JP2002034502A - New method for producing stevia and new stevia sweetener - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing a stevia plant having improved taste and a method for producing a sweetener having improved taste in high efficiency. SOLUTION: A stevia plant growing indoors or outdoors or a culturing stevia cell tissue is irradiated with radiation, especially γ-rays at various radiation doses to induce the mutation of the genetic character of stevia relating to rebaudioside A to efficiently obtain a genetically stable stevia having high content of rebaudioside A relative to stevioside and/or rebaudioside C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing stevia (Stevia rebaudiana BERTONI) with improved taste.
More specifically, the present invention relates to a method for producing stevia with improved taste quality by irradiating stevia with mutation and breeding.

[0002]

2. Description of the Related Art Stevia is a perennial herb belonging to the Asteraceae family native to Paraguay. It produces sweet components such as stevioside, rebaudioside A and rebaudioside C as main components and stores them mainly in leaves. Stevia extract is widely used in soft drinks and confectionery instead of sugar as a natural sweetener due to its low calorie.

[0003] Stevioside has a sweetness about 300 times that of sugar and is relatively similar to sugar, but is not preferred because a unique bitterness remains in the aftertaste. Further, rebaudioside C has a sweetness about 40 to 60 times that of sugar, but has a strong astringent taste and leaves an unpleasant aftertaste.

[0004] On the other hand, rebaudioside A has a sweetness level equal to or higher than those thereof, exhibits a mellow prior taste similar to sugar, and does not leave an unpleasant aftertaste such as bitterness or astringency. Since the stevia extract is a mixture of these, it has a mellow sweet taste similar to sugar, but has a unique taste of aftertaste.

[0005] Therefore, in order to improve the taste quality, various improvement methods such as increasing the relative ratio of rebaudioside A have been studied.

[0006] For example, as a measure for improving the quality of sweetness in stevia extract, multiples of chromosomes of a stevia plant,
Breeding methods such as creation of aneuploid lines and crossing between lines having good traits are known (Japanese Patent No. 2748141,
Japanese Patent Publication No. 6-95866). However, aneuploid plants, such as polyploids, generally have an increased plant mass and a higher yield of plants, but also tend to have a higher content of stevioside. Thereafter, a step of removing this is required, and a considerable amount of rebaudioside A may be removed in that step.

[0007] In addition, there is known a food industry method in which α-glucosidase is allowed to act on stevia extract to increase the number of α-glucosyl groups added thereto. However, new production costs are required besides cultivation, harvesting and extraction. And increase production costs.

[0008] Stevia is highly self-incompatible, and it is difficult to fix a pure line by selfing, and systematized cross-breeding is extremely low in probability and practically difficult.

As described above, in order to obtain a sweetener having a high content of rebaudioside A in a large amount and at a low cost, it is extremely important to increase the content and amount of rebaudioside A in the stevia plant itself. Therefore, development of a new method is desired.

On the other hand, the use of radiation as a method for mutating and breeding plants is one of the well-known breeding methods. In particular, since gamma rays are photons, they do not activate the irradiation target and are edible. It can be safely used for things. Japanese Patent Application Laid-Open No. 7-2649 is an example used for edible plants.
No. 42, an example used for ornamental plants is disclosed in
No. 749.

Turning to the same Asteraceae plant as Stevia, a large number of various mutants relating to the type and color of petals are isolated by gamma irradiation in chrysanthemum (Dendranthema grandiflorum), and are breeding (radiation breeding). Field Technical News No. 36). However, knowledge on the relationship between the type of mutagen, the method of application, and the mutation that appears is poor, and even in the case of plants of the same family, various conditions such as radiation sensitivity are usually different if the genus or species is different, The mutagen application conditions must be determined for each plant species to be treated.

As described above, the taste quality is improved by irradiating the stevia plant with radiation, and more specifically, the content ratio of rebaudioside A to stevioside and rebaudioside C is improved. No increase was known at all.

