JPS62210920A - Production of bitter component of wasabi - Google Patents
Production of bitter component of wasabiInfo
- Publication number
- JPS62210920A JPS62210920A JP61052671A JP5267186A JPS62210920A JP S62210920 A JPS62210920 A JP S62210920A JP 61052671 A JP61052671 A JP 61052671A JP 5267186 A JP5267186 A JP 5267186A JP S62210920 A JPS62210920 A JP S62210920A
- Authority
- JP
- Japan
- Prior art keywords
- wasabi
- medium
- hair roots
- culturing
- pungent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 235000000760 Wasabia japonica Nutrition 0.000 title claims description 49
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 241001293164 Eutrema japonicum Species 0.000 title 1
- 244000195452 Wasabia japonica Species 0.000 claims description 48
- 238000000034 method Methods 0.000 claims description 18
- 241000589156 Agrobacterium rhizogenes Species 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 235000000346 sugar Nutrition 0.000 claims description 5
- 240000003291 Armoracia rusticana Species 0.000 claims description 3
- 235000011330 Armoracia rusticana Nutrition 0.000 claims description 3
- 239000004615 ingredient Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 230000003115 biocidal effect Effects 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- ZOJBYZNEUISWFT-UHFFFAOYSA-N allyl isothiocyanate Chemical compound C=CCN=C=S ZOJBYZNEUISWFT-UHFFFAOYSA-N 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 239000006870 ms-medium Substances 0.000 description 7
- 235000019633 pungent taste Nutrition 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000003988 headspace gas chromatography Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 241000589158 Agrobacterium Species 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 238000004817 gas chromatography Methods 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004186 food analysis Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000008164 mustard oil Substances 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- PHZOWSSBXJXFOR-UHFFFAOYSA-N 2-Propenyl glucosinolate Natural products OCC1OC(SC(CC=C)=NOS(O)(=O)=O)C(O)C(O)C1O PHZOWSSBXJXFOR-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 235000009419 Fagopyrum esculentum Nutrition 0.000 description 1
- 240000008620 Fagopyrum esculentum Species 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 108010040201 Polymyxins Proteins 0.000 description 1
- PHZOWSSBXJXFOR-MYMDCHNCSA-N Sinigrin Natural products S(=O)(=O)(O/N=C(\S[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)/CC=C)O PHZOWSSBXJXFOR-MYMDCHNCSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000007799 cork Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000004508 fractional distillation Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- QKFJKGMPGYROCL-UHFFFAOYSA-N phenyl isothiocyanate Chemical compound S=C=NC1=CC=CC=C1 QKFJKGMPGYROCL-UHFFFAOYSA-N 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- QKFAFSGJTMHRRY-OCFLFPRFSA-M potassium;[(e)-1-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]sulfanylbut-3-enylideneamino] sulfate Chemical compound [K+].OC[C@H]1O[C@@H](S\C(CC=C)=N\OS([O-])(=O)=O)[C@H](O)[C@@H](O)[C@@H]1O QKFAFSGJTMHRRY-OCFLFPRFSA-M 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000017291 sinigrin Nutrition 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 108010058651 thioglucosidase Proteins 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Seasonings (AREA)
Abstract
(57)【要約】本公報は電子出願前の出願データであるた
め要約のデータは記録されません。(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は植物組織の培養の分野に属するものであり、わ
さびの新鮮なｍ織にアグロバクテリウムリゾゲネス（Ａ
ｇｒｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ）を
接種することにより、毛根及びその培地にわさび辛味成
分を産生せしめる新規なわさび辛味成分の製造方法に関
するものである。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention belongs to the field of plant tissue culture, and involves the use of Agrobacterium rhizogenes (A.
The present invention relates to a novel method for producing a wasabi pungent component in which the wasabi pungent component is produced in hair roots and their culture medium by inoculating them with grobacterium rhizogenes.
わさびはその組織に上品な辛味があるので香辛料として
用いられる。この辛味はシニグリン（Ｓｉｎｉｇｒｉｎ
）と言われる配糖体が酵素ミロシナーゼ（Ｍｙｒｏｓｉ
ｎａｓｅ）によって分解され、からし油（辛味成分の主
体はアリルからし油）が出来る為である。この辛味は刺
身、そば、或いは菓子用として珍重されているため、わ
さびを栽培してその根茎から得ている。Wasabi is used as a spice because its structure has a refined spiciness. This spiciness is called Sinigrin.
