JP2002262822A - Sweetener obtained from plant body of variety of stevia rebaudiana cultivatable from seed - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sweetener prepared from dried leaves of a plant body belonging to a variety of Stevia Rebaudiana Bertoni cultivatable from seed and to provide a method for sweetener production. SOLUTION: This sweetener is produced from dried leaves of a plant belonging a variety of Stevia Rebaudiana Bertoni capable of growing leaves containing rebaudioside A of >=2.56 times as much as stevioside. This method for producing the sweetener is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a plant cultivated from a seed of a plant belonging to the Stevia rebaudiana Bertney variety capable of growing seeds capable of growing leaves exhibiting a high rebaudioside A content. Sweetener and a method for producing the sweetener.

[0002]

BACKGROUND OF THE INVENTION Stevia is a perennial plant of the Chrysanthemum family originating in Paraguay, South America, and its scientific name is Stevia rebaudiana Bertney (St.
evia Rebaudiana Bertoni). Since stevia contains a sweet component having a sweetness 300 times or more that of sugar, stevia is cultivated to extract the sweet component and use it as a natural sweetener.

[0003] Stevia sweeteners include stevioside (C ₃₈ H ₆₀ O ₁₈ , molecular weight 804), rebaudioside A (C ₄₄ H ₇₀ O ₂₃ , molecular weight 966), rebaudioside C, D, E, Zurcoside A and the like are known. Stevia varieties generally cultivated contain stevioside (ST) as the main component in the above sweetness components, and the content of rebaudioside A (RA) is about 3/10 to 4 of stevioside, and the content of rebaudioside C Is slightly less than that, but there are various types such as those not containing rebaudiosides A and C, and those containing rebaudioside C as a main component, depending on the variety.

Among the tastes perceived by the tongue such as astringency and spiciness, the quality of sweetness is very subtle. Stevioside has a sweetness 300 times that of sugar and is therefore used as a natural sweetener in the food industry. Its sweetness is relatively similar to sugar, but has the disadvantage that unpleasant tastes such as bitterness remain in the aftertaste.
Therefore, including a large amount of stevioside is not preferable as a sweetener. On the other hand, rebaudioside A has a sweetness of good quality and 1.3 times that of stevioside.
It has 1.5 times the sweetness.

[0005] The inventors of the present invention repeated breeding and selection from conventional varieties to improve varieties, and obtained stevia varieties having a high ratio of rebaudioside A (RA) to stevioside (ST). A sweet component is extracted to produce an excellent sweetener having a high content ratio of rebaudioside A to stevioside (JP-A-59-045).
848 and JP-A-61-202667).

However, plants cultivated from seeds of these varieties have a reduced content of rebaudioside A.
For example, a plant cultivated from a seed of a variety having a sweetness component ratio of ST: RA = 1: 4 has a component ratio of about ST: RA = 1: 1 to 1.5. Therefore, in order to maintain a high content ratio of rebaudioside A, it was necessary to rely on cutting seedlings.

In addition, Stevia rebaudiana Bertney is self-incompatible and harvested seeds do not always germinate. That is, the germination rate is very low.
For these reasons, stevia containing a high proportion of rebaudioside A has been propagated in cuttings, and those skilled in the art did not consider cultivating with seeds.

[0008] However, the cutting seedlings need to secure a large amount of the mother tree temporarily, because the number of cuttings obtained from the mother tree is limited.
Has the disadvantage that mass seedling raising is difficult. The present inventors have enthusiastically developed a variety of Stevia genus capable of seed cultivation while maintaining a high content ratio of rebaudioside A to stevioside in order to improve these disadvantages. .

[0009]

Means for Solving the Problems Then, Stevia rebaudiana Berney, which has a gene containing rebaudioside A in a high ratio and can transmit this gene to the next generation by seed propagation, is selected and fixed. Thus, the present invention has been completed.

[0010] The leaves of a plant cultivated from seeds for cultivation of a plant belonging to the Stevia rebaudiana Bertney variety capable of growing leaves containing rebaudioside A at least 2.56 times the amount of stevioside are used to produce a sweetener. Used for

A first aspect of the present invention is to extract dried leaves of a plant belonging to the cultivar Stevia rebaudiana Bertney, which is capable of cultivating seeds, with water or a water-containing solvent, and separating and collecting a sweet component from the obtained extract. A sweetener characterized by a rebaudioside A content of at least 2.56 times that of stevioside.

A second aspect of the present invention is characterized in that the dried leaves of the plant belonging to Stevia rebaudiana Bertney are extracted with water or a water-containing solvent, and a sweet component is separated and collected from the obtained extract. This is a method for producing a sweetener containing rebaudioside A that is at least 2.56 times stevioside.

