Chemical Components of Matcha and Powdered Green Tea

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1 日本調理科学会誌日本調理科学会誌 Vol. 50,No. Vol. 5,182~188(2017) 50 No. 5(2017) Note Chemical Components of Matcha and Powdered Green Tea Hideki Horie* Kaori Ema* Osamu Sumikawa** Matcha is a type of powdered tea; it was previously regarded as a precious beverage and is served at tea ceremonies in Japan. Today, powdered teas labeled matcha are used in the food industry as well as in cooking; both are referred to as industrial-grade matcha in this report. Moreover, powdered green teas that are not labeled as matcha are also sold in the market. However, there is little information on the quality of these powdered teas. Therefore, in this study, we compared the chemical components of matcha and powdered green tea. Ten grades of Tencha (milled Tencha is matcha)were collected and their chemical components were analyzed. Based on the results, the contents of theanine and chlorophyll a (Chl-a)and the ratio of epigallocatechin gallate/epigallocatechin (EGCG/EGC)were compared among the powdered teas sold on the market. Tea ceremony-grade matcha was found to contain >1.8 g/100 g DW of theanine and exhibited an EGCG/EGC ratio >3.2 (weight-based). Most industrial-grade matcha and powdered green tea samples exhibited lower values; however, it was difficult to distinguish industrial-grade matcha from powdered green tea by using only these parameters. The Chl-a content of matcha for tea ceremonies was more than 250 mg/100 g DW. Powdered green tea samples, except for Gyokuro powders, exhibited low Chl-a contents relative to both ceremonial and industrial grade matcha samples. Keyword:high-performance liquid chromatography, quality evaluation, Tencha, food industry, cooking INTRODUCTION Several types of green tea are produced in Japan. Chu (1997)reported the production procedure for several * Institute of Fruit Tree and Tea Science, NARO ** National Agriculture and Food Research Organization Inquiry Division of Tea Research, Institute of Fruit Tree and Tea Science, NARO 2769 Kanaya-Shishidoi, Shimada, Shizuoka , Japan horie@affrc.go.jp types of green tea. Sencha, the most popular tea in Japan, is produced as follows. Harvested tea leaves are steamed to inactivate the enzymes present in the leaves and subsequently, rolled and dried. On the other hand, matcha is a powdered tea for tea ceremonies and is produced from shaded leaves. The shading process is important and affects the quality of matcha. Shade treatment prior to harvest is known to increase the amino acid and caffeine contents and decrease the catechin content (Saijo, 1999). In authentic matcha production, the steamed leaves are dried without rolling using a special drier called a Tencha-ro. The dried leaves are called Tencha and powdered Tencha ground with exclusive stone mills is termed matcha. Gyokuro is another high-quality tea with a manufacturing process similar to that of Sencha; however, shaded leaves are used for the production of Gyokuro (Tokunaga, 2004). Consumption of green tea as a beverage used to be very popular in Japan; use of green tea as a food ingredient, however, has rapidly increased recently and the production of this tea is estimated to be >3,800 t in Japan (Kuwabara, 2016). Powders prepared from tea leaves are often found in food products including cakes, noodles, and ice creams. As mentioned above, authentic matcha is the powdered tea from Tencha and is used for tea ceremonies; however, so-called matcha is popularly used as a food ingredient. These matcha teas are sold as matcha for cooking or matcha for the food industry. Moreover, powdered green tea is also sold as a food ingredient or for drinking. Powdered green tea is the powder of Sencha or green teas other than Tencha. Several kinds of powdered teas, namely authentic matcha for tea ceremonies, matcha for ingredients, and powdered green tea, are produced and circulated in Japan. These teas all look similar, so it is not easy to differentiate them intuitively. There is little information on the chemical components of powdered teas other than authentic matcha. Thus it is interesting to compare