) is a glycoside called enzyme myrosinase (Myrosi
This is because the mustard oil (the main component of the pungent component is allyl mustard oil) is decomposed by the mustard oil. This spiciness is prized for use in sashimi, soba, and sweets, so wasabi is cultivated and obtained from its rhizomes.
わさびは低温（１０〜１３℃）を好み、清澄な有機含有
量の少ない流水のあるところを好む。このため栽培を行
う場所はゆるい傾斜地で冬でも湧水のある谷間に限られ
ている。栽培期間は１５〜２５ケ月を要し、通常足かけ
３年の栽培期間を要する。又、収穫は秋から冬の寒冷期
が多く、寒気をおしての収穫はきつい作業である。この
ように天然のわさびを栽培することは、栽培に長時間を
要するだけでなく、栽培する場所も限られ、又、その作
業も困難なものである。Wasabi prefers low temperatures (10-13°C), and prefers clear running water with low organic content. For this reason, cultivation is limited to valleys with gentle slopes and spring water even in winter. The cultivation period takes 15 to 25 months, and usually takes three years. Also, most of the harvest is done during the cold season from autumn to winter, and harvesting through the cold weather is difficult work. Cultivating natural wasabi in this way not only requires a long time to cultivate, but also has limited cultivation areas and is difficult to cultivate.
このため、わさびの根茎をカルスにし、このカルスを培
養することによってわさび辛味成分を取得しようという
試みが長年なされているが、いまだ成功していない。For this reason, attempts have been made for many years to obtain the pungent component of wasabi by turning the rhizome of wasabi into a callus and culturing this callus, but no success has been achieved so far.
本発明は上述のように限定された方法のために生産性が
低いわさびの生産を栽培期間を短くし、栽培場所が限定
されることなく、高収率で天然わさびと同等の品質をも
つわさび辛味成分を製造することを目的としたものであ
る。The present invention aims to shorten the cultivation period for the production of wasabi, which has low productivity due to the limited method described above, and produce wasabi with high yield and quality equivalent to natural wasabi without being limited in cultivation location. The purpose is to produce pungent ingredients.
本発明者等はわさびの組織を培養することにより、わさ
び辛味成分を取得する方法を研究し、わさび組織にアグ
ロバクテリウムリゾゲネス（Ａｇｒ。The present inventors researched a method for obtaining wasabi pungent components by culturing wasabi tissue, and found that Agrobacterium rhizogenes (Agr.
ｂａｃｔｅｒｉｕｎ＋ ｒｈｉｚｏｇｅｎｅｓ）を接種
し、その結果発生する毛根を培養することにより、容易
に大量のわさび辛味成分を毛根に産生させることができ
ることを見出して本発明を完成したのである。The present invention was completed based on the discovery that a large amount of wasabi pungent components could be easily produced in the hair roots by inoculating the hair roots with the resulting hair roots.
即ち、本発明は、わさびの組織にアグロバクテリウム
リゾゲネス（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏ
ｇｅｎｅｓ）を接種することにより発根した毛根を、糖
を含む培地で培養することによりわさび辛味成分を取得
する方法を特徴とする。That is, the present invention allows Agrobacterium to be present in wasabi tissue.
Agrobacterium rhizo
This method is characterized by a method for obtaining the wasabi pungent component by culturing hair roots that have grown by inoculating the horseradish (wasabi) in a sugar-containing medium.
わさび組織としては、根茎、茎、葉等の組織を用いるこ
とができる。As the wasabi tissue, tissues such as rhizomes, stems, leaves, etc. can be used.
次に、本発明の詳細な説明する。Next, the present invention will be explained in detail.
わさび辛味成分を含有する新鮮な根茎等の組織を水洗・
消毒したのち、これを約５〜１０ｍｍの厚みになるよう
に円板形に切断する。この片面にアグロバクテリウム
リゾゲネス（＾ｇｒｏｂａｃｔａｒｉｕｍｒｈｉｚｏｇ
ｅｎｅｓ）の懸濁液を塗布する。バクテリヤを接種した
面を上にして円板状のわさびの根茎等をアガープレート
の上に乗せ、２５℃の暗所で培養する。約２Ｍ間培養を
続けるとバクテリヤを接種した面から不定形の毛根が出
てくるので、これを先端から１０〜２０１の長さに切断
する。切断した毛根を１重量％アガーＭＳ培地（３重量
％スクロース含有）で２〜３日間、２５℃で前培養する
。Fresh rhizomes and other tissues that contain the pungent components of wasabi are washed with water.
