JPH0768A - Method for separating active ingredient from new kind of japanese horseradish - Google Patents

Abstract

PURPOSE:To obtain a sharp taste ingredient having the sharp taste flavors of both Wasabi Japonica Matumura and Armoracia rusticana and further obtain a peroxidase in a good yield by separating and recovering the sharp taste ingredient and the peroxidase, contained in a specific kind of Japanese horseradish (wasabi). CONSTITUTION:A sharp taste ingredient and an enzyme, peroxidase, are separated and recovered from a wasabi called Hilliella shuangpaiensis Z.Y.Li as a raw material. The wasabi is somewhat larger than Wasabi Japonica Matumura, but its other properties are quite similar. Namely, the sharp taste ingredient contained in the wasabi is the flavors of both the Wasabi japonica Matumura and Armoracia rusticana, and the of the peroxidase is remarkably high.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the use of plants belonging to a new cultivar of horseradish, and in particular, it has characteristic components of both Japanese wasabi and horseradish and is used as a powdered wasabi or raw grated horseradish product or enzyme production. As a raw material,
The present invention relates to a method for separating an active ingredient from a new horseradish variety that can be used as an antibacterial agent.

[0002]

2. Description of the Related Art Wasabia has traditionally been the scientific name of Wasabia as a variety.
The Japanese wasabi from Japan called japonika Matumura and the horseradish from the scientific name Armoracia rusticana are mainly known.

The former Sawa Wasabi is a characteristic component of Sawa Wasabi, in addition to allyl pepper oil as the main component of spiciness, ω-S-.
Contains a methyl compound. (Details will be described later with reference to FIG. 2 attached.)

Further, the latter horseradish, like Sawa wasabi, contains, in addition to allyl pepper oil as a main component of spiciness, aromatic pepper oil having a radish-like aroma, which is a characteristic component of horseradish. (Details will be described later with reference to the attached FIG. 3.)

[0005]

However, although all of these wasabi and horseradish commonly contain allyl pepper oil, which is the main component of spiciness, the omega-S-methyl compound, which is a characteristic component of them, is common. The aromatic pepper oils are contained individually and not individually.

Therefore, the object of the present invention is to combine the characteristic components of both Japanese wasabi and horseradish, with the scientific name Hil.
liella shuangpaiensis ZY Li A plant belonging to a new cultivar of wasabi different from the above-mentioned known variety is used as a raw material, and when the active ingredient contained therein is separated and recovered, its utility as a raw material is that of conventional wasabi. It is to provide a very high separation method compared to horseradish and horseradish.

[0007]

[Means for Solving the Problems] In order to achieve the above-mentioned object, according to the present invention, a new cultivar of horseradish called scientific name Hilliella shuangpaiensis ZY Li is used as a raw material, and the spicy ingredient and enzyme contained in this raw material are used. It is characterized in that the active ingredient of peroxidase is separated and collected separately or simultaneously.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention is described in detail below. [Spicy ingredient] In Japan, wasabi has traditionally been
Wasabia japonika Matu-mura strain and the horseradish Armoracia rusticana strain are classified into two types. The new breed of horseradish used in the present invention (hereinafter referred to as the present invention wasabi) is scientific name Hilliellashuangpaiensis ZY Li.
It was recently discovered in Sichuan, China.
The appearance is slightly larger than Japanese-made wasabi, but the others are quite similar. However, it has a slightly different spiciness and also has a horseradish-like flavor. Therefore, the spicy components of the wasabi of the present invention were analyzed, and the quality and quantity of the existing spicy components of Japanese wasabi and horseradish were compared to examine which one belongs to chemotaxonomy.

Experimental method 1. Collection of Pungency Components Fresh stalks and leaves of the present invention were removed, and rhizomes made into a mud-like state with a wasabi wholesaler were left at room temperature with stirring for about 1 hour in order to sufficiently perform an enzymatic reaction. To 100 g of the sludge, 300 ml of ether [containing 0.06 mg / 100 ml of ethyl caprate (internal standard substance)] was added and left overnight. The next day, the mixture was filtered using gauze and extracted twice with 100 ml of ether each time, the extracts were combined, dehydrated with Glauber's salt, and concentrated under atmospheric pressure with a concentrator equipped with a Widmer distillation column to give a spicy ingredient concentrate.