the components in these different teas. In this study, we compared the chemical components of authentic matcha and other powdered green tea samples available from local markets. Before comparing matcha and other teas, we analyzed Tencha samples to determine the chemical indicators. As several lots of Tencha are blended and milled into commercial matcha, the prices of Tencha directly reflect the quality of the tea itself. The most important components of Japanese green tea are catechins, caffeine, and amino acids (Goto et al., 1996). The bitter and astringent taste of tea is attributed to catechins, while the umami taste is attributed to amino acids. 16 (182)

2 Chemical Components of Matcha and Powdered Green Tea Caffeine is a stimulant that acts on the central nervous system and is abundant in high-quality teas (Chu & Juneja, 1997). The content of four major catechins, epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), as well as caffeine, was analyzed using high-performance liquid chromatography (HPLC). Seven major amino acids, namely aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), arginine (Arg), and theanine, were also analyzed using HPLC. Theanine is a unique amino acid that constitutes almost half of the total amino acids in green teas (Chu & Juneja, 1997). On the other hand, the green color of the leaf powder in matcha and powdered green teas is also important (Tokunaga, 2004). Tachibana and Shoyama (1979)previously reported the relationship between leaf color and chlorophyll content; therefore, in this study, we evaluated the content of chlorophyll and related components using HPLC. MATERIALS AND METHODS Sample Table 1. Tencha sampels (Sample Set 1) Price (Yen/kg) season plucking method A1 30,000 first* hand A2 15,000 first hand A3 10,000 first hand + plucker A4 7,500 first plucker A5 6,000 first plucker A6 4,500 first plucker A7 3,000 first + second plucker A8 2,500 second** plucker A9 2,000 second plucker A10 1,000 autumn*** plucker * First crop, ** Second crop *** Harvested in autumn without shading. Tea samples Ten grades of Tencha were purchased from a wellestablished matcha company in Uji, Kyoto, Japan (Sample Set 1, Table 1). The Tencha samples were powdered using the exclusive stone mill for matcha at the company. Matcha and powdered green tea samples were purchased from tea companies (Sample Set 2, Table 2); some teas were labeled matcha for the food industry or matcha for cooking, and one matcha for cooking (G) indicated the addition of chlorella. In this report, the matcha samples for cooking or the food industry are treated as industrial grade and other matcha samples used just for drinking are described as ceremonial grade. Among the powdered green tea samples, sample J1 comprised Gyokuro powder while samples I, J2, and J3 were Sencha powders. On the other hand, there was no indication of the nature of the tea on the label of sample H. All the teas were used for the analysis without any further preparation. Analytical methods The catechin and caffeine contents of the tea powders were measured using an HPLC system (Shimadzu, LC- 10ADvp)according to previous reports (Maeda- Yamamoto et al., 2004, Maeda-Yamamoto et al., 2005). Briefly, 250 mg of powdered tea sample were extracted with 25 ml of a 2% phosphoric acid/ethanol (1:1)mixture at 30 for 60 min. The filtrate was diluted with water and injected into the HPLC system. The conditions for the reversed-phase HPLC analysis have been described in the literature (Maeda-Yamamoto, 2005). For the analysis of free amino acids, 100 mg of powdered tea were extracted with 10 ml of distilled water at 80 for 30 min. The resultant filtrate was collected and used for further analysis. The free amino acids were analyzed using HPLC (Shimadzu, LC-20AD)connected to an auto-injector (SIL-30AC). A pre-column derivatization method, using o-phthalaldehyde, was employed and the Table 2. Matcha and powdered green tea samples (Sample Set 2) Sample* Use Price (Yen/100 g) B1 Matcha tea ceremony 8,100 B2 Matcha tea ceremony 4,320 B3 Matcha cooking 1,890 B4 Matcha food indusutry 1,468 B5 Matcha food indusutry 1,292 C Matcha cooking 972 D1 Matcha tea ceremony 6,250 D2 Matcha tea ceremony 3,750 D3 Matcha tea ceremony 3,250 E1 Matcha tea ceremony 4,280 E2 Matcha food indusutry 1,500 E3 Matcha food indusutry 1,050 E4 Matcha food indusutry 630 F1 Matcha food indusutry 950 F2 Matcha food indusutry 600 G Matcha cooking** 1,400 H Powdered Green Tea 580 I Powdered Green Tea*** 300 J1 Powdered Green Tea**** 560 J2 Powdered Green Tea*** 200 J3 Powdered Green Tea*** 170 *: The same letter indicate the samples are bought from the same company. **: Chlorella is mixed wih matcha. ***: Powder of Sencha. ****: Powder of Gyokuro. (183) 17