The plants belonging to the Stevia rebaudiana Bertney variety according to the present invention were grown according to the protocol shown in Table 1 below. The parent plant, SF
The seeds of the three varieties are owned by the inventor, and are guaranteed for sale in accordance with the provisions of Article 27-3 of the Patent Act Enforcement Regulations.

[Table 1]

Cultivation of plants belonging to the Stevia rebaudiana Bertney variety of the present invention
No. 2667, a strain containing rebaudioside A 2.56 times or more the seed obtained by crossing between SF3 (S
(F3 class) and repeated mating and selection.
As a result of crossing only with a strain (SF3 grade) containing twice or more rebaudioside A, strain SF5-1 (No. 103) having excellent disease resistance to Septoria and strain SF5-2 having excellent sweetness component content No. (No. 109) was selected. 1
By cultivating using the seeds obtained from No. 03,109, 80% or more of the strains containing rebaudioside A 2.56 times or more of stevioside were obtained, and were almost stable. In addition, although SF3 was crossed with No. 103 and No. 109, a strain having a stable component ratio was not obtained by cultivation using seeds fruiting on SF3.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Stevia cultivation method It is better to prepare a seedbed, put fine soil or sand well drained in the lower part, and add a small amount of fertilizer. A finer soil of about 10 mesh or a vermiculite of SS size is layered on the upper part, and then sufficiently sprinkled and moistened. The temperature of the sowing bed is kept at 20 ° C. to 25 ° C. before sowing. The seeds may be in a state with a crown, but it is preferable to remove the crown in order to enhance the adhesion to the soil.

After soaking the seeds in water all day and night, in order to enhance the dispersibility, the soil is as fine as possible about 10 mesh or SS size vermiculite (Asahi Kogyo)
And mix it with soup. After sowing, the seeds are settled by dispersing enough water to cover the seeds, then covered with cold water to avoid direct sunlight, keeping the surface humidity and germinating. It takes 7-14 days to germinate, but may take longer if germination is slow or the temperature is low.

Careful attention must be paid to temperature control and moisture control until germination is completed. After germination, gradually expose to sunlight and apply diluted liquid fertilizer as needed. In the case of temporary planting, the seedlings are temporarily planted when they reach about 2 to 3 cm. When a seedling pot, a seedling tray, or the like is used as the seeding bed, it is not necessary to plant the plantings temporarily.

When the seedlings have grown to about 10 cm, they are transplanted to a field. The field is preliminarily fertilized and ridges are formed according to the number of plants planted. The number of plants planted per hectare is preferably about 100,000 to 150,000, but may be increased or decreased as necessary.

The amount of fertilization varies depending on the fertility of the field, but as a rough guide, each component of nitrogen, potassium and potassium is about 200 kg per hectare. After transplanting, water well enough to help the root and soil adhere and reduce transplant pain. In low-latitude areas, flowers may bloom early in the growing period, but when they bud and flowering phenomena are observed, pinching is performed. During the growing period, weeding and moisture management are performed, topdressing is performed as necessary, and fungicides are sprayed during hot and humid periods.

After growing to about 30 cm or more in height, when buds and flowering are observed, the lower leaves are harvested while leaving 3 to 5 pairs. After harvesting, the leaves are separated or sun-dried or hot-air-dried in the form of grass. The leaf part is divided into a branch and a stem, and the leaf part is used as a sweetener material as dried leaves.

Sample Preparation 20 g of the dried leaves were extracted several times with 10 to 20 times the amount of water until the sweetness was no longer felt, and the extract was extracted with a column filled with 20 ml of a cation exchange resin (Amberlite IR-120B), 20 ml of ion exchange resin (Durite A-4)
Slowly pass through a column filled with water, pass the passing liquid through a column filled with 100 ml of adsorption resin (Amberlite XAD-2) to adsorb the sweet components, wash thoroughly with water, drain water and elute the sweet components with 300 ml of methanol. I do. The eluate is concentrated under reduced pressure and dried under reduced pressure to obtain a pale yellow powder.

Analysis method 1) High performance liquid chromatography method A sweetener powder is dissolved in an eluent and put into a column. Column packing Lichrosolve NH ₂ 5μ Flow rate 1.5 ml / min Eluent Acetonitrile: water = 80: 20 Measurement wavelength 210 nm

2) Thin-layer chromatography The sweetener powder is dissolved in water and placed on a thin-layer plate. Measurement model Shimadzu Chromatoscanner 910 Thin plate Merck silica gel 60F254 Developing solvent chloroform: methanol: water = 30: 20: 4 Color former 50% sulfuric acid Measurement method Reflective zigzag scanning method Measurement wavelength 350 nm Scan speed 20 mm / min