2. Analysis of pungent components 1) Gas chromatograph (GC) Hitachi 163 gas chromatograph, fused silica capillary column (0.23mmφ, 25m), liquid phase Silicon OV-101
The column temperature was set to 60 to 270 ° C. (first temperature was kept at 60 ° C. for 30 minutes, and then the temperature was raised to 3 ° C./minute), and the injector and detector were operated at 230 and 250 ° C., respectively. The splitlation is 100: 1 and the carrier gas is N ₂ : 1.0m
l / min.

2) Gas Chromatograph Mass Spectrometer (GC-MS) JEOL302 type GC-MS manufactured by JEOL Ltd. has the same column in the case of GC analysis, and the analysis temperature condition is 60 to 250.
℃ (3 ℃ / min temperature rise), carrier gas is He: 1.0ml /
In addition, the analysis was performed at an ionization voltage of 70 ev.

Experimental Results 1) GC The results of the GC analysis of the spicy ingredient concentrate of wasabi of the present invention are shown in FIG. Table 1 shows the ratio of each peak area to the peak area of the internal standard substance (ethyl caprate). 2) GC-MS A component corresponding to each component peak of GC was obtained from MS.
The results are shown in Table 1.

[0013]

[Table 1]

Discussion 1) Pungency component of wasabi of the present invention The analysis results of the pungency component are shown in FIG. 1 and Table 1. Interesting results are obtained from this FIG. 1 and Table 1. That is, the contained pungency ingredient system has both the characteristics of Japanese wasabi and horseradish.

Further, FIGS. 2, 3 and 4 and Table 2
The volatile components of Nisawa horseradish, horseradish and horseradish of the present invention and their content balances are shown. 2 is a horseradish, FIG. 3 is a horseradish, and FIG. 4 is a graph showing the relative area of the main volatile components of the wasabi of the present invention measured by gas chromatography. The experiment was performed as in FIG. Table 2 is also
These are the relative area ratios of the main volatile components of Sawa Wasabi, Horseradish and Wasabi of the present invention measured by gas chromatography. These are the peak area ratios to the area of the internal standard substance (0.3 mg of ethyl caprate in 100 g of the sample). expressed.

[0016]

[Table 2] Note: (1) Allyl isothiocyanate with peak No. 1 is allyl pepper oil. (2) Peak Nos. 8, 12, 13, and 16 are ω-S-methyl compounds. (3) Peak Nos. 9 and 11 are aromatic pepper oils.

The following can be seen from FIGS. 2 to 4 and Table 2 described above. The main component of spiciness is allyl pepper oil, which is not different among the three, but Sawa Wasabi has a ω-S-methyl compound (peak NO. 12, 13) as a characteristic component, which is the characteristic scent of Sawa Wasabi. It is considered to be an ingredient of. On the other hand, horseradish is characterized by having aromatic spicy oil (peak NO. 9) and therefore having a radish-like scent. The wasabi of the present invention contains a relatively large amount of ω-S-methyl compound as in the case of peaks Nos. 8, 12, 13, and 16, while aromatic pepper oil (peaks No. 9, 11) also contains In particular, NO.9 2-phenylethylisothiocyanate is contained in about the same amount as or more than allyl pepper oil. This tendency shows exactly the same tendency as the composition ratio of the pungent component of horseradish. Judging from the above results, the wasabi of the present invention is considered to be a new variety because it has both the characteristics of Japanese wasabi and horseradish based on the type and content of the constituent spicy ingredients.

[Content of Enzyme Peroxidase] Peroxi
Dase (peroxidase) is an enzyme that catalyzes the oxidation of various substances by using hydrogen peroxide as a hydrogen acceptor. It is known that peroxidase exists widely in the kingdoms of animals, plants and microorganisms. Among them, horseradish is currently used as a raw material for the extraction of peroxidase because it is contained in a large amount in the root of horseradish. Therefore,
The peroxidase content of the horseradish of the present invention was also measured and compared with that of conventional wasabi and horseradish.

Measuring method <Guayacol method> 2 g of a sample from which stains have been removed (cut to a size of 5 mm square on one side) is placed in a test tube and mixed with 0.1 M potassium phosphate and 0.1 M sodium hydroxide. Prepared
Add 20 ml of 0.1 M phosphate buffer (PH 6.0) and mix,
Intoxicated with Ultra Disperser, at 22,000G, 4 ℃
The peroxidase was extracted by centrifugation for 15 minutes. The obtained supernatant liquid (extract liquid) was appropriately diluted with 0.1 M phosphate buffer and used as a sample liquid.