After sterilization, it is cut into discs with a thickness of approximately 5 to 10 mm. Agrobacterium on this one side
Rhizogenes (^grobactarium rhizog
enes) suspension. Place a disc-shaped wasabi rhizome or the like on an agar plate with the side inoculated with bacteria facing up, and culture in a dark place at 25°C. When the culture is continued for about 2M, an amorphous hair root emerges from the surface inoculated with bacteria, and this is cut into a length of 10 to 20 cm from the tip. The cut hair roots are precultured in 1% by weight agar MS medium (containing 3% by weight sucrose) at 25°C for 2 to 3 days.
次にこのものをＭＳ培地に移植し、２５℃で２〜３週間
培養すると、根は活発に成長して、組織中にわさび辛味
成分が生成する。Next, this plant is transplanted to MS medium and cultured at 25°C for 2 to 3 weeks, and the roots grow actively and the wasabi pungent component is produced in the tissue.
以上の操作は雑菌の混入を起こさないように無菌状態で
行うことが必要である。The above operations must be performed under aseptic conditions to avoid contamination with bacteria.
アグロバクテリウム リゾゲネス（Ａｇｒｏｂａｃｔｅ
ｒｔｕｍ ｒｈｉｚｏｇｅｎｅｓ）を接種するわさび組
織は、辛味成分を多く含有するものを用いる方が望まし
い。Agrobacterium rhizogenes (Agrobacterium rhizogenes)
It is preferable to use a wasabi tissue that contains a large amount of pungent components to inoculate the horseradish (Rtum rhizogenes).
又、収穫してからあまり時間の経過していない新鮮なも
のを用いるほうが、発根の発生およびわさび辛味成分の
収量の点から望ましい。このわさび組織を無菌状態にす
るための消毒は、例えば、次亜塩素酸ソーダの２重量％
水溶液に１０〜１５分間浸漬することで十分に行われる
。滅菌の終わったわさび組織は、無菌水で十分に洗浄を
行う。In addition, it is preferable to use fresh wasabi that has not been harvested for a long time in terms of rooting and the yield of the pungent wasabi component. Disinfection to make this wasabi tissue sterile is, for example, 2% by weight of sodium hypochlorite.
Immersion in an aqueous solution for 10 to 15 minutes is sufficient. Once the wasabi tissue has been sterilized, wash it thoroughly with sterile water.
又、バクテリヤを接種する原料のわさびの組織としては
、その形状のまま、あるいは塊状、薄板、粒状にしたも
のが用いられ、特にその形状に制限はない。本発明に用
いられる発根を促すための菌種はアグロバクテリウム
リソ゛ゲネス（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒｈｉｚ
ｏｇｅｎｅｓ）が特によい・この菌種は、通常の培養に
よって得られたもので十分である。接種に用いるバクテ
リヤ懸濁液中の菌体数は多い方が望ましいが、１０６〜
１０３個／ｍ７程度で行うことができる。Furthermore, the wasabi tissue used as the raw material for inoculating bacteria may be used in its original shape, or in the form of lumps, thin plates, or granules, and there is no particular restriction on its shape. The bacterial species used in the present invention to promote rooting is Agrobacterium
Agrobacterium rhiz
This type of bacteria is particularly good. - This type of bacteria obtained by normal culture is sufficient. It is desirable that the number of bacterial cells in the bacterial suspension used for inoculation be as large as possible;
This can be done at a rate of about 103 pieces/m7.
本発明による毛根の培養は糖類および無機塩類を含む培
地で行われる。糖類としては、特にスクロースおよびグ
ルコースの群から選ばれたものが好ましい。The hair root culture according to the present invention is carried out in a medium containing sugars and inorganic salts. As sugars, those selected from the group of sucrose and glucose are particularly preferred.