[0013]

SUMMARY OF THE INVENTION Accordingly, the present invention provides a method for producing stevia that improves the taste of conventional stevia extract, and a method for producing a sweetener that efficiently obtains a sweetener with improved taste. The task is to

[0014]

As described above, the improvement of the taste quality of stevia sweetener is to increase the mellow taste and suppress the unpleasant aftertaste accompanied by garnish. In order to obtain efficiently, it was thought that the content of rebaudioside A in the stevia leaves should be increased to produce a stevia plant in which the content was relatively increased relative to stevioside or rebaudioside C.

As a result of intensive studies, the present inventors have found that in order to obtain such a plant, radiation, in particular, gamma rays, of Stevia plants grown indoors and outdoors or Stevia cells grown in culture. Irradiating the tissue at various doses induces mutations in the genetic trait for Stevia rebaudioside A, and is genetically stable and rebaudioside relative to stevioside and / or rebaudioside C. It has been found that stevia having a high content of side A can be efficiently produced, and the present invention has been completed.

That is, the present invention provides: (1) a method of producing stevia with improved taste, characterized by irradiating radiation;

(2) The method for producing stevia according to (1), wherein the radiation is gamma rays.

(3) The irradiation dose of radiation is 30 to 900
The method for producing stevia according to (1) or (2), wherein the method is gray (Gy).

(4) Stevia according to any one of (1) to (3), wherein the improvement of taste quality increases the content ratio of rebaudioside A to stevioside to 3.0 or more. How to create

(5) The method according to any one of (1) to (4), wherein the content ratio of rebaudioside A to the sum of stevioside and rebaudioside C is 5.0 or more. How to make stevia,

(6) The content ratio of rebaudioside A to the sum of stevioside and rebaudioside C is 5 using the stevia plant or the dried leaf according to any one of (1) to (5). 0.0 or more stevia sweeteners,

(7) The content of rebaudioside A to the sum of stevioside and rebaudioside C is 5 using the stevia plant or the dried leaf according to any one of (1) to (5). And a method for producing a stevia sweetener of 0.0 or more.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the improvement of the taste quality of stevia is to increase the mellow taste similar to sugar contained in a so-called stevia sweetener obtained from plant stevia, and to increase the taste derived from stevioside. It means to suppress an unpleasant aftertaste accompanied by a peculiar harshness in which a bitter taste and astringency derived from rebaudioside C are mixed.

More specifically, in stevia leaves,
The content ratio (in terms of dry weight) of rebaudioside A to stevioside is 3.0 or more, preferably 3.0 to 3.0.
10, more preferably 7.0 to 9.0, or a rebaudioside A content ratio (in terms of dry weight) to the sum of stevioside and rebaudioside C of 5.0 or more, preferably 5.0 to 8 0.0, more preferably 5.0 to 6.0.
It means to increase to zero.

As the stevia to be subjected to the radiation irradiation of the present invention, those in a state such as those cultivated in soil and the like and those cultured in tissue can be used. Tissues with high cell division activity are more susceptible to gamma rays and are more advantageous.When using those grown in soil, those from the growing part of the aerial part to the flowering period are preferable. Also when using the thing, the thing of the time when it is actively growing and growing is preferable.

When a plant cultivated in soil or the like is used, it is preferable to use a plant that has been sterilized and washed.

In practicing the present invention, first, the above-mentioned stevia is irradiated with radiation. Examples of the radiation include alpha rays, beta rays, gamma rays, and x-rays, and preferably include gamma rays that have a high transmission power and do not activate the irradiation target.

Irradiation is performed on plants or cultures. Irradiation dose is 30 to 900 gray (G
The range of y) is preferable, and the range of 30 to 600 Gy is more preferable. When the irradiation dose falls below this range, ie, 30 Gy, the probability of mutation of the genetic trait for rebaudioside A becomes very low.