Then, the aforementioned 0.1M phosphate buffer solution (PH
6.0) 1.00 ml, hydrogen peroxide (30% on the market) 1 ml with distilled water
0.10 ml of 0.3% hydrogen peroxide prepared by adjusting the volume to 100 ml,
And 1.68 ml of distilled water were pre-incubated at 37 ° C and 2.8 g of guaiacol in distilled water.
2.8% guaiacol 0.12ml prepared to a fixed volume of 00ml
And a total of 3.00 ml of the sample solution 0.10 ml are mixed in this order, transferred to a spectrophotometer cell, and the absorbance immediately after mixing and the absorbance after 1 minute are measured by the spectrophotometer (470 nm).
The peroxidase activity value was calculated using this measurement value, and the results are shown in Table 3.

Calculation of the peroxidase activity value

[Formula 1] A: Absorbance one minute after the start of the reaction B: Absorbance at the start of the reaction Q: Volume of sample solution (ml) L: Length of light path in liquid layer (cm) ε: Guaphcol molecular extinction coefficient (M · cm) It was done using.

[0022]

[Table 3]

As is clear from Table 3, the wasabi of the present invention showed an average value of 875.22 U per gram in its rhizome, which was about 4 as compared with 205.81 U of the root of horseradish.
The activity value was twice as high. The leaves and petioles were also higher than those of horseradish and wasabi.

As described above, the horseradish of the present invention was found to have a considerably high activity as compared with the content of horseradish that was conventionally considered to be high, and it will replace horseradish in the future.
It is expected to be a raw material for peroxidase extraction.

[Characteristics of Plant] The characteristics of the above-described wasabi of the present invention as a plant are as follows. It is a hairless perennial herb with thick cylindrical rhizomes, reaching a diameter of 4 cm. Root leaves spreading like rosettes are feather-shaped 7-9
Rip, petioles 10 cm long. Each leaflet has an oval oval shape with a blunt tip and a truncated or rounded base, about 8 × 4 cm, with a clear petiole except for the upper one. The flower stalk always grows on the aerial leaves, erects or slopes up, and reaches a height of 80 cm. The leaves are similar to rooted leaves, and the lower ones are feather-shaped 7
It splits, becoming smaller and scale-like toward the top. The inflorescence is basically an axillary raceme, and the foliage is small on the upper part of the flower stem, resulting in a panicle. The flowers are white and have a pedicel pattern of about 7 mm with no bracts. The sepal is a wide boat, about 2 mm long, and falls off just before flowering. Petals open, wide elliptical, base narrows abruptly. Stamen has 6 and 2 short stamens with dense glands at the base. The ovary is elliptical and has one room,
There is a style with a length of about 1 mm and about 1 mm. Two to four ovules hang with a long handle. Sakuka is flat and oblivious.

Table 4 is a comparison of the above-mentioned wasabi of the present invention with plants of conventional wasabi and horseradish.

[Table 4]

[Cultivation] The above-mentioned wasabi of the present invention is cultivated as follows. 1. Proliferation method 1) Shoot top culture method The shoot top (0.2 mm) was extracted and BA 0.1 ppm was added 1
/ 2MS agar plate, after precultured for about 3 months,
Transfer to 1/2 MS medium supplemented with 1.0 ppm of BA. After that, since it grows 5 to 6 times a month, growth and transplantation are repeated in the same medium. 2) Splitting method The axillary buds formed on the rhizome at the base of petiole are split into separate seedlings. 2. Raising seedlings The grown seedlings are acclimatized and raised in a greenhouse for 2 to 3 months with vermukilite using a polypot. 3. Cultivation method The seedlings produced by the above method were planted in the spring in a corner of a wasabi interforestation cultivation test plant in Toyone-mura, Kitashitara-gun, Aichi Prefecture, and were cultivated in the same manner as wasabi. It was confirmed that rhizomes were formed.

[0028]

The present invention is to separate and collect the spicy ingredient and the active ingredient of the enzyme peroxidase contained in the above-mentioned wasabi of the present invention, and the spicy ingredient grated the new horseradish cultivar to cause an enzymatic reaction. After that, it is extracted by steam distillation at a temperature of about 110 ° C., and the enzyme peroxidase is extracted and separated by grazing the new horseradish variety and immersing it in a saline solution with stirring. These extraction separations may be performed separately or simultaneously.

