JP6990909B2 - Pepper with high capsiate content and its manufacturing method - Google Patents
Pepper with high capsiate content and its manufacturing method Download PDFInfo
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Description
トウガラシの辛味成分カプサイシンの類縁物質であるカプシエイトは、ジヒドロカプシエイト、ノルジヒドロカプシエイトとともにカプシノイドと総称される成分である。このカプシノイド（以下カプシエイトとする）は、辛味成分がカプサイシンの１０００分の１程度でありながら、体熱亢進作用といったカプサイシンと同様の機能をもつことから、近年、機能性成分として注目されている。
カプシエイトは、矢澤らにより選抜固定されたトウガラシの無辛味固定品種である「ＣＨ－１９甘」に含有することが知られており（非特許文献１）、その機能性としては、免疫賦活作用、エネルギー代謝作用等が報告されている（特許文献１）。
Capsaicin, a pungent component of capsicum, is a component collectively called capsinoid along with dihydrocapsiate and nordihydrocapsiate. This capsaicin (hereinafter referred to as capsiate) has been attracting attention as a functional component in recent years because its pungent component is about 1/1000 of that of capsaicin, but it has the same function as capsaicin such as body heat enhancing action.
Capsiate is known to be contained in "CH-19 sweet", which is a pungent fixed variety of red pepper selected and fixed by Yazawa et al. (Non-Patent Document 1). Energy metabolism and the like have been reported (Patent Document 1).
しかし、カプシエイトは含有量が少なく、多量に含有するとされるＣＨ－１９甘であっても、１トンあたり１００グラム程度と言われており、こうした少ない収量が、カプシエイトの事業化を困難にする要因となっている。本発明は、トウガラシに含有するカプシエイトの量を増加させ、カプシエイトを安定的に供給することを可能にする、カプシエイト高含有トウガラシ及びその製造方法を提供することを目的とする。 However, the content of capsiate is low, and even CH-19 sweet, which is said to be contained in a large amount, is said to be about 100 grams per ton, and such a small yield makes it difficult to commercialize capsiate. It has become. It is an object of the present invention to provide a capsicum high content capsicum and a method for producing the same, which can increase the amount of capsiate contained in the capsicum and enable a stable supply of the capsiate.
発明者らは、上記課題を解決するべく鋭意研究を行い、対象となる植物に所定の処理を施すことによって、処理をしない植物と比較して著しくカプシエイトの収量が増加することを見出し、本発明を完成させるに至った。 The inventors have conducted diligent research to solve the above-mentioned problems, and found that by applying a predetermined treatment to a target plant, the yield of capsiate is significantly increased as compared with a plant not treated, and the present invention has been made. Has been completed.
即ち、本発明に係る請求項１に記載のカプシエイト高含有トウガラシの製造方法は、カプシエイトを含有し、putative aminotransferase(pAMT)遺伝子が変異しCS(pun1)遺伝子が変異していないCapsicum属のトウガラシの植物を単為結果させる工程を含むことを特徴とする、カプシエイト高含有トウガラシの製造方法である。
また、本発明に係る請求項２に記載のカプシエイト高含有トウガラシの製造方法は、前記トウガラシがひもとうがらしであることを特徴とする、請求項１記載のカプシエイト高含有トウガラシの製造方法である。
That is , the method for producing a capsicum-rich capsicum according to claim 1 according to the present invention is a capsicum genus capsicum containing capsiate , in which the putative aminotransferase (pAMT) gene is mutated and the CS (pun1) gene is not mutated . It is a method for producing a capsicum-rich capsicum, which comprises a step of parthenocarpy of a worm plant.
Further, the method for producing a capsicum-rich capsicum according to claim 2 according to the present invention is the method for producing a capsiate-rich capsicum according to claim 1, wherein the capsicum is a capsicum.
また、本発明に係る請求項３に記載のカプシエイトの製造方法は、カプシエイトを含有し、putative aminotransferase(pAMT)遺伝子が変異しCS(pun1)遺伝子が変異していないCapsicum属のトウガラシの植物を単為結果させる工程と、前記植物を粉砕し、溶媒に浸漬することでカプシエイトを抽出する工程と、を含むことを特徴とする、カプシエイトの製造方法である。
また、本発明に係る請求項４に記載のカプシエイトの製造方法は、前記トウガラシがひもとうがらしであることを特徴とする、請求項３記載のカプシエイトの製造方法である。
Further, the method for producing a capsiate according to claim 3 according to the present invention is to obtain a capsicum plant of the genus Capsicum, which contains the capsiate and has the putative aminotransferase (pAMT) gene mutated and the CS (pun1) gene not mutated . A method for producing a capsiate, which comprises a step of parthenocarpy and a step of crushing the plant and immersing the plant in a solvent to extract the capsiate.