At 900 Gy or more, mutations of genes other than rebaudioside A are severe, and mutations occur in genes required to maintain Stevia plants at a minimum. Is not preferred.

The irradiation dose (dose rate) per unit time and the irradiation time are 0.1 to 500 Gy / day and 0.1 to 2,000.
Days, preferably 0.4 to 13 at 0.1 to 300 Gy / day
Although it is 00 days, it is most preferable to irradiate a small irradiation dose for a long period of time, and it is 300 to 0.1 to 0.7 Gy / day.
It is most desirable to do 1300 days.

When the concentration is less than 0.1 Gy / day, the probability of mutation of a gene related to an increase in rebaudioside A becomes extremely low, which is not preferable. However, when the rate is 500 Gy / day or more, mutation of genes other than rebaudioside A becomes remarkable, and radiation damage is caused, which is not preferable.

The combination of the dose rate and the irradiation time is arbitrary, but the irradiation time is shortened when the dose rate is high, and the irradiation time is lengthened when the dose rate is low, so that the irradiation dose ranges from 30 to 900 Gy. So that

As a gamma ray source, for example, cobalt 6
0 ⁽⁶⁰ Co), it is possible to use cesium 137 ^{(13 7} Cs) and the like.

Immediately after irradiating Stevia, the irradiated tissue is a so-called chimera composed of variously mutated cells. It is preferable to eliminate the chimera by cutting back, or inducing callus or multiple buds in tissue culture and redifferentiating them into plants, and it is possible to easily handle a large amount of tissue, Therefore, it is more preferable to use a tissue culture system because of its high yield.

Stevia calli and polyblasts can be obtained by a general plant tissue culture method. Specifically, the stem apex, axillary buds, leaves, etc. of Stevia plants are 70% ethanol,
After sterilization using sodium hypochlorite, etc., wash well and place on a medium. When using the culture, the sterilization operation can be omitted. The medium used at this time is Murashige
Examples include a Skoog (MS) medium, a white medium, a Rinsmeier-Skoog medium, and a Gamborg B5 medium, and the salts of each medium may be appropriately diluted.

A solidifying agent such as agar and gellan gum, and auxins and / or cytokinins can be added to any of these media. Examples of the auxin include naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy acetic acid (2,4-D), and are generally 0.01 to 10 mg / l, preferably 0.1 to 1.0 mg. / L range. As cytokinins, 0.01 to 10 mg of 6-benzyladenine (BA), kinetin, thidiazuron (TDZ), etc.
/ L, preferably in the range of 0.1 to 5.0 mg / l.

The callus and multiple buds thus obtained can be subcultured and propagated in the same medium, and can be regenerated individuals for the purpose of obtaining seedlings for planting in a field.
To obtain a regenerated individual, a medium containing cytokinins more than auxins was used,
1 mg / l, 0.01 mg / l of cytokinins
Callus or multi-blasts are transplanted to a medium supplemented with 0.2 mg / l. By culturing this for 2 to 3 weeks, a rooted regenerated individual is obtained, and by acclimating it with care for drying, it is possible to obtain a seedling that can be planted in a flowerpot or a field.

Cultivation is carried out by planting seedlings grown by the above-described method on soil that has been previously disinfected and fertilized. When cultivating outdoors, it must be carried out at a time when there is no frost.

By cultivating the mutant population thus cultivated, the content ratio of rebaudioside A to stevioside (in terms of dry weight) is 3.0 to 8.0,
Alternatively, it is possible to obtain a stevia plant in which the content ratio (in terms of dry weight) of rebaudioside A to the sum of stevioside and rebaudioside C is 5.0 to 8.0. The upper limits of the ranges of these content ratios are not particularly limited, and may be slightly affected by the content ratio of the mother book used.

The obtained stevia plant or dried leaf is
Extract with water or aqueous alcohol, use the extract as it is, remove any ionic impurities with various ion-exchange columns as necessary, further adsorb to an adsorption resin column, wash, dissolve after washing, etc. And can be used as a sweetener.