培養はわさび組織から発根した毛根を培養するもので、
その培養方法に特に制限はなく、組織を付けたままでの
毛根の培養、および毛根のみの、培養方法があるが、毛
根のみを用いると効率が良い。Cultivation involves culturing hair roots that have grown from wasabi tissue.
There are no particular restrictions on the culture method, and there are methods for culturing hair roots with tissues attached and for culturing only hair roots, but it is more efficient to use only hair roots.
又、毛根のみをカラムに充填し、通気しつつ培養液を循
環する方式、あるいはタンク培養の方式をとってもかま
わない。Alternatively, a method in which only the hair roots are packed in a column and the culture solution is circulated while aeration is performed, or a tank culture method may be used.
培養は１５〜３５℃で行うが、好ましくは２０〜３０℃
である。Cultivation is carried out at 15-35°C, preferably 20-30°C.
It is.
毛根中に生成・蓄積したわさび辛味成分の分析・定量法
については大きく分けて滴定法とガスクロマトグラフィ
ー法とがある。その操作の詳細については「食品分析法
日本食品工業学会・食品分析法編集委員会曙 先車、
７９１〜７９４頁」に記載がある。ガスクロマトグラフ
ィーによる簡易定量法については「Ｂ本食品工業学会誌
、第２４巻（第２号）９０〜９３頁（１９７７年）“ヘ
ッドスペースガスクロマトグラフィーによるわさび辛味
成分の簡易定量法”」にその具体的方法が記載されてい
る。There are two main methods for analyzing and quantifying wasabi pungent components produced and accumulated in hair roots: titration and gas chromatography. For details on its operation, see "Food Analysis Methods, Japan Society of Food Industry/Food Analysis Methods Editorial Committee Akebono Sakisha,"
791-794''. A simple quantitative method using gas chromatography is described in "Simple quantitative method of wasabi pungency using headspace gas chromatography", Journal of Japan Food Industry Association, Vol. 24 (No. 2), pp. 90-93 (1977). The specific method is described.
本発明により生成したわさび辛味成分はヘッドスペース
ガスクロマトグラフィーにより確認し、定量を行った。The wasabi spiciness component produced according to the present invention was confirmed and quantified by headspace gas chromatography.
即ち、得られた毛根を乳鉢でよく磨砕したものを用いヘ
ッドスペースガスクロマトグラフィーによる簡易定量法
に従って分析した結果、アリルからし油の含有量は０．
２〜０．３重量％、乾物換算で０．８〜１．２重量％で
あった。That is, as a result of analyzing the hair roots obtained by thoroughly grinding them in a mortar according to a simple quantitative method using headspace gas chromatography, the content of allyl mustard oil was found to be 0.
It was 2 to 0.3% by weight, and 0.8 to 1.2% by weight in terms of dry matter.
磨砕したペーストは鼻にランとくるわさび特有の刺激性
辛味を持っていた。The ground paste had the characteristic pungent taste of wasabi that hit the nose.
毛根中に生成・蓄積、したわさび辛味成分の利用にあた
っては、毛根を培養液から分離・洗浄した後、ペースト
状に磨砕した形で用いても良いし、あるいは磨砕したペ
ーストから溶媒抽出法、分溜あるいはカラムクロマト等
を使って辛味成分のみを分離して用いても良い。又、毛
根を乾燥後、粉砕して用いることもできる。To use the wasabi pungent components produced and accumulated in the hair roots, the hair roots can be separated from the culture solution and washed, then ground into a paste and used, or the ground paste can be used by solvent extraction. Only the pungent component may be separated and used using fractional distillation or column chromatography. Alternatively, the hair roots can be used by drying and then pulverizing them.
〔実施例〕
以下、本発明を実施例に基づいて説明するが、本発明は
これに限定されるものではない。[Example] The present invention will be described below based on Examples, but the present invention is not limited thereto.
