham, USA). Preparation of calibration solutions and samples solutions were described by Lukšič et al. (2016). Rutin and quercetin were determined using an Agi- lent 1100 Series high performance liquid chromatograph (Agilent Technologies, Santa Clara, USA) with quater- nary solvent pump (G1311A) coupled with degasser (G1379A), sample manager (G1329A), column manag- er (G1316A), autosampler (G1329A) and DAD detector (G1315B). All HPLC analyses were performed on a Zor- bax Eclipse XDB-C18 column (4.6 mm x 250 mm x 5 µm) (Agilent Technologies, Santa Clara, USA). The mobile phase consisted of acetonitrile (gradient) (A) and 0.1% phosphoric acid in dH2O (B). The gradient elution was as follows: 0-1 min isocratic elution (20% A and 80% B), 1-5 min linear gradient elution (25% A and 75% B), 5-15 min (30% A and 70% B) and 20-25 min (40% A and 60% B). The initial flow rate was 1 mL min-1 and the injection volume was 10 µL. Column oven tem- perature was set up to 25 °C and the samples were kept at 4 °C in the sample manager. The detection wavelengths were conducted at 265 nm (rutin) and 372 nm (querce- tin). The data were collected and proceed using Agilent Chemstation 9.01 software. Statistical evaluation The data are expressed as means, and standard devia- tion, from three independently prepared samples. ANO- VA was performed, the data were considered to be signif- icantly different at p <0.05. RESULTS AND DISCUSSION Results are presented in Fig. 1. During 20 minutes soaking at 51 °C or 61 °C the con- centration of rutin decreased. This effect was most pro- nounced by soaking at 70 °C and 75 °C, where instead of missing amount of rutin, some quercetin appeared. After soaking at 80 °C, 85 °C, 90 °C, 93 °C, 97 °C and 99 °C concentration of rutin was not significantly different in comparison to the concentration of rutin after soak- ing 20 minutes at 21°C. It is suggested that exposure to water at 21°C is similar to natural conditions, where rutin degrading enzymes remain mainly inactive and in Brajčič et al. (2020): Rutin and quercetin in Tartary buckwheat 38 grains separated from its potential substrate. Further is suggested that at the soaking temperatures 51 °C, 61 °C, 70 °C and 75 °C, grain structures are partly degrad- ed and rutin degrading enzymes got contact to the sub- strate. By soaking at 80 °C, 85 °C, 90 °C, 93 °C, 97 °C and 99 °C, rutin degrading enzymes lose their activity. Thus wet treatment of Tartary buckwheat grains for 20 min- utes at temperature at 80 °C or above this threshold is enough to preserve the content of rutin in the samples. This is of importance for nutritional quality of Tartary buckwheat food products. CONCLUSION Rutin degrading enzymes were most active during soaking Tartary buckwheat grain at the temperatures 70 °C and 75. At temperatures 80 °C, 85 °C, 90 °C, 93 °C, 97 °C and 99 °C concentration of rutin was not signif- icantly decreased, and no quercetin appeared, meaning that the rutin degrading enzymes were under this condi- tion inactivated. ACKNOWLEDGEMENT Authors are thankful to prof. Ivan Kreft (ARRS pro- ject L4-9305, co-financed by Ministry of Agriculture, Forestry and Food), to Mr. Anton Rangus (Mill Rangus, Šentjertnej, Slovenia) and to Gimnazija Novo mesto. This research was performed in the frame of activities for tal- ented students of Gimnazija Novo mesto, Novo mesto »Razmišljam, iščem, (se) razvijam«, supported by EU and MIZŠ, in the educational project »RaST – Razvojno središče talentov«, coordinated by II. gimnazija Maribor (number of project 20.02054). Fig. 1 Concentration of rutin and quercetin in Tartary buckwheat samples (in mg/g dry mass), treated at given temperatures Fagopyrum 37(2):37-40 (2020) 39 REFERENCES Anand David A., R. Arulmoli, S. Parasuraman. 2016. Overviews of Biological Importance of Quercetin: A Bioactive Flavo- noid. Pharmacogn Rev., 10 (20): 84–89. Bonafaccia G., L. Gambelli, N. Fabjan, I. Kreft. 2003. Trace elements in flour and bran from common and Tartary buck- wheat. Food Chemistry, 83: 1–5. Li S., Q.H. Zhang. 2001. Advances in the development of functional foods from buckwheat. Critical Reviews in Food Science and Nutrition, 451–464. Lukšič L., J. Árvay, A. Vollmannová, T. Tóth, V. Škrabanja, J. Trček, M. Germ, I. Kreft. 2016. Hydrothermal treatment of Tartary buckwheat grain hinders the transformation of rutin to quercetin. Journal of Cereal Science, 72: 131–134. MetaCyc, 2011. Enzyme: rutinase. https://biocyc.org/META/NEW-IMAGE?type=NIL&object=MONOMER-16764. (21. 2. 2019). IZVLEČEK Ekstrakcija rutina in kvercetina iz hidrotermično obdelanih zrn tatarske ajde pri različnih temperaturah Namen raziskave je bil določiti pogoje, pod katerimi se rutin ohrani v ajdi pri hidrotermični obdelavi zrn tatarske ajde. Zrna tatarske ajde so bila hidrotermično obdelana v vodi pri temperaturah 51 °C, 61 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 93 °C, 97 °C in 99 °C s kontrolno skupino pri 21 °C. Pri 51 °C in 61 °C se je koncentracija rutina znižala. Pri 70 °C in 75 °C se je znaten del rutina pretvoril v kvercetin. Pri 80 °C, 85 °C in 90 °C se je pretvorba rutina v kvercetin zman- jšala predvidoma zaradi denaturacije encimov, ki razgrajujejo rutin. Pri višjih temperaturah 93 °C, 95 °C in 99 °C je bila koncentracija kvercetina izrazito manjša, ohranil pa se je velik delež rutina. Glede na ugotovljeno imajo hidrotermično obdelana zrna tatarske ajde višjo koncentracijo rutina, kar je pomembno s stališča prehrane. Brajčič et al. (2020): Rutin and quercetin in Tartary buckwheat 40 41 INSTRUCTIONS FOR AUTHORS FAGOPYRUM accepts scientific papers, and information and bibliographies on buckwheat. SCIENTIFIC PAPERS Manuscript should be written in standard English and submitted to the Editorial office as a .doc document. Figures (photographs) should be IN SEPARATE FILE each in jpg or other original file, not imbeded in word .doc document. Submission for the 2021 issue shall be sent latest on November 30, 2020 to the email ivan.kreft@guest.arnes.si. After accepting the paper, the editorial office will ask the authors to provide the original figures if the first submission will not be adequate. 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