These can be used as a concentrated liquid or powder by using a known and commonly used concentration method or drying method. The content of rebaudioside A in the obtained stevia sweetener is desirably as high as possible, and there is no particular upper limit. However, in practice, those obtained by extraction and purification from stevia are generally 3.0 to 1 with respect to stevioside.
2. It is up to about 3.0 to 8.0 with respect to the sum of stevioside and rebaudioside C.

Further, α is added to recrystallization and stevioside.
It is also possible to increase the content of rebaudioside A by a known and commonly used technique such as a glycosyltransferase such as a glucosyltransferase.

According to the present invention, the content ratio of rebaudioside A to stevioside and / or rebaudioside C in stevia leaves is relatively increased, the mellow taste is increased, and unpleasantness accompanied by harshness is caused. It is possible to obtain a stevia sweetener having an improved taste with a reduced aftertaste. In particular, since the content ratio of rebaudioside A contained in the plant stevia is relatively high, on the industrial production of a stevia sweetener, extraction of rebaudioside A becomes easy, and the cost of the production process is reduced. be able to.

[0044]

Next, the present invention will be described with reference to examples, but is not limited to the scope of the examples. The following percentages are based on weight.

(Example 1) The top buds, axillary buds and buds of a Stevia plant cultivated in a greenhouse were collected and collected with 70% ethanol for 1 minute and then with a sodium hypochlorite solution containing Tween 20 at 0.05%. After sterilizing for 3 minutes, sucrose, 3%, NA
A, containing 0.2 mg / l and BA, 0.2 mg / l, and / or TDZ, 0.2 mg / l, and having a pH of 5.
8, and the cells were inoculated on an MS medium in which the salt concentration was 1/3 of the usual formulation. When this was cultured at 25 ° C. for 3 weeks, it differentiated into multiple buds. The multiple shoots were irradiated with a gamma ray using ⁶⁰ Co as a radiation source at 120 Gy and a dose rate of 20 Gy / hour in the incubator. This equipment can be used at the Agricultural and Biological Resources Research Institute, Ministry of Agriculture, Forestry and Fisheries, radiation breeding station.

The buds after irradiation were sucrose, 3%, B
A, 0.2 mg / l and / or TDZ, 0.2 mg
The medium was replaced with a 1/3 salt strength MS medium containing / l to differentiate into individuals. The obtained individuals were planted in vermiculite, rooted and acclimated to the open air, then potted, transplanted into a vinyl flowerpot filled with soil and having a diameter of 8 cm, and used as a seedling to be planted in soil. The 29 strains of Stevia seedlings thus grown were disinfected in advance with methyl bromide, and placed on an outdoor field where fertilizer was applied with 408 (30 kg / 10a), which was exclusively used for compounding fertilizers, and mixed granular mica carbonate (30 kg / 10a). Planting was carried out at 30 cm between plants.

On the way, the cultivation was carried out while cultivating the roots of the roots appropriately. In early August, an upper portion of about 15 cm was cut off from the roots of the roots, dried at 70 ° C. for 24 hours with a hot air drier, and only leaves were harvested from the plants. . The harvested dried leaves are each 100
g was extracted twice with 20 times the volume of hot water (50 ° C.), and then adsorbed resin Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei Corporation)
The solution was passed through a 400 mL column to adsorb sweet components.
This was sufficiently washed with water to elute impurities, and then desorbed with 80% methanol.

After recovering methanol under reduced pressure, a cation exchange resin column (IR-120B: trade name, manufactured by Organo, 100 mL), an anion exchange resin column (IR
A-93: trade name (manufactured by Organo Corporation, 100 mL), decolorized and concentrated under reduced pressure to obtain a dry powder. These were analyzed and excellent line 1 and excellent line 2 having excellent sweetness component composition were selected. Stevioside of each dried leaf (shown as Stv in the table), rebaudioside A (shown as Reb-A in the table), rebaudioside C (shown as R in the table)
eb-C. ) Is shown in Table 1, and the analytical values of the dry powder (sweetener) obtained from the superior line are shown in Table 2. Excellent system 1
Obtained 12.9 g of dry powder as a sweetener, and 12.6 g of dry powder from Superior Line 2.

