JP3352860B2 - α-Glucosylated Stevia sweetener - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a stevia sweetener having excellent sweetness.

[0002]

2. Description of the Related Art Stevia is a perennial plant of the chrysanthemum family originating in Paraguay, South America, and its scientific name is Stevia 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. Stevioside (C ₃₈ H ₆₀ O ₁₈ , molecular weight 804) as a sweet component of stevia,
Rebaudioside A (C ₄₄ H ₇₀ O ₂₃ , molecular weight 966),
Rebaudiosides C, D, E, zurcoside A and the like are known. In stevia varieties that are generally cultivated, the content of rebaudioside A is about 3/10 to 4 of stevioside, and the content of rebaudioside C is slightly less than that of stevioside A, which is the main component of the above sweetness components, Some varieties do not contain rebaudiosides A and C, and others contain rebaudioside C as a main component.

[0003] 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. In contrast, rebaudioside A has a sweetness 1.3 to 1.5 times that of stevioside, and its sweetness is similar to sugar and does not leave a mellow and unpleasant taste, but it is only slightly present in stevia leaves. Only included
There is also a drawback that the body is not sharp and the taste is sharp.

[0004] In order to improve the taste quality of these sweeteners, particularly to improve the bitterness of stevia extract containing stevioside as a main component, α-added by adding α to glucose.
A sweetener mainly comprising glucosyl stevioside has been put into practical use (Japanese Patent Publication No. 57-18779). α-Glucosylation gives a rich sweet taste,
Glycosyl stevioside has a unique aftertaste different from stevioside with a decrease in sweetness. In order to improve these, there is a method of adding glucose to a stevia extract containing stevioside and then adjusting the sugar chain of glucose (Japanese Patent Application Laid-Open No. 2-163056). Stevioside is isolated from the extract by recrystallization, and α-addition of glucose is also available and commercially available. However, although this method slightly improves the unique aftertaste, it produces a mother liquor containing a sweet component after the isolation of stevioside, and this mother liquor cannot be used as a sweetener in terms of sweetness. , Which increases production costs.

[0005] It is impossible on an industrial scale to isolate rebaudioside A from a stevia extract containing stevioside as a main component, and even if rebaudioside A is isolated, recovery of rebaudioside A is possible. Crystallization causes an increase in cost with decreasing rate. For this reason, it has been difficult to industrialize α-glucosyl rebaudioside A obtained by α-adding glucose to rebaudioside A using cyclodextrin glycosyltransferase.

In addition, when an extract from a stevia plant or a dried leaf containing rebaudioside A that is 1.5 times or less of stevioside is subjected to α-addition of glucose using cyclodextrin glucosyltransferase, rebaudioside is added. The rate at which glucose α-adds to stevioside is faster than the rate at which glucose α-adds to side A. Therefore, in order to add glucose α to rebaudioside A, an excess enzyme, α-glucosyl saccharified product, and reaction time are required. In addition, an increase in the reaction time leads to the addition of a large amount of glucose to stevioside, which results in a decrease in sweetness. In order to prevent this, a large amount of unreacted rebaudioside A remained, and as a result, a sweetener containing α-glucosyl rebaudioside A as a main component could not be obtained.

[0007]

Therefore, the present inventors efficiently and inexpensively produce a high-sweetness natural sweetener having a sweetness similar to the above-mentioned sugar, having a kokumi, and a sharp aftertaste. As a result of intensive studies, the present invention has been completed by extracting a sweet component from a new variety of Stevia rebaudiana Bertney having an improved sweet component ratio.

[0008]

SUMMARY OF THE INVENTION We have worked to create an extract based on rebaudioside A that does not require the removal of stevioside, and have developed α-glucosyl rebau v. A sweetener containing 1.2 times or more of Dioside A was completed. This sweetener is obtained by extracting a plant or dried leaf of Stevia rebaudiana Bertney containing rebaudioside A at least 1.5 times the amount of stevioside with water or a water-containing solvent, and separating it from the obtained extract. It is obtained by adding glucose α to the sweet component using cyclodextrin glycosyltransferase. Furthermore, a sweetener in which an added sugar chain is regulated by allowing α-1,4-glucosidase to act on the above sweetener can be obtained.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A new variety of Stevia rebaudiana Bertney containing rebaudioside A at least 1.5 times that of stevioside (Japanese Patent Laid-Open No. 60-160)
No. 823, JP-A-63-173531).
These plants or dried leaves are extracted with water or a water-containing solvent, and glucose is α-added to the sweet component (rebaudio extract) separated from the resulting extract (hereinafter referred to as stevia extract) using cyclodextrin glycosyltransferase. Thus, a sweetener containing at least 1.2 times α-glucosyl rebaudioside A with respect to α-glucosyl stevioside is obtained. Furthermore, a sweetness containing α-glucosyl rebaudioside A at least 1.2 times that of α-glucosyl stevioside obtained by reacting α-1.4-glucosidase on an α-glucosylated product and controlling an added sugar chain. Get the fee.