Production Example 1 Production and Analysis of SF4 At the end of February, 1994, a seedbed was prepared by laying a heating wire on a greenhouse in the Niimi Plant of Morita Chemical Industry Co., Ltd. The lower part was fine soil, sand and chemical fertilizer ( 2 g of Chisso, Rinsan and Kali 8% each)
On the upper part, a soil obtained by mixing soil having passed through a 10-mesh sieve and vermiculite of the SS size in a ratio of 1 to 1 was covered to a thickness of 3 cm, and then sufficiently sprinkled with water to make it moist.
C. After removing the hairs of the seeds collected from SF3 in 1993, the seeds were soaked in water for 24 hours, mixed with SS size vermiculite, and sown. After sowing, the seeds were settled by sufficiently dispersing water, and then covered with cold water to avoid direct sunlight, and the humidity and temperature of the sowing bed were maintained. 1 after sowing
Sunlight was gradually applied from the fourth day, and a liquid fertilizer diluted 500-fold was given on the third week of sowing.

When the seedlings became about 2 to 3 cm, the same seedling bed as above was placed in a nursery box, and 1500 seedlings were temporarily planted at an interval of 4 cm and kept warm in a vinyl tunnel. In early May when the seedlings grew to about 10 cm, they were transplanted to the field at intervals of 25 × 30 cm. In the field, make a 60cm wide ridge in advance and add
Fertilization was performed at 250 kg per 0 ares.

After transplanting, water was sufficiently sprinkled. During the growing period, weeding work and water management were performed, and in the middle of June, topdressing was performed at 40 kg per 10 ares. Fungicide daisen (Kumiai Chemical, MANSEN), orthoside every two weeks during rainy season
(Takeda Horticultural Materials Co., Ltd., CAPTAN) diluted 1000 times was sprayed.

After July 20, 900 well-growing plants were selected, the leaves were cut off leaving 5 pairs of lower leaves, and the leaves were separated.
The leaves were dried in the sun and the dried leaves were analyzed by thin-layer chromatography.

According to the thin layer chromatography method, 21% of the strains had a sweet component ratio of stevioside: rebaudioside A = 1 or less: 2.54 or more. Among them, stevioside: rebaudioside A = 1 Below: The ratio of the strains of 3 or more is 5.7%, and the average ratio of stevioside: rebaudioside A of the obtained dried leaves is 1:
It turned out to be 1.43.

Stevioside: Rebaudioside A =
1 or less: 4 of the strains having a sweetness component ratio of 2.54 or more
Five strains having the same growth and yield as SF3, which was cut in the middle of the month and transplanted in the same manner in early May, were selected and designated as SF4.

Production Example 2 Production and Analysis of SF5-1 and SF5-2 At the end of July 1994, five of the above SF4 strains were propagated by cuttings of 20 each, grown in a greenhouse, and crossed with SF4. After the beginning of November, 2000 seeds were obtained from SF4.

Seed 15 obtained from SF4 in 1995
00 seeds were sown and cultivated in the same manner as described above.
From 0 plants, 650 plants with good growth were selected from July 20 onward, and the lower leaves were cut off, the leaves were separated, the leaves were separated, and then dried in the sun. The dried leaves were analyzed by thin layer chromatography. .

Only those strains having a sweet component ratio of stevioside: rebaudioside A = 1 or less: 2.54 or more were selected by thin layer chromatography. The obtained strain was 39%. Among them, the ratio of stevioside: rebaudioside A = 1 or less: 3 or more strains was 6.5%, and the average stevioside of the obtained dried leaves was 6.5%. : The ratio of rebaudioside A was 1: 1.21. Stevioside: Rebaudioside A = 1 or less: Two strains having particularly excellent growth, yield and disease resistance were selected from among three or more strains, and SF5-1 (No. 103, disease resistance) and SF5-2 were selected.
(No. 109, yield of sweet component).

Production Example 3 Production and Analysis of SF6-1 SF5-1 and SF5-2 were cut from cuttings at the end of July.
Proliferate one by one and grow in a greenhouse.
After the beginning of January, SF5-1 × SF5-2 was crossed and seeds were collected.

The seed 6 obtained by sowing and cultivating the seed obtained from SF5-1 × SF5-2 in 1996 in the same manner as described above was obtained.
From September 00 to September 13, five pairs of lower leaves were cut off, the leaves were separated, dried in the sun, and the dried leaves were analyzed by thin-layer chromatography. 79% of the strains had the same sweetness ratio as SF3. Among them, Stevioside: Rebaudioside A = 1 or less: The ratio of strains of 3 or more is 65
% And the average stevioside of the resulting dried leaves is:
The ratio of rebaudioside A was 1: 3.

Production Example 4 Production and Analysis of SF6-2 SF5-1 and SF5-2 were cut from cuttings at the end of July using cuttings.
Proliferate one by one and grow in a greenhouse.
SF3x (SF5-1 and SF5-
2) was crossed and seeds were collected.