3 日本調理科学会誌 Vol. 50 No. 5(2017) instrument manufacturer guidelines were followed. A reversed phase column (GL Sciences, Inesrtsil ODS-4, 5 μm, 4.6 mm 250 mm)was employed. Mobile phases A and B were 20 mm potassium phosphate buffer (ph 6.4) and a water/acetonitrile/methanol (15/45/40 V/V/V) mixture, respectively. The linear gradient elution was performed as follows: 10% mobile phase B for the first 3 min, programed to 30% mobile phase B for 27 min; 40% mobile phase B for the next 0.5 min, maintained at 40% mobile phase B for 3.5 min; and finally, back to 10% mobile phase B. The seven major amino acids (Asp, Glu, Asn, Ser, Gln, Arg, and theanine)were analyzed with glycylglycine as internal standard (Goto et al., 1993). The color pigments chlorophyll a (Chl-a), chlorophyll b (Chl-b), pheophytin a (Phy-a), and pheophytin b (Phyb)were analyzed using HPLC (Shimadzu, LC-10ADvp) connected to a column (Tosoh, TSKgel ODS-80TM, 5 μm, 4.6 mm 150 mm). The sample solution for the analysis of these pigments was prepared as follows: 100 mg of powdered tea were extracted with 10 ml of 85% acetone by centrifugation. The resultant supernatant was transferred to a 25 ml volumetric flask. The precipitate was reextracted with 10 ml of 85% acetone and the supernatant was added to the contents in the volumetric flask. Subsequently, 85% acetone was added to fill to the 25 ml mark. HPLC analysis was performed according to the procedure described by Kohata et al. (1998). Each analysis was repeated and the average analytical data were described as dry weight-based in the tables and figures. RESULTS AND DISCUSSION Chemical components of Tencha Matcha sold on the market is powdered tea that is blended and milled from several lots of Tencha by various tea companies. Tencha samples of known origins and grades were purchased from a reliable tea company. Among the Tencha samples listed in Table 1, A1 was of the highest grade; the grade decreased in numerical order, with A10 being the lowest. Tencha from the first crop is more valuable than that of the second crop and handplucked teas are of higher quality than those obtained via mechanical plucking. Matcha is usually made from shaded leaves, whereas the lowest-grade tea, A10, was made from non-shaded leaves. The amino acid contents were compared in Table 3a. Higher-grade teas contained greater amounts of total amino acids, theanine, and other individual amino acids. In the literature, high-grade green teas including matcha are rich in amino acids (Goto et al., 1996). Tsuji (2001)suggested that the total amino acid content could be used as a quality indicator for Tencha. Our results also indicated that the total amino acid or theanine contents would be good indicators for the quality of Tencha. On the other hand, the high-grade teas contained lower amounts of total catechins than lower-grade teas (Table 3b). Goto et al. (1996)also reported that high-grade matcha contained a lower total catechin content than lowgrade matcha. Moreover, the EGC and EC contents were greater in lowergrade teas, while those of EGCG and ECG did not seem to correlate with tea grade (Table 3b). It is well known that younger leaves of tea shoots contain less EGC or EC than older leaves (Miwa et al., 1978)and strong shade treatment decreases the EGC and EC contents (Saijo & Osawa, 1981; Wang et al., 2012). Highgrade green teas are made from younger leaves and shading is a requirement for the production of matcha. The EGC or EC contents may be an indicator of the quality of matcha. The analysis of EGC is easier than that of EC because tea leaves are rich in the former. Thus it seems Table 3a. Free amino acids contents in Tencha (Sample Set 1, n=2) g/100 g DW Sample Asp Glu Asn Ser Gln Arg Theanine total-a* A A A A A A A A A A * Total of Asp, Glu, Asn, Ser, Gln, Arg and Theanine. Asp: aspartic acid, Glu: glutamic acid, Asn: asparagine, Ser: serine, Gln: glutamine, Arg: arginine. 18 (184)