[0049]

[Table 1]

[0050]

[Table 2]

The method of analyzing the components is as follows. Analysis method: High performance liquid chromatography Column used: Lichrosolve NH ₂ 10μ 4φ250mm Solvent: acetonitrile: water = 80: 20 (volume ratio) Flow rate: 2 mL / min

Example 2 A commercially available potting medium was filled in a vinyl flowerpot having a diameter of about 20 cm, and two stevia plants were prepared. Two gamma rays having ¹³⁷ Cs as a radiation source were prepared. Irradiation was performed at a dose of 252 Gy in a glass chamber capable of irradiation at a rate of 0.5 Gy / day for 42 months.

The apical buds, axillary buds and buds were collected from the irradiated Stevia plant, sterilized with 70% ethanol for 1 minute, and then sterilized with a sodium hypochlorite solution containing 0.05% Tween 20 for 5 minutes. Sucrose, 3%, NAA, 0.2
Inoculated on MS medium containing mg / l and BA, 0.2 mg / l, and / or TDZ, 0.2 mg / l, pH adjusted to 5.8 and salt concentration 1/3 of normal formulation Was.

When this was cultured at 25 ° C. for 3 weeks, it regenerated into multiblasts. The redifferentiated buds were sucrose, 3%, B
A, 0.2 mg / l and / or TDZ, 0.2 mg
The medium was replaced with a 1/3 salt strength MS medium containing / l to differentiate into individuals. The obtained individuals were planted in vermiculite, rooted and acclimated to the open air, then potted, transplanted into a vinyl flowerpot filled with soil and having a diameter of 8 cm, and used as a seedling to be planted in soil.

The 20 strains of Stevia seedlings thus grown were disinfected in advance with methyl bromide, and fertilized with 408 (30 kg / 10a), a chemical fertilizer dedicated to mixed vegetables and 30 kg / 10a, mixed granular carbonated lime (30 kg / 10a). The plants were planted in an outdoor field at a distance between plants of 30 cm. On the way, the cultivation was carried out while cultivating the roots of the roots appropriately. In early August, an upper portion of about 15 cm was cut off from the roots of the roots, dried at 70 ° C. for 24 hours with a hot air drier, and only leaves were harvested from the plants.

The harvested dried leaves are each independently 100
g was extracted twice with 20 times the volume of hot water (50 ° C.), and then adsorbed resin Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei Corporation)
The solution was passed through a 400 mL column to adsorb sweet components.
This was sufficiently washed with water to elute impurities, and then desorbed with 80% methanol.

After recovering methanol under reduced pressure, a cation exchange resin column (IR-120B: trade name, manufactured by Organo Corporation, 100 mL), an anion exchange resin column (IR
A-93: trade name (manufactured by Organo Corporation, 100 mL), decolorized and concentrated under reduced pressure to obtain a dry powder. These were analyzed and excellent line 3 having an excellent sweetness component composition was selected. Table 3 shows the composition of the dried leaves of the excellent line 3 and Table 4 shows the analysis values of the dry powder (sweetener) obtained from the excellent line. From superior line 3, 13.9 g of dry powder was obtained as a sweetener.