The method for producing a capsiate according to claim 4 according to the present invention is the method for producing a capsiate according to claim 3, wherein the capsicum is a capsicum.
また、本発明に係る請求項５に記載のカプシエイトの製造方法は、前記単為結果させる方法として、前記トウガラシ類の植物の子房を植物ホルモンによる浸漬処理を行うことを特徴とする、請求項３または４記載のカプシエイトの製造方法である。
The method for producing a capsiate according to claim 5 according to the present invention is characterized in that, as a method for producing the parthenocarpy, the ovary of the capsicum plant is immersed in a plant hormone. 3 or 4 is the method for producing a capsiate.
また、本発明に係る請求項６に記載のカプシエイトの製造方法は、前記植物ホルモンが、オーキシンであることを特徴とする、請求項５記載のカプシエイトの製造方法である。
The method for producing a capsiate according to claim 6 according to the present invention is the method for producing a capsiate according to claim 5 , wherein the plant hormone is auxin.
また、本発明に係る請求項７に記載のカプシエイトの製造方法は、前記オーキシンが、２、４-ジクロロフェノキシ酢酸であることを特徴とする、請求項６記載のカプシエイトの製造方法である。
The method for producing a capsiate according to claim 7 according to the present invention is the method for producing a capsiate according to claim 6 , wherein the auxin is 2,4-dichlorophenoxyacetic acid.
また、本発明に係る請求項８に記載のカプシエイトの製造方法は、前記溶媒が、メタノール、エタノール，ｎ－プロパノール、イソプロパノール、ｔ－ブタノール、エチルアセテート、アセトン、およびこれらの混合物から成る群から選択されている、請求項３から７のいずれか一項記載のカプシエイトの製造方法である。
Further, in the method for producing a capsiate according to claim 8 , the solvent is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, t-butanol, ethyl acetate, acetone, and a mixture thereof. The method for producing a capsiate according to any one of claims 3 to 7 .
本発明に係るトウガラシ有用物質の製造方法によれば、トウガラシ類植物に含有されるカプシエイトの量を著しく増加させることが可能になり、有効成分として期待の高いカプシエイトを安定して供給することが可能になる。 According to the method for producing a useful substance for capsicum according to the present invention, it is possible to significantly increase the amount of capsiate contained in a capsicum plant, and it is possible to stably supply capsiate, which is highly expected as an active ingredient. become.
以下、本発明の実施の形態について説明する。 Hereinafter, embodiments of the present invention will be described.
本発明に係るトウガラシ有用物質の製造方法は、トウガラシ類の植物に所定の処理を施すことによって、当該植物に含有するカプシエイトの量を増加させ、それにより目的とするカプシエイトの収量を増加させるものである。 The method for producing a useful substance for capsicum according to the present invention is to increase the amount of capsiate contained in a plant of capsicum by subjecting it to a predetermined treatment, thereby increasing the yield of the target capsiate. be.
本発明に係るトウガラシ有用物質の製造方法に用いるトウガラシ類の植物としては、特に制限はなく、現在当業者に知られているトウガラシ類の植物から任意のものを選択可能である。ただし、本発明においてトウガラシ類の植物は、カプサイシン、カプシエイト等の有用物質を含有する種類であることが望ましく、特にputative aminotransferase(pAMT)遺伝子が変異したCapsicum属植物が望ましい。さらに、CS(pun1)遺伝子が変異していないCapsicum属植物であるとなお好適である。トウガラシ類の植物の例としては、ししとう、ひもとうがらし、伏見甘長、パドロンペッパーが挙げられるが、この中では、特にpAMT遺伝子に変異があるひもとうがらしが好適に適用できる。 The capsicum plant used in the method for producing a useful substance for capsicum according to the present invention is not particularly limited, and any capsicum plant currently known to those skilled in the art can be selected. However, in the present invention, the capsicum plant is preferably a species containing useful substances such as capsaicin and capsiate, and a Capsicum genus plant in which the putative aminotransferase (pAMT) gene is mutated is particularly desirable. Furthermore, it is still preferable that the plant belongs to the genus Capsicum in which the CS (pun1) gene is not mutated. Examples of capsicum plants include shishito, himoto-garashi, Fushimi-kancho, and padron pepper, and among these, himoto-garashi with a mutation in the pAMT gene is particularly applicable.