実施例１゜
収穫した直後の新鮮な沢わさびの根茎をよぐ水洗し泥等
の付着物を除去した後、厚さ約１ｃｍに輪切りする。こ
れを７０重量％エタノールに３０秒間漬け、滅菌水で洗
い、次亜塩素酸ナトリウム２重量％水溶液に１０分間漬
け、滅菌水で２回洗った後、コルクポーラ−で直径１ｃ
＋ｎに打抜き、０．９重量％ＭＳ寒天培地（蔗糖３％含
有）上に置き、上面をアグロバクテリウム リゾゲネス
（＾ｇｒｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ
）の菌体懸濁液（菌体濃度は約１０”個／ｄ）を付着さ
せた針で数回穿刺する。これを１５℃の暗所でインキュ
ベートする。Example 1 Immediately after harvesting, the rhizomes of fresh wasabi were washed with water to remove dirt and other deposits, and then cut into rounds about 1 cm thick. This was soaked in 70% ethanol for 30 seconds, washed with sterile water, soaked in a 2% sodium hypochlorite aqueous solution for 10 minutes, washed twice with sterile water, and then wrapped with cork polar to a diameter of 1 cm.
+n, placed on a 0.9 wt% MS agar medium (containing 3% sucrose), and the top surface was covered with Agrobacterium rhizogenes (^grobacterium rhizogenes).
) is punctured several times with a needle attached with a suspension of bacterial cells (concentration of approximately 10" cells/d). This is incubated in the dark at 15°C.
２週間後、根茎表面から無数の毛根が発生してくるが、
この毛根を約２ｃｍの長さになるように切断する。切断
した毛根を蔗糖３重量％、カルベニシリン：Ｏ，ＯＳ重
量％、ペニシリンＧ：Ｑ、Ｑ１６重量％、テトラサイク
リン：０．００２重世％、ポリミキシン：０．００１重
量％：バンコマイシン：０．００１重量％を含むＭＳ液
体培地が４０−人った１００−容マイヤーフラスコで６
゜ｒｐｍで１５℃で３日間暗所で振盪培養する。活発に
成育している毛根をＭＳ培地が２５０ｍｊ入っている１
１容マイヤーフラスコに移植し、１５℃で３週間暗所で
振盪培養する。After two weeks, countless hair roots will emerge from the surface of the rhizome,
Cut this hair root into a length of approximately 2 cm. The cut hair roots were mixed with 3% by weight of sucrose, carbenicillin: O, OS, 16% by weight, penicillin G: Q, Q 16% by weight, tetracycline: 0.002% by weight, polymyxin: 0.001% by weight, vancomycin: 0.001% by weight. The MS liquid medium containing 40-6 liters was prepared in a 100-volume Meyer flask.
Incubate with shaking at 15°C for 3 days in the dark at 15°C. Actively growing hair roots containing 250 mj of MS medium 1
Transplant into a 1-volume Meyer flask and culture with shaking at 15°C for 3 weeks in the dark.
培養後、増殖した毛根を濾別する。湿重量で４２ｇの毛
根が得られた。この毛根をがら５ｇを取り、これを磨砕
し、ヘッドスペースガスクロマトグラフィーにより、ア
リルからし油の確認と定量を行った。アリルからし油の
含を量は、あり姿で０．２２重量％であり、磨砕したペ
ーストはわさび特有の刺激性辛味を有していた。After culturing, the proliferated hair roots are filtered out. A hair root weighing 42 g in wet weight was obtained. Five grams of this hair root was taken and ground, and allyl mustard oil was confirmed and quantified by headspace gas chromatography. The content of allyl mustard oil was 0.22% by weight in the fresh form, and the ground paste had a pungent pungent taste characteristic of wasabi.
ガスクロマトグラフィーの測定条件； カラム５％ＰＥ
Ｇ （２０Ｍ−ＣＨＡＭＥＬ ＩＴＥ−Ｃ８）、キャリ
アガス：窒素、浴温ニア０〜１８０℃、昇温速度：５℃
／分、内部標準：フェニルがらし油（東京化成製）。Measurement conditions for gas chromatography; Column 5% PE
G (20M-CHAMEL ITE-C8), carrier gas: nitrogen, bath temperature near 0 to 180°C, heating rate: 5°C
/min, internal standard: phenyl mustard oil (manufactured by Tokyo Kasei).