The breeding process of a new varieties of stevia for obtaining the rebaudio extract used in the present invention is as follows, but is not limited to this breeding process, and is 1.5 times or more to stevioside. Any breeding method and cultivation method can be used to obtain stevia containing rebaudioside A.

[0011] From October to December 1979, a stevia native variety S having a rebaudioside A content of 6/10 of stevioside was obtained from Morita Chemical Industry Co., Ltd. in Ashimi, Niimi City, Okayama Prefecture. The seeds obtained are artificially crossed at the same time, and the obtained seeds are sown in the nursery greenhouse at the same place in early March, 1980.
The seedlings germinated and grown in early May of the same year were transplanted to the field, and
The sweet component content was investigated at the beginning of the month, and seedlings containing at least 1: 1 rebaudioside A with respect to stevioside were selected and designated as SF1.

[0012] SF1 was propagated in cuttings, and 10-12
The artificial seeds were crossed in a plastic greenhouse in May, and the obtained seeds were sown in a nursery plastic greenhouse in February 1981, and the seedlings that germinated and grew in early May of the same year were transplanted to a field, and in early August of the same year The sweet component content was investigated, and seedlings containing rebaudioside A at a ratio of 1: 1.5 or more with respect to stevioside were selected and designated as SF2.

[0013] SF2 was similarly propagated in cuttings, and
The artificial seeds were crossed artificially in a plastic greenhouse from December to December, and the obtained seeds were sown in a nursery plastic greenhouse in February 1981, and the seedlings that had germinated and grown in early May of the same year were transplanted to a field, and in August of the same year The sweetness component content was investigated in the early stage, and seedlings containing rebaudioside A at a ratio of 1: 2.56 or more to stevioside were selected and designated as SF3.

The seedlings obtained in SF2 and SF3 were each propagated in 100 cuttings, and the content of the sweetness component in the dried leaves was examined. The dried leaves A (Stevioside 3.6)
%, Rebaudioside A5.6%, rebaudioside C1.1%), dried leaves B (stebioside 2.7%, rebaudioside A7.1%, rebaudioside C1.1)
%) And dried leaves C (Stevioside 1.0%, Rebaudioside A 9.1%, Rebaudioside C 0.9%).

The Stevia plant or its dried leaves are extracted with water or a hydrophilic solvent. Next, the extract is subjected to removal of ionic impurities with a cation exchange resin or an anion exchange resin, if necessary, and then the sweet component is adsorbed on the adsorption resin and eluted with a hydrophilic solvent to concentrate the eluate. Alternatively, conventional purification means such as adsorption to an adsorption resin, elution with a hydrophilic solvent, concentration of the eluate, dilution with water again, and treatment with an ion exchange resin can be appropriately performed.

The α-glucosyl rebaudioside A used in the present invention is obtained by α-adding glucose to a rebaudio extract using cyclodextrin glycosyltransferase. Further, α-glucosyl rebaudioside A in which the added sugar chain is regulated can be obtained by reacting α-glucosyl rebaudioside A obtained above with α-1.4 glucosidase.

The cyclodextrin glucosyltransferase used in the present invention is Bacillus macerans
(Bacillus macerans), Bacillus megaterium (Baci
llusmegaterium), Bacillus stearothermophilus
(Bacillus stearothermophilus). α-1.4 glucosidase has α-
There are α-amylase that randomly cleaves the sugar chain of the 1.4 glucosyl sugar compound, β-amylase that cleaves the non-reducing terminal in maltose units, and glucoamylase that cleaves the non-reducing terminal in glucose units. The transfer reaction between cyclodextrin glucosyltransferase and α-1.4 glucosidase is carried out at pH 4 to 7, preferably at pH 4.
5-6, reaction temperature is 20-80 ° C, preferably 40-70
The temperature is good and the amount of the enzyme is not limited, but an appropriate amount may be added in consideration of the reaction time. Other sweeteners, diluents and the like can be added to the sweetener obtained according to the present invention.

The sweetener obtained according to the present invention has less sweetness linger than α-glucosyl stevioside obtained from an extract containing stevioside as a main component. And the sweetness was excellent. In addition, the sweetener of the present invention uses a rebaudioside A obtained by crystallization from a stevia extract instead of a rebaudio extract to produce α-glucosyl rebaudioside A as compared with a case where α-glucosyl rebaudioside A is produced. This is advantageous in that crystallization costs required for isolating the vaudioside A and an increase in production costs due to a decrease in the recovery rate can be avoided.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1 Rebaudio extract A 40 g of dried stevia leaves A (3.6% stevioside, 5.6% rebaudioside A, 1.1% rebaudioside C) are sweetened with 10 to 20 times the amount of water. The extract was extracted several times until no more was obtained, and the extract was packed with a column filled with 200 ml of a cation exchange resin (Amberlite IR-120B) and an anion exchange resin (Amberlite IRA-94) 2.
Pass through a column filled with 00 ml, and filter the passing solution through a synthetic adsorption resin.
(Diaion HP-20) The sweet component is adsorbed through a column filled with 200 ml, washed thoroughly with water, and methanol 40
Elute at 0 ml. The eluate is concentrated under reduced pressure and dried to give a pale yellow powder. The results of the analysis are shown in the table below.