4 Chemical Components of Matcha and Powdered Green Tea Table 3b. Catechins and caffeine in Tencha (Sample Set 1, n=3) g/100 g DW Sample EGC EC EGCG ECG Caffeine total-c* EGCG/EGC A A A A A A A A A A * Total of EGC, EC, EGCG, ECG. Table 3c. Chlorophylls and pheophytins in Tencha (Sample Set 1, n=2) mg/100 g DW Sample Chl-a Chl-b Pheo-a Pheo-b A A A A A A A A A A Chl-a: Chlorophyll a, Chl-b: Chlorophyll b, Pheo-a: Pheophytin a, Pheo-b: Pheophytin b. feasible to estimate the quality based on EGC content. However, it is technically possible to mix the powders of the leaves of some catechin-less plants or algae (as with G in Table 2)with those of matcha; the EGC content should be low in such teas. Since the EGCG content did not exhibit any correlation with the quality of the teas in this sample set, we concluded that the EGCG/EGC ratio reflected the quality of matcha more effectively than the EGC or total catechin contents (Table 3b). Caffeine contents are also compared in Table 3b. The relationship between the grade of Tencha and caffeine contents seemed low; however, it is notable that the caffeine content of A10 (harvested in autumn without shading)was extremely low among these samples. The chlorophylls and pheophytins contents are listed in Table 3c. The highest-grade Tencha samples, A1 and A2, did not contain the greatest amounts of Chl-a or Chl-b; however, the chlorophyll contents were greater in the higher-grade teas among samples A3 to A10. Kohata et al. (1999)analyzed chlorophylls and pheophytins in four types of green tea sold on the market and discussed the relationship between tea quality and the contents of these components. In their report, the Chl-a contents of matcha samples were greater than those of Gyokuro or Sencha samples and higher-grade matcha samples were rich in Chl-a. Therefore, the Chl-a content indicates the quality of Tencha or matcha samples, in spite of some exceptions such as A1 or A2. Since it has been reported that excessive shading decreases the chlorophyll content (Kobayashi et al., 2011), the relatively lower Chl-a amounts in A1 and A2 were likely related to the strong shading used to cultivate high-quality Tencha. Kohata et al. (1999)also reported that the pheophytin content (Phy-a and Phy-b)in high-grade matcha was lower than that of low-grade matcha. However, in this study, the lowest-grade sample A10 contained as much pheophytin content as the second highest-grade sample, A2. A10 was harvested in autumn without shading and the chlorophyll content was extremely low in this sample set. Since pheophytins are the degradation products of chlorophylls, the low pheophytin content in A10 was caused by the low chlorophyll content. It is difficult to use the contents of pheophytin as an indicator of grade among samples with a wide range of quality. Chemical components of matcha and powdered green tea Sample Set 1 comprised Tencha whose quality was determined by specialists. Usually several lots of Tencha are blended and milled to matcha. Teas sold as matcha and powdered green tea in the market were collected as Sample Set 2 and are listed in Table 2. The same letters in the table show that the tea samples were sold at the same company and the use (for tea ceremonies, cooking, or the food industry)labeled on the samples is shown in the table. The sizes of the packages collected were different among the samples, thus the prices in Table 2 are quoted per 100 g. The prices of matcha were >600 Yen (185) 19