実施例２゜
実施例１と同様に滅菌処理した根茎を約５ｍｍ角の粒状
にカットする。カットした根茎を１重量％アガープレー
ト（スクロース３１重量％含有ＭＳ培地）上に重ならな
いように敷き詰める。次にこの敷き詰めた根茎の上面に
予め１重量％ヌトリエントブロース培地で液体培養した
アグロバクテリウム リゾゲネス（Ａｇｒｏｂａｃｔｅ
ｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ）の菌体懸濁液（菌体
濃度は約１０’個／ｒｎｌ）を塗布する。バクテリヤを
接種した面を上にして２５℃の暗所でインキユベートす
る。Example 2 The rhizomes were sterilized in the same manner as in Example 1 and cut into particles of approximately 5 mm square. Spread the cut rhizomes on a 1% by weight agar plate (MS medium containing 31% by weight of sucrose) so that they do not overlap. Next, on the upper surface of the spread rhizomes, Agrobacterium rhizogenes (Agrobacterium rhizogenes), which had been liquid cultured in a 1% by weight nutrient broth medium, was placed on top of the spread rhizomes.
A suspension of microbial cells (concentration of microbial cells: about 10' cells/rnl) of M.rium rhizogenes is applied. Incubate in the dark at 25°C with the side inoculated with bacteria facing up.
２週間後、根茎表面から無数の毛根が発生してくるが、
この毛根を約１５〜２０１−の長さになるように切断す
る。切断した毛根を再び１重量％７ガープレート（スク
ロース３重量％含有ＭＳ培地）で２５℃にて３日間暗所
で培養する。活発に成育している毛根をＭＳ培地が２０
０ｍｊ入っている１１のフラスコに移植し、２５℃で３
週間暗所で振盪培養する。After two weeks, countless hair roots will emerge from the surface of the rhizome,
This hair root is cut to a length of approximately 15 to 20 cm. The cut hair roots are again cultured on 1% by weight 7 Gar plates (MS medium containing 3% by weight of sucrose) at 25°C in the dark for 3 days. Actively growing hair roots are grown in MS medium at 20%
Transferred to 11 flasks containing 0 mj and incubated at 25°C for 3
Culture with shaking in the dark for a week.
培養後、増殖した毛根を濾別する。湿重量で３５ｇの毛
根が得られた。この毛根をから５ｇを取り、これを碧砕
し、ヘッドスペースガスクロマトグラフィーにより、ア
リルからし油の確認と定量をｆｒ’った。アリルからし
油の含有量は、あり姿で０．２０重量％であった。After culturing, the proliferated hair roots are filtered out. A hair root weighing 35 g in wet weight was obtained. Five grams of this hair root was taken, crushed, and allyl mustard oil was confirmed and quantified by headspace gas chromatography. The content of allyl mustard oil was 0.20% by weight as is.
実施例３゜
試料として沢わさびの葉を葉柄と共に除去し、根茎から
葉柄の分化している部分、すなわち根茎を含む葉柄基部
（以後葉柄基部と称する）を用いた。良く水洗して泥等
の付着物を除去した後、厚さ約５ｍｍの円板状にカット
する。カットした葉柄基部を根茎と同様に滅菌、洗浄す
る。洗浄後、葉柄基部の片面にナイフで約２ｍＩＩ＋間
隔で格子状に切目を入れる。切目を入れたものを根茎同
様、アガープレート上にセットし、菌体懸濁液を塗布し
た。Example 3 As a sample, a wasabi leaf was removed along with its petiole, and the part where the petiole was differentiated from the rhizome, that is, the petiole base containing the rhizome (hereinafter referred to as the petiole base) was used. After thoroughly washing with water to remove dirt and other deposits, cut into discs with a thickness of about 5 mm. Sterilize and wash the cut petiole base in the same way as the rhizome. After washing, use a knife to make grid-like cuts at approximately 2 mII+ intervals on one side of the petiole base. The cut pieces were set on an agar plate in the same way as the rhizomes, and a bacterial suspension was applied.
１０℃の暗所で３週間インキュベートした。インキユベ
ートすると、葉柄基部表面にカル人様の組織および毛根
が発生してくる。発生の確認された葉柄基部を滅菌水で
洗浄し、菌を洗い流す。葉柄基部を抗生物質を含むアガ
ープレート（スクロース２重量％含有ＭＳ培地）上にセ
ットし、１週間暗所で約１０℃の培養温度にて培養した
後、毛根を切断し、前記実施例と同様の操作を行い、！
音饗毛根を２０ｇ得た。It was incubated in the dark at 10°C for 3 weeks. After incubation, Karu-like tissue and hair roots develop on the basal surface of the petiole. Wash the base of the petiole where the infestation is confirmed with sterile water to wash away the bacteria. The petiole base was set on an antibiotic-containing agar plate (MS medium containing 2% sucrose) and cultured in the dark at a culture temperature of about 10°C for one week, and then the hairy roots were cut and cultured in the same manner as in the previous example. Do the following!