本発明に係るトウガラシ有用物質の製造方法で製造する物質としては、カプサイシンの類縁物質であるカプシエイトが好適に適用可能である。また、カプシエイトと同様、カプサイシンの類縁体であれば、カプシエイトに限らず適用可能であると予想され、例として、ジヒドロカプシエイト、ノルジヒドカプシエイトが挙げられる。 As the substance produced by the method for producing a useful substance for red pepper according to the present invention, capsiate, which is a related substance of capsaicin, is suitably applicable. Further, as with capsaicin, any analog of capsaicin is expected to be applicable not only to capsaicin, and examples thereof include dihydrocapsaicin and nordihidecapsiate.
本発明においては、トウガラシ類の植物に含有するカプシエイトの量を増加させる方法として、対象となるトウガラシ類植物を単為結果させる方法を採用する。単為結果とは、植物において、受精が行われずに子房壁や花床が肥大して果実を形成する現象をいう。単為結果させる方法の例としては、対象植物の子房を植物ホルモンによる浸漬処理を行う方法、対象植物に単為結果遺伝子を導入する方法、対象植物の三倍体を製造する方法等が挙げられる。本発明においては、植物ホルモンを用いる方法または対象植物に単為結果遺伝子を導入する方法が望ましく、植物ホルモンを用いる方法が特に望ましい。 In the present invention, as a method for increasing the amount of capsiate contained in a capsicum plant, a method for parthenocarpy of a target capsicum plant is adopted. The parthenogenetic result is a phenomenon in which the ovary wall and the flower bed are enlarged to form fruits without fertilization. Examples of methods for producing parthenocarpy include a method of dipping the ovary of the target plant with a plant hormone, a method of introducing a parthenocarpy gene into the target plant, a method of producing a triploid of the target plant, and the like. Be done. In the present invention, a method using a plant hormone or a method of introducing a parthenocarpy gene into a target plant is desirable, and a method using a plant hormone is particularly preferable.
本発明において、対象植物を単為結果させる方法として植物ホルモンを用いる場合、使用する植物ホルモンの種類に特に制限はなく、当業者であれば、公知の植物ホルモンから任意のものを選択することが可能である。使用する植物ホルモンの例としては、オーキシン、ジベレリン等が挙げられ、特にオーキシンが望ましい。使用するオーキシンは、天然および合成のいずれも適用可能であるが、特に合成オーキシンが好適である。合成オーキシンの例としては、ナフタレン酢酸、ナフトキシ酢酸、フェニル酢酸、2,4-ジクロロフェノキシ酢酸（2,4-D）、2,4,5-トリクロロフェノキシ酢酸（2,4,5-T）が挙げられ、特に2,4-ジクロロフェノキシ酢酸（2,4-D）が好適に適用できる。 In the present invention, when a plant hormone is used as a method for parthenocarpy of a target plant, the type of plant hormone to be used is not particularly limited, and a person skilled in the art can select any known plant hormone. It is possible. Examples of the plant hormone to be used include auxin, gibberellin and the like, and auxin is particularly desirable. The auxin used can be either natural or synthetic, but synthetic auxin is particularly preferred. Examples of synthetic auxins include naphthalene acetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In particular, 2,4-dichlorophenoxyacetic acid (2,4-D) is preferably applicable.
本発明において、対象となるトウガラシ類植物を単為結果させる方法のほかに、トウガラシ類の植物に含有するカプシエイトの量を増加させる方法としては、例えば、対象となるトウガラシ類植物と、単為結果性の高いトウガラシ類植物系統とを交配させることによって、カプシエイト高含有トウガラシの品種を作出する方法が挙げられる。 In the present invention, in addition to the method of parthenocarpy of a target capsicum plant, as a method of increasing the amount of capsiate contained in the capsicum plant, for example, a target capsicum plant and a parthenocarpy result. A method of producing a varieties of capsicum-rich capsicum by crossing with a highly sexual capsicum plant line can be mentioned.