5 日本調理科学会誌 Vol. 50 No. 5(2017) and those of the matcha for tea ceremonies were >3,000 Yen. On the other hand, those of powdered green tea were <600 Yen. According to the results of sample set 1, higher-grade Tencha contains higher amounts of theanine. The theanine contents of matcha and powdered green tea samples were compared (Fig. 1a). The teas with higher prices showed high contents of theanine; the contents in matcha for tea ceremonies were >1.8 g/100 g DW. The industrial-grade matcha and powdered green teas had contents <1.7 g/100 g DW. We hypothesized in the previous section that EGCG/ EGC rates would reflect the quality of matcha. The EGCG/EGC ratios (weight-base)are displayed in Fig. 1b. The samples of matcha for tea ceremonies exhibited ratios >3.2, while ratios exhibited by the other teas theanine (g/100 g DW) J Price (Yen/100 g) Fig. 1a. Relationship between the prices and the contents of theanine in matcha and powderd green tea (Sample Set 2, n=2) :ceremonial grade, :industrial grade, :powdered tea were <3.3. The matcha for tea ceremonies could be characterized by the high contents of theanine and the high EGCG/EGC ratios. On the other hand, it was not easy to distinguish between the industrial-grade matcha and powdered green tea by using these parameters. Chl-a contents are compared in Fig. 1c. Those of ceremonial-grade matcha samples were higher than 250 mg/100 g DW, while those of powdered green tea samples, except J1, were less than 60 mg/100 g DW. The powdered tea sample J1 was labeled as powdered Gyokuro. Gyokuro is cultivated under the shade like matcha, so the chemical constituents are expected to resemble those of matcha. In Fig. 1a and 1b, the content of theanine and the rate of EGCG/EGC of J1 were higher than those of the other powdered green tea samples. On the other hand, the contents of Chl-a of industrial-grade matcha were divided into two groups: contents of more than 660 mg/100 g DW (shown as B3, B4, and B5 in Fig. 3)and less than 260 mg/100 g DW. It has been reported that chlorophylls are fragile in the manufacturing process. Samples B3 B5 were produced in the same company, so it can be supposed that the process of manufacturing of these teas is different from that of the other industrialgrade matcha teas. The contents of Chl-a of industrialgrade matcha samples were higher than those of powdered green tea samples, although there was one exception: powdered Gyokuro (J1). Since Chl-a contenst is related to the green color of the tea powder, matcha (both ceremonial and industrial grades)is expected to be recognized as the tea powder with the strongest green color by the tea companies. In our results, samples of matcha for tea ceremonies were characterized by high contents of theanine (> EGCG/EGC J1 G Chl-a (mg/100 g DW) B5 B4 B3 J Price (Yen/100g) Price (Yen/100 g) Fig. 1b. Relationship between the prices and the rate of EGCG/ EGC in matcha and powderd green tea (Sample Set 2, n= 3) :ceremonial grade, :industrial grade, :powdered tea Fig. 1c. Relationship between the prices and the contents of Chlorophyll a in matcha and powderd green tea (Sample Set 2, n=2) :ceremonial grade, :industrial grade, :powdered tea 20 (186)

6 Chemical Components of Matcha and Powdered Green Tea g/100 g DW), and high ratios of EGCG/EGC (>3.2). On the other hand, for the industrial-grade matcha samples and powdered green teas, the theanine contents and EGCG/EGC ratios were <1.7 g/100 g DW and 3.3, respectively. The contents of Chl-a of matcha for tea ceremonies were >250 mg/100 g DW and of most of the other samples were <260 mg/100 g DW. It is hypothesized that the matcha for tea ceremonies can be characterized with minimum contents of theanine and Chl-a and a minimum ratio of EGCG/EGC. It will be necessary to compare the analytical data of hundreds of matcha and powdered teas before determining the minimum values necessary to distinguish ceremonial-grade matcha. Although we could not find any difference between the theanine contents and EGCG/EGC ratios of industrialgrade matcha and powdered green teas, the Chl-a contents in industrial-grade matcha tended to be higher than those of powdered green tea. It is suggested that matcha is the powdered tea that has the greenest particles. In this report, we compared chemical components only. A comparison of physical properties, such as particle size, would also be interesting to determine the characteristics of matcha and other powdered teas. This research was supported by grants from the Project of the Biooriented Technology Research Advancement Institution, NARO (the special scheme project on advanced research and development for next-generation technology). REFERENCES Chu, D.