20 g of Otogi hair roots were obtained.
わさび辛味成分は、天然のわさびを栽培することにより
生産されているが、この生産は栽培地が限定され、苦痛
な作業が伴い、更に生産性が良くない点で問題があった
。しかし、本発明の方法によれば、組織培養によるわさ
びの生産を容易に行うことができ、前述の問題点を全て
解決し、品質の良いわさび辛味成分を大量に供給するこ
とを可能にした。Wasabi pungent ingredients are produced by cultivating natural wasabi, but this production has problems in that cultivation areas are limited, laborious work is involved, and productivity is poor. However, according to the method of the present invention, wasabi can be easily produced by tissue culture, all of the above-mentioned problems have been solved, and it has become possible to supply a large quantity of high-quality wasabi pungent components.
Claims (1)
Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ）
を接種することにより発根した毛根を、糖を含む培地で
培養することによりわさび辛味成分を製造する方法。 ２、わさびが沢わさび、畑わさび、或いは西洋わさびで
ある特許請求の範囲第１項記載の方法。 ３、わさびの組織にアグロバクテリウム リゾゲネス（
Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ）
を接種するに当たり、わさび組織を塊状、薄片状、粒状
に切断したものを用いる特許請求の範囲第１項記載の方
法。 ４、わさびにアグロバクテリウム リゾゲネス（Ａｇｒ
ｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ）を接種
することにより発根した毛根を切り、毛根のみを培養す
る特許請求の範囲第１項記載の方法。 ５、毛根を培養する培地がスクロース及びグルコースの
群から選ばれた糖、及び無機塩を含む培地である特許請
求の範囲第１項記載の方法。 ６、培地が液体、或いは固体培地である特許請求の範囲
第１項記載の方法。 ７、毛根を培養する培地が抗生物質を含む培地である特
許請求の範囲第１項記載の方法。 ８、毛根を培養することにより、毛根及び培地に生成し
たわさび辛味成分を、毛根或いは培地中より抽出する特
許請求の範囲第１項記載の方法。[Claims] 1. Agrobacterium rhizogenes (
Agrobacterium rhizogenes)
A method for producing wasabi pungent ingredients by culturing hair roots that have grown by inoculation with a sugar-containing medium. 2. The method according to claim 1, wherein the wasabi is Sawa wasabi, Hata wasabi, or horseradish. 3. Agrobacterium rhizogenes (
Agrobacterium rhizogenes)
2. The method according to claim 1, in which wasabi tissue cut into chunks, flakes, or granules is used for inoculating the wasabi tissue. 4. Wasabi and Agrobacterium rhizogenes (Agr)
2. The method according to claim 1, wherein the hair roots that have grown by inoculation with B. obacterium rhizogenes are cut and only the hair roots are cultured. 5. The method according to claim 1, wherein the medium for culturing hairy roots is a medium containing a sugar selected from the group of sucrose and glucose, and an inorganic salt. 6. The method according to claim 1, wherein the medium is a liquid or solid medium. 7. The method according to claim 1, wherein the medium for culturing hair roots is a medium containing an antibiotic. 8. The method according to claim 1, wherein the wasabi pungent component produced in the hair roots and the medium is extracted from the hair roots or the medium by culturing the hair roots.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61052671A JPS62210920A (en) | 1986-03-12 | 1986-03-12 | Production of bitter component of wasabi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61052671A JPS62210920A (en) | 1986-03-12 | 1986-03-12 | Production of bitter component of wasabi |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62210920A true JPS62210920A (en) | 1987-09-17 |
Family
ID=12921332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61052671A Pending JPS62210920A (en) | 1986-03-12 | 1986-03-12 | Production of bitter component of wasabi |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62210920A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5236544A (en) * | 1989-06-26 | 1993-08-17 | Canon Kabushiki Kaisha | Process for growing crystal |
-
1986
- 1986-03-12 JP JP61052671A patent/JPS62210920A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5236544A (en) * | 1989-06-26 | 1993-08-17 | Canon Kabushiki Kaisha | Process for growing crystal |
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