As the method of this simultaneous separation, for example, a spicy ingredient is produced from the above-mentioned wasabi raw material of the present invention, this is extracted with an extract, and then the spicy ingredient is separated and recovered from the extract, A method of separating and recovering the enzyme peroxidase from the residue is adopted.

Specifically, in this method, the above-mentioned wasabi raw material of the present invention undergoes an enzymatic reaction in an extraction tank to produce a pungent component, and supercritical carbon dioxide or liquefied carbonic acid is introduced into this tank to produce the pungent taste. The components are extracted into the supercritical carbon dioxide or liquefied carbonic acid, then the supercritical carbon dioxide containing the pungent component or liquefied carbonic acid is introduced into a separation tank to separate and recover the pungent component, and further in the extraction tank. In this method, the enzyme peroxidase is separated and recovered from the residual.

Hereinafter, this method will be described in detail with reference to FIG. 5 of the accompanying drawings. First, raw wasabi of the present invention is put into the cooling tank 1 and frozen at a low temperature below the freezing point, and then freeze-pulverized by the pulverizer 2 while maintaining this frozen state. The coolant in the cooling tank 1 is liquid nitrogen or the like. The crusher 2 is an ordinary crusher or a grater.

Next, the frozen material pulverized by the pulverizer 2 is filled in the extraction tank 3 and heated to cause an enzymatic reaction to produce a pungency component (allyl isothiocyanate). When the dried wasabi of the present invention is used as a raw material, water is introduced into the extraction tank 3 by an entrainer device so that the water content is 10%.
Above, preferably 30 to 50% (both by weight) is adjusted to cause an enzymatic reaction. Further, in the case of crushing at room temperature or grazing at room temperature, the enzyme reaction is caused as it is.

Furthermore, at the same time as the above-mentioned enzymatic reaction, the extraction tank 3
A solvent of supercritical carbon dioxide or liquefied carbonic acid is introduced therein, and the pungency component produced by the enzyme reaction is extracted into the solvent of supercritical carbon dioxide or liquefied carbonic acid. At the time of introduction, in the case of supercritical carbon dioxide, the temperature is 30 ° C.
As described above, it is introduced under the conditions of a pressure of 75 atm or more, and in the case of liquefied carbonic acid, for example, it is introduced under the conditions of a temperature of 20 ° C. and a pressure of 200 atm.

The solvent containing the pungent component thus obtained is introduced into the separation tank 4 and is decompressed to be phase-separated into a gas phase and a liquid phase, which is discharged from the separation tank 4 as a gas to be a useful substance. Only certain spicy ingredients are selectively separated and recovered.

Further, in this method, the enzyme peroxidase is extracted and separated from the residue remaining in the extraction tank 3 by a usual method. Since this method uses supercritical carbon dioxide or liquefied carbonic acid as the extraction solvent, the enzyme peroxidase in the residue in the extraction tank 3 remains without being killed. By recovering this from the residue, the pungency component and the enzyme peroxidase are simultaneously detected. Extraction and recovery becomes possible.

[0036]

Since the wasabi of the present invention has the characteristic components of both the conventional wasabi and horseradish, it is very useful as a raw material when the active ingredient contained therein is separated and recovered. .

The obtained spicy ingredient is used not only as a raw material for powdered wasabi products and raw grated wasabi products, but also as an antibacterial agent, and the enzyme peroxidase is widely used for clinical test reagents, analytical reagents, etc. , Its demand is increasing.

[0038]

[Example] 100 Kg of a new cultivar of horseradish (wasabi of the present invention) (water content of about 70%) was grated at room temperature and filled in an extraction tank. The temperature in this extraction tank was kept at 40 ° C., supercritical carbon dioxide at 40 ° C. and 200 atm was introduced, and extraction was performed for about 2 hours while stirring. Next, this supercritical carbon dioxide was introduced into a separation tank through a pressure reducing valve and depressurized to 50 atm to release the supercritical carbon dioxide as carbon dioxide gas to obtain 0.3 kg of allyl isothiocyanate as a pungency component. Then, the paste-like residue remaining in the extraction tank was transferred to another extraction tank, and separated by peroxidase extraction. This extraction separation was carried out by the following method.

