JPS62210920A - Production of bitter component of wasabi - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

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.

[Conventional technology and its problems]

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.

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.

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.

[Means for solving problems]

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.

The present inventors researched a method for obtaining wasabi pungent components by culturing wasabi tissue, and found that Agrobacterium rhizogenes (Agr.

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.

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.

As the wasabi tissue, tissues such as rhizomes, stems, leaves, etc. can be used.

Next, the present invention will be explained in detail.

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.

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.

The above operations must be performed under aseptic conditions to avoid contamination with bacteria.

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).

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.

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.

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.

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.

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.

Cultivation is carried out at 15-35°C, preferably 20-30°C.
It is.

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.

The wasabi spiciness component produced according to the present invention was confirmed and quantified by headspace gas chromatography.

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.

The ground paste had the characteristic pungent taste of wasabi that hit the nose.

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.

[Example] The present invention will be described below based on Examples, but the present invention is not limited thereto.

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.

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.

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.

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).

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.

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.

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.

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.

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.

〔Effect of the invention〕

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)

[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.