SF3 × (SF5-1 and SF5-2)
The obtained seeds were sown and cultivated in 1996 in the same manner as described above, and on September 13, five lower leaves were cut off from the 600 obtained strains, the leaves were separated, and then dried in the sun and dried. Leaves were analyzed by thin layer chromatography.

The strain having the same sweetness component ratio as SF3 is 4
9% was obtained. Among them, Stevioside: Rebaudioside A = 1 or less: The ratio of strains of 3 or more is 12%,
The average ratio of stevioside: rebaudioside A of the obtained dried leaves was 1: 2.17.

The production and analysis results of the plants of Production Examples 1 to 4 are summarized in Table 2.

[Table 2] The ratio in () is the ratio of stevioside: rebaudioside A (ST: RA), and the ratio in (average) is the ratio of ST: RA of the present hybrid plant cultivated in the field. is there.

Production Example 5 Dried leaves of 115 strains obtained from seeds (= SF6-I) which had set on SF5-1 strain SF5-1 × SF5-2, SF
Dried leaves (SF6-II) and SF3 of 131 strains obtained from seeds that settled on SF5-2 strain of 5-1 × SF5-2
× (SF5-1 and SF5-2), dried leaves of 99 strains obtained from seeds (= SF6-III) fruiting to the SF3 strain were each crushed and mixed, and 50 g of each was used as a sample to prepare a sample. The sweetness component content was measured by a photographic method.

The measurement results are as follows.

TABLE 3 STRA RC ST: RA SF6-I strain 3.6% 12.7% 1.6% 1: 3.53 SF6-II strain 4.1% 10.5% 1.5% 1: 2 .56 SF6-III strain 3.6% 7.8% 1.4% 1: 2.16

From the above results, SF5 has a genetic factor containing high rebaudioside A, and even when crossed with SF3 having a weak genetic factor containing high rebaudioside A, high rebaudioside A is obtained. Was found to be inherited predominantly. The average value of ST: RA of SF-6 obtained by crossing SF5-1 and SF5-2 is 1: 3.
Thus, a sweetener having a ratio of stevioside: rebaudioside A of 1: 2.54 or more can be produced only from a plant belonging to Stevia rebaudiana bertney obtained by seed cultivation. Even after SF6, varieties with ST: RA of 1: 2.54 or more are selected and the varieties can be fixed by selfing, that is, the obtained seeds inherit the same genetic factors.

The sweetener of the present invention can be produced in the same manner as described in the above sample preparation, and the yield from dried leaves is about 12 to 13%. The cation exchange resin and the anion exchange resin used in the present invention may be those which are generally known, and as the aqueous solvent, an aqueous solvent such as methanol or ethanol can be used.

The sweetener of the present invention can be used together with an appropriate amount of additives such as dextrin, starch, glucose and other commonly used diluents. The sweetener of the present invention can be added to foods, beverages, medicines, and the like in any amount.

Example 1 Production of a sweetener 40 g of dried leaves were extracted several times with 10 to 20 times the amount of water until the sweetness was no longer felt, and the extract was filled with 40 ml of a cation exchange resin (Amberlite IR-120B). Column and 40 ml of anion exchange resin (Durite A-4)
Slowly pass through a column filled with water, pass the passing liquid through a column filled with 200 ml of adsorption resin (Amberlite XAD-2) to adsorb sweet components, wash thoroughly with water, drain water and elute sweet components with 600 ml of methanol. I do. The eluate was concentrated under reduced pressure and dried under reduced pressure to obtain a pale yellow powder of 5.0.
g (12.5%).

Example 2 Preparation of a sweetener composition 5.0 g of the powder obtained in Example 1 and 5.0 g of dextrin
Was mixed to prepare a sweetener composition.

[0046]

According to the present invention, since the number of cuttings obtained from the mother tree is limited, it is necessary to secure a large amount of mother trees, complicated cutting and preparation of cuttings from cuttings, and At the same time, the disadvantages of cutting seedling raising, such as the difficulty of mass seedling raising, can be overcome by sowing the new plant of the present invention, which enables a significant reduction in cultivation work and costs.

Claims (2)

[Claims] 1. Stevia which can grow a leaf containing rebaudioside A at least 2.56 times that of stevioside.
Extracting dried leaves of a plant cultivated from cultivated seeds of a plant belonging to the Rebaudiana Bertney variety with water, or a water-containing solvent, and separating and collecting a sweet component from the obtained extract, to stevioside. 2.56
Sweetener containing rebaudioside A twice or more times. 2. Stevia, which is capable of growing leaves containing rebaudioside A in an amount of at least 2.56 times that of stevioside.
Extracting dried leaves of a plant cultivated from cultivated seeds of a plant belonging to the Rebaudiana Bertney variety with water, or a water-containing solvent, and separating and collecting a sweet component from the obtained extract, to stevioside. 2.56
A method for producing a sweetener containing rebaudioside A twice or more times.