To the extraction residue, 140 l of water was added and well stirred, and after standing overnight, 54 l of a mixed solution of ethanol and chloroform (2: 1) was added, and the mixture was stirred at 0 ° C for 20 minutes and centrifuged (3,000 rp).
m 15 minutes) to obtain the supernatant. Then, it is adjusted to 5.5 with 2N caustic soda and concentrated under reduced pressure at 30 ° C. or lower to 40 liters. Ammonium sulfate was added to this (250 g / l) and the resulting precipitate was collected by centrifugation. This precipitate is suspended in 1 l of 0.2 M phosphate buffer (PH7) and dialyzed overnight. The precipitate is removed by centrifugation to obtain a supernatant. It is cooled and 1.5 volumes of ethanol are added and the resulting precipitate is collected. This is dissolved in water and purified by alcoholic precipitation. That is, the first time, the sections that precipitate by adding ethanol (1.2 to 2.0 volumes) are collected, and the sections that dissolve in water and precipitate again by adding ethanol (1.3 to 1.9 volumes) are collected. Dissolve this in 1 liter of water, add ammonium sulfate and take a section where the degree of saturation precipitates at 0.5-0.70. Dissolve in a small amount of water and add saturated ammonium sulfate solution dropwise to give a slight turbidity. After soaking, the mixture was cooled and crystal seeds were added, and if the mixture was left at room temperature, crystals were precipitated in several hours to obtain peroxidase.

[0040]

The above-mentioned present invention uses a new variety of wasabi, which has the characteristic components of both wasabi and horseradish, as a raw material. Therefore, when the active ingredient contained therein is separated and recovered, it is used as a raw material. The utility of the invention is extremely higher than that of conventional wasabi and horseradish, and it is widely used in various fields as a raw material for enzyme production and as an antibacterial agent, and is a practically useful invention.

[Brief description of drawings]

FIG. 1 is a graph showing a component analysis result of a wasabi gas chromatograph according to the present invention.

FIG. 2 is a graph showing the relative area of the wasabi component of Sawa by a gas chromatograph.

FIG. 3 is a graph showing the relative area of horseradish components by gas chromatography.

FIG. 4 is a graph showing a relative area of wasabi according to the present invention by a gas chromatograph.

FIG. 5 is a block diagram showing a specific example of a separation method according to the present invention.

[Explanation of symbols]

 1 Cooling tank 2 Crusher 3 Extraction tank 4 Separation tank

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hideo Eto 857-11 Miho, Shimizu City, Shizuoka Prefecture (72) Inventor Kazuo Ina 3-13-7 Kasuga, Shizuoka City, Shizuoka Prefecture (72) Inventor Seto Sakurai, Seto Aichi Prefecture 1-211, Kitamatsuyama-cho, Ichi, Japan (72) Noriyoshi Sato 2-6-15, Kami Nagoya, Nishi-ku, Nagoya, Aichi (72) Inventor, Kumi 1-23, Kanayama, Naka-ku, Nagoya, Aichi 701 issue

Claims (5)

[Claims] 1. Scientific name Hilliella shuangpaiensis ZY
A method for separating an active ingredient from a new cultivar of wasabi, which comprises using as a raw material a new cultivar of wasabi called Li and separately and simultaneously separating and recovering a pungency component and an active ingredient of the enzyme peroxidase contained in the raw material. 2. The method for separating an active ingredient according to claim 1, wherein the pungency component contained in the raw material is extracted by steam distillation. 3. The method for separating an active ingredient according to claim 1, wherein the enzyme peroxidase contained in the raw material is extracted and separated by immersing it in a saline solution. 4. A method of producing a spicy ingredient from the raw material, extracting the spicy ingredient with an extract, separating and recovering the spicy ingredient from the extract, and further recovering the enzyme peroxidase from the residue. Claim 1
Method for separating active ingredients of. 5. The raw material is subjected to an enzymatic reaction in an extraction tank to generate a spicy ingredient, and supercritical carbon dioxide or liquefied carbonic acid is introduced into the tank to liquefy the spicy ingredient. Extraction in carbonic acid, then introducing supercritical carbon dioxide or liquefied carbonic acid containing the pungency component into a separation tank to separate and recover the pungency component, and further separate and recover the enzyme peroxidase from the residual in the extraction tank. The method for separating an active ingredient according to claim 4, characterized in that