本発明において、所定の処理を施すことで含有量が増加したトウガラシ類の植物から、カプシエイトを抽出する方法に特に制限はなく、当業者が公知の方法から任意に選択可能である。抽出方法の例としては、溶媒を用いて溶出させる方法が挙げられ、使用する溶媒の例としては、メタノール、エタノール，ｎ－プロパノール、イソプロパノール、ｔ－ブタノール、エチルアセテート、アセトン、およびこれらの混合物から成る群が挙げられる。 In the present invention, there is no particular limitation on the method for extracting capsiate from the capsicum plant whose content has been increased by subjecting it to a predetermined treatment, and those skilled in the art can arbitrarily select from known methods. Examples of the extraction method include a method of eluting with a solvent, and examples of the solvent used include methanol, ethanol, n-propanol, isopropanol, t-butanol, ethyl acetate, acetone, and a mixture thereof. The group consists of.
本実施例における実験には、市販の‘ししとう’2 品種、‘ひもとうがらし’3 品種、‘伏見甘長’2 品種，スペインの‘パドロンペッパー’2 品種の計9 品種を供試した。 In the experiment in this example, a total of 9 varieties of commercially available "Shishito" varieties, "Himoto Ugarashi" 3 varieties, "Fushimi Kancho" 2 varieties, and Spanish "Padron Pepper" 2 varieties were tested.
前記の品種を2015年にビニルハウス内で慣行栽培し、それぞれの開花盛期に単為結果処理として非特許文献２の方法に従い、柱頭切除および2,4-ジクロロフェノキシ酢酸(2,4-D)の塗布を行い、得られた果実を単為結果果実とした。対照として。放任受粉させた、種子を十分に持つ果実を受精果実として供試した。受精果実は官能試験により辛味の有無で分けて分析を行った。果実は成熟して赤くなった直後に収穫し、形態調査、官能試験を行った後、それぞれ数果実を混合し、HPLC分析によるカプシエイト含量を測定した。 The above varieties were conventionally cultivated in a greenhouse in 2015, and stigma resection and 2,4-dichlorophenoxyacetic acid (2,4-D) were performed according to the method of Non-Patent Document 2 as parthenocarpy treatment at each flowering peak. ) Was applied, and the obtained fruit was used as a parthenocarpy fruit. As a contrast. Fruits with sufficient seeds, which were pollinated freely, were tested as fertilized fruits. Fertilized fruits were analyzed separately according to the presence or absence of pungent taste by a sensory test. The fruits were harvested immediately after they matured and turned red, and after morphological investigation and sensory test, several fruits were mixed and the capsiate content was measured by HPLC analysis.
カプシエイトの抽出方法は、既知の方法を利用した。まず、凍結乾燥機(FDU-200 EYELA)で乾燥させたトウガラシをミキサーで粉砕した。そのトウガラシ粉末0.1 gを測りとってサンプル瓶に入れ、アセトン4 mLを加えて室温(25℃)で15分静置した。上清をナスフラスコに回収し、残渣にアセトン1 mLを加えた。そして軽く振った後、上清を先ほどのナスフラスコに回収し、さらにその残渣に酢酸エチルを1 mL加えて軽く瓶を振り、同じナスフラスコに上清を回収した。これら6 mLほどの抽出液をエバポレーター(SIBATA ROTAVAPOR RE120)で完全に蒸発させた。残った抽出物をメタノール5 mLで溶解させ、この溶液をHPLCに供試した。 As the extraction method of the capsiate, a known method was used. First, the capsicum dried in a freeze-dryer (FDU-200 EYELA) was crushed with a mixer. 0.1 g of the red pepper powder was weighed and placed in a sample bottle, 4 mL of acetone was added, and the mixture was allowed to stand at room temperature (25 ° C.) for 15 minutes. The supernatant was collected in an eggplant flask, and 1 mL of acetone was added to the residue. Then, after shaking lightly, the supernatant was collected in the eggplant flask, 1 mL of ethyl acetate was added to the residue, the bottle was shaken lightly, and the supernatant was collected in the same eggplant flask. About 6 mL of these extracts were completely evaporated by an evaporator (SIBATA ROTAVAPOR RE120). The remaining extract was dissolved in 5 mL of methanol and this solution was tested for HPLC.