-C. (1997), Green tea -its cultivation, processing of the leaves for drinking materials, and kinds of green tea, Chemistry and Applications of Green Tea, Yamamoto, T., Juneja, L.R., Chu, D.-C. and Kim, M., Ed., CRC Press, New York, pp Chu, D.-C. and Juneja L. R. (1997), General chemical composition of green tea and its infusion, Chemistry and Applications of Green Tea Yamamoto, T., Juneja, L. R., Chu, D.-C. and Kim, M., Ed., CRC Press, New York, pp Goto, T., Horie, H. and Mukai, T. (1993), Analysis of major amino acids in green tea by high-performance liquid chromatography coupled with OPA precolumn derivatization, Tea Res. J. (in Japanese), 77, Goto, T., Yoshida, Y., Amano, I. and Horie, H. (1996), Chemical composition of commercially available Japanese green tea, Foods Food Ingredients J., 170, Kobayashi, E., Nakamura, Y., Suzuki, T., Oishi, T. and Inaba, K. (2011), Influence of light intensities on the color and ingredients of new shoots in tea plants, Tea Res. J. (in Japanese), 111, 9-41 Kohata, K., Hanada, K. and Horie, H. (1998), High performance chromatographic determination of pheophorbide-a and its related chlorophyll derivatives in tea leaves, Food Sci. Technol. Int. Tokyo, 4, Kohata, K., Yamashita, Y., Yamauchi, Y. and Horie, H. (1999), Investigations of qualities and properties of commercially available green teas on the basis of chlorophyll and its derivatives contents, Tea Res. J. (in Japanese), 87, Kuwabara, H. (2016), O-Matcha no subete (All about Matcha), The Tea (in Japanese), 69, Maeda-Yamamoto, M., Nagai, H., Asai, K., Moriwaki, S., Horie, H., Kohata, K., Tachibana, H., Miyasa, T. and Sano, M. (2004), Changes in epigallocatechin-3-o-(3-o-methyl)gallate and strictinin contents of tea (Camellia sinensis L.) cultivar Benifuki in varous degree of maturity and leaf order, Food Sci. Technol. Res., 10, Maeda-Yamamoto, M., Nagai, H., Suzuki, Y., Ema, K., Kanda, E. and Mitsuda, H. (2005), Changes in O-methylated catechin and chemical component contents of Benifuuki green tea (Camellia sinensis L.)beverage under various extraction conditions, Food Sci. Technol. Res., 11, Miwa, E., Takayanagi, H. and Nakagawa, M. (1978), Distribution of the chemical constituents in different position of tea shoot, Tea Res. J. (in Japanese), 47, Saijo, R. (1999), Japanese green tea Characteristics and manufacture, Global Advances in Tea Science, Jane, J. K., Ed., Aravali Books International, New Delhi, pp Saijo, R. and Osawa, K. (1981), Regulation of products of catechins and nitrogenous compounds by shade treatment on tea plants, Tea Res. J. (in Japanese), 54, Tachibana, H. and Shoyama, T. (1979), Studies on the relation between color of tea leaves and its chlorophyll content, Tea Res. J. (in Japanese), 49, Tokunaga, M. (2004), Types of Japanese Green Tea, New Tastes in Green Tea, a novel flavor for familiar Drinks, dishes, and desserts, Kodansha International, Tokyo, pp Tsuji, M. (2001), The relationship between chemical components and quality of tencha tea, Tea Res. J. (in Japanese), 90, 1-7 Wang, Y., Gao, L., Shan, Y., Liu, Y., Tian, Y. and Xia, T. (2012), Influence of shade on flavonoid biosynthesis in tea (Camellia sinensis (L.)O. Kuntze), Scientia Horticulturae, 141, 7-16 (Received Jan, 23, 2017 Accepted Jun, 20, 2017) (187) 21

7 日本調理科学会誌 Vol. 50 No. 5(2017) 抹茶および粉末緑茶の化学成分 堀江秀樹 * 江間かおり * 角川修 ** 和文抄録茶道用の抹茶以外に, 抹茶 と表示された粉末状の茶が食品産業や料理用に流通しており, 本報ではこれらを業務用抹茶と記載する さらには, 抹茶とは表示されていない粉末緑茶も市販されている しかしながら, これら粉末状の茶については品質に関する情報が乏しい そこで, 本研究においては, これらの粉末状の緑茶について化学成分を比較した まず, 抹茶原料であるてん茶について, 品質の異なるものを 10 種類収集し化学分析した 分析結果に基づき, 市販の抹茶や粉末緑茶のテアニン, クロロフィル a(chl-a) の含量, エピガロカテキンガレート / エピガロカテキン (EGCG/EGC) の重量比について比較した 茶道用抹茶のテアニン含量および EGCG/EGC 比は, それぞれ乾物 100 g 当たり 1.8 g および 3.2 以上であった 業務用抹茶と粉末緑茶の大部分ではより低い値を示した しかしながら, これらのパラメータだけでは加工用抹茶と粉末緑茶の判別はできなかった 茶道用抹茶の Chl-a 含量は 250 mg/100 g 乾物重以上であった 玉露粉以外の粉末緑茶試料では, 茶道用や業務用の抹茶よりも Chl-a 含量が低かった キーワード : 高速液体クロマトグラフィ, 品質評価, てん茶, 食品産業, 料理 * 農研機構果樹茶業研究部門 ** 農研機構本部 連絡先農研機構金谷茶業研究拠点 静岡県島田市金谷猪土居 horie@affrc.go.jp 22 (188)