[0058]

[Table 3]

[0059]

[Table 4]

The method of analyzing the components is as follows. Analysis method: High performance liquid chromatography Column used: Lichrosolve NH ₂ 10μ 4φ250mm Solvent: acetonitrile: water = 80: 20 (volume ratio) Flow rate: 2 mL / min

(Comparative Example 1) Stevia native varieties were cut, rooted ears were picked up, and soil was filled to a diameter of 8 cm.
The seedlings were transplanted into a vinyl flower pot and planted in soil. The seedling thus grown is disinfected in advance with methyl bromide, and 408 (30
kg / 10a) and intermeshing granular carbonated mica lime (30k
g / 10a) was planted in an outdoor field to which fertilization was applied at a distance of 30 cm between plants. On the way, cultivate while cultivating soil at the roots of the roots, cut off about 15 cm from the roots of the roots at the beginning of August, and use a hot air drier.
After drying at 0 ° C. for 24 hours, only the leaves were harvested from the plants.

Each of the harvested dried leaves is independently 100
g was extracted twice with 20 times the volume of hot water (50 ° C.), and then adsorbed resin Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei Corporation)
The solution was passed through a 400 mL column to adsorb sweet components.
This was sufficiently washed with water to elute impurities, and then desorbed with 80% methanol.

After recovering methanol under reduced pressure, a cation exchange resin column (IR-120B: trade name, manufactured by Organo, 100 mL), an anion exchange resin column (IR
A-93: trade name, manufactured by Organo Corporation, 100 mL), decolorized and concentrated under reduced pressure to obtain 19.1 g of dry powder. Show.

[0064]

[Table 5]

[0065]

[Table 6]

The method of analyzing the components is as follows. Analysis method: High performance liquid chromatography Column used: Lichrosolve NH ₂ 10μ 4φ250mm Solvent: acetonitrile: water = 80: 20 (volume ratio) Flow rate: 2 mL / min

(Test Example 1) Panel Test Ten panelists evaluated the taste quality of the stevia sweeteners obtained in Examples 1 and 2 and Comparative Example 1 with respect to the fore taste such as mellowness and the aftertaste such as harshness. Was determined by a sensory test according to Evaluation method: good taste: +3, slightly better: +
A score of 2, normal: +1 and bad: 0 was used and quantified according to the following equation.

Evaluation point = (3 × N3 + 2 × N2 + 1 × N1
+ 0 × N0) ÷ N, where N: the number of panelists, N3: the number of persons determined to be +3, N2: the number of persons determined to be +2, N1: +1
And the number of persons determined to be N0: 0.

[0069]

[Table 7]

[0070]

According to the present invention, a stevia plant having improved taste quality by relatively increasing the ratio of rebaudioside A to stevioside and / or rebaudioside C in stevia leaves, and An improved stevia sweetener having improved taste obtained from the plant can be obtained.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mika Takahashi 1-27-1, Osakidai, Sakura City, Chiba Prefecture (72) Inventor Taku Fujii 6-5-7 Takahama, Mihama-ku, Chiba City, Chiba Prefecture (72) Inventor Honami 7-13-19 Maehara Nishi, Funabashi-shi, Chiba Pref. (72) Inventor Masato Kitazume 6-37-2 Asumigaoka Midori-ku, Chiba-shi, Chiba F-term (reference) 4B047 LB08 LB09 LG32 LP20

Claims (7)

[Claims] 1. A method for producing stevia with improved taste, comprising irradiating radiation. 2. The method according to claim 1, wherein the radiation is a gamma ray. 3. The method for producing stevia according to claim 1, wherein the irradiation dose of the radiation is 30 to 900 gray (Gy). 4. The production of stevia according to claim 1, wherein the improvement of taste quality increases the content ratio of rebaudioside A to stevioside to 3.0 or more. Method. 5. Stevioside and rebaudioside C
Of rebaudioside A relative to the sum of
The method for producing stevia according to any one of claims 1 to 4, wherein: 6. The content ratio of rebaudioside A to the sum of stevioside and rebaudioside C of 5.0 using the stevia plant or the dried leaf according to any one of claims 1 to 5, is 5.0. Stevia sweetener over. 7. The content ratio of rebaudioside A to the sum of stevioside and rebaudioside C of 5.0 using the stevia plant or dried leaf according to any one of claims 1 to 5, is 5.0. The above-mentioned method for producing a stevia sweetener.