カプシエイトの定量は、HPLC(LC-10AD、送液ユニット：SPD-M10A、検出器：CTO-10AD、カラムオープン：DGU-10AD、デガッサ：SIL-10AD、オートインジェクタ、島津製作所社製)を用いた。各システムの管理はLC solutionで行った。HPLC分析に用いる前に、サンプル液1 mLをPTFEメンブランシリンジフィルター(Whatman社製、膜孔径0.45μm)でろ過してHPLC用試料とした。既知の分析条件を参考にし、ODSカラム(YMC-Pack ODS-A、75×4.6 mm、YMC社)、カラム温度40℃、移動相は70％メタノール、0.1％トリフルオロ酢酸、流速1 mL/min、吸光度280 nmで行った。カプサイシンの標品として、和光純薬工業社製のHPLC用カプサイシン(純度99.0％以上)を用い、62.5μg/mL、125μg/mL、250μg/mL、500μg/mLの4段階のカプサイシン標品を調整して分析し、検量線を作成した。この際、8分前後に検出されるピークをカプシエイトのピークとして測定した。 For the quantification of capsiate, HPLC (LC-10AD, liquid feed unit: SPD-M10A, detector: CTO-10AD, column open: DGU-10AD, degassa: SIL-10AD, auto injector, manufactured by Shimadzu Corporation) was used. .. Each system was managed by LC solution. Before using for HPLC analysis, 1 mL of the sample solution was filtered through a PTFE membrane syringe filter (manufactured by Whatman, membrane pore size 0.45 μm) to prepare a sample for HPLC. ODS column (YMC-Pack ODS-A, 75 × 4.6 mm, YMC), column temperature 40 ° C, mobile phase 70% methanol, 0.1% trifluoroacetic acid, flow rate 1 mL / min, referring to known analytical conditions. , The absorbance was 280 nm. Using capsaicin for HPLC (purity 99.0% or more) manufactured by Wako Pure Chemical Industries, Ltd. as a capsaicin standard, prepare a four-stage capsaicin standard of 62.5 μg / mL, 125 μg / mL, 250 μg / mL, and 500 μg / mL. And analyzed, and a calibration curve was created. At this time, the peak detected around 8 minutes was measured as the capsiate peak.
HPLC分析を行った結果の表を表１に示す。表中の数字は、トウガラシの胎座の乾物重量1gあたりのカプシエイトの含有量（単位：μg）である。供試した9品種のうち‘伏見甘長’以外の7品種で、単為結果処理によるカプシエイトの発現および増加が確認された。特に‘ひもとうがらし’3品種では、カプシエイト含量が単為結果処理により大幅に増加しており、その増加率は受精果実の8.3倍、101.7倍および37.6倍であった。
Table 1 shows a table of the results of HPLC analysis. The numbers in the table are the capsiate content (unit: μg) per 1 g of dry matter in the placenta of capsicum. Of the 9 varieties tested, 7 varieties other than'Fushimi Kancho'were confirmed to express and increase capsiate by parthenogenetic treatment. In particular, in the'Himoto Ugarashi'3 varieties, the capsiate content was significantly increased by parthenogenetic treatment, and the rate of increase was 8.3 times, 101.7 times and 37.6 times that of fertilized fruits.
Claims (8)
The capsiate according to any one of claims 3 to 7 , wherein the solvent is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, t-butanol, ethyl acetate, acetone, and mixtures thereof. Production method.
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| JP4089306B2 (en) | 2002-06-14 | 2008-05-28 | 味の素株式会社 | Capsinoid extract production method and capsinoid extract obtained by the method |
| JP2010528670A (en) | 2007-06-13 | 2010-08-26 | ゼライム ゲデラ リミティド | Improved pepper plant |
| WO2015108185A1 (en) | 2014-01-17 | 2015-07-23 | 独立行政法人農業・食品産業技術総合研究機構 | Parthenocarpy regulation gene and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP4089306B2 (en) | 2002-06-14 | 2008-05-28 | 味の素株式会社 | Capsinoid extract production method and capsinoid extract obtained by the method |
| JP2010528670A (en) | 2007-06-13 | 2010-08-26 | ゼライム ゲデラ リミティド | Improved pepper plant |
| WO2015108185A1 (en) | 2014-01-17 | 2015-07-23 | 独立行政法人農業・食品産業技術総合研究機構 | Parthenocarpy regulation gene and use thereof |
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| Journal of Agricultural and Food Chemistry, 2010, Vol.58, pp.1761-1767 |
| 特産種苗, 2015, Vol.20,pp.13-17 |
| 特産種苗, 2015, Vol.20,pp.18-21 |
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