Example 2 Rebaudio extract B In place of dried stevia leaf A in Example 1, dried leaf B
(2.7% stevioside, 7.1% rebaudioside
%, Rebaudioside C1.1%) was used in the same manner as in Example 1. The results are shown in the table below.

Example 3 Rebaudio extract C In place of dried stevia leaf A in Example 1, dried leaf C
(Stevioside 1.0%, Rebaudioside A9.1
%, Rebaudioside C0.9%) was used in the same manner as in Example 1. The results are shown in the table below.

[0022] ST: Stevioside RA: Rebaudioside

Example 4 α-Glucosylation Each of the rebaudio extract A ~ obtained in the above Examples 1-3
C: 4 g each and DE (starch decomposition rate) as α-glucosyl saccharified product; 8 g of dextrin of 10 were dissolved in 20 ml of water by heating, cooled to 70 ° C., and calcium chloride was added to 1 mmol based on the total amount of the substrate. At the same time, the pH was adjusted to 6.0, 100 units of cyclodextrin glucosyltransferase was added, and the mixture was reacted at a temperature of 70 ° C. for 24 hours. Each reaction solution was kept at 95 ° C. for 30 minutes to inactivate the enzyme.

Each reaction solution was divided into two equal parts, and the reaction solutions were filtered to remove suspended solids. Each filtrate was passed through a column filled with 100 ml of synthetic adsorption resin DIAION HP-20 and thoroughly washed with water. Then elute with 300 ml of methanol. The eluent was a cation exchange resin (Amberlite IR-120)
B), anion exchange resin (Amberlite IRA-94)
And subjected to desalting and decoloring, and then concentrated and dried under reduced pressure to obtain white powders α-glucosylated sweeteners A-a, Ba-a and Ca-a.

Example 5 Modulation of Additional Sugar Chain One half of each other was prepared using commercially available glucoamylase (glucozyme).
1.0% based on the solid content was added thereto, and reacted at a temperature of 50 ° C. for 5 hours. After the reaction, the enzyme was inactivated by heating to 95 ° C. for 30 minutes. Each reaction solution was filtered to remove suspended matter, and then each filtrate was subjected to synthesis adsorption resin Diaion HP.
-20 was passed through a column packed with 100 ml, washed thoroughly with water and eluted with 300 ml of methanol. The eluent is passed through a column packed with a cation exchange resin (Amberlite IR-120B) and an anion exchange resin (Amberlite IRA-94), desalted and decolorized, and then concentrated and dried under reduced pressure. The obtained white powders were designated as added sugar chain-regulated α-glucosylated sweeteners Ab, Bb and Cb.

The α-glucosylated sweeteners Aa, Ba and Ca obtained in Example 4 and the three types of additional sugar chain-regulated α-saccharides obtained in Example 5 in which the sugar chains are further controlled. The yields of glucosylated sweeteners Ab, Bb and Cb are shown in Tables 2 and 3 below. Table 2 A-a 2.79 g B-a 2.80 g C-a 2.75 g A-b 2.60 g B-b 2.55 g C-b 2.60 g

A-a and A- obtained in Examples 4 and 5
b, Bb, and Cb were subjected to thin-layer chromatography. Measurement conditions Thin plate HPTLC Fertingplatten kieselgel 60F 254 (manufactured by MELK) Developing solvent chloroform: methanol: water = 20: 30: 8 Color former 50% sulfuric acid The thin plate that has been colored is Shimadzu Chromatoscanner 91
Type 0 (reflective zigzag scanning method, manufactured by Shimadzu Corporation),
The reading was performed at a measurement wavelength of 350 nm. A-a, Ab, B
FIGS. 1 to 4 show the thin-layer chromatograms of -b and Cb read by a chromato-scanner.

Further, Cb obtained in Example 5 was subjected to high performance liquid chromatography. Measurement conditions Column Shodex RS pak DC-613 Eluent Acetonitrile: water = 84: 16 → 50: 50 Linear gradient Flow rate 1 ml / min Column temperature 40 ° C. Measurement wavelength UV 210 nm The high-performance liquid chromatogram of Cb is shown in FIG.

[0029]

[Table 3] Analytical values A-a Ab-B-a B-b C-a C-b ST 1.2 1.2 0.6 0.6 0.5 0.5 RA 12.2 12.2 1.5 1.5 2.2 2.2 STG1 3.0 4.5 1.5 2.5 2.0 2.0 RAG1 6.6 6.6 10.2 11.0 12.1 15.1 STG2 7.0 10.5 3.0 7.0 2.4 2.6 RAG2 9.6 13.4 14.5 22.2 19.2 27.2 STG3 5.0 12.0 3.8 6.3 1.5 1.7 RAG3 5.4 11.6 13.4 18.3 16.8 21.0 STG4 5.5 2.0 6.5 5.4 1.2 1.2 RAG4 4.8 3.2 8.5 6.2 9.5 6.6 STG5 4.5 0 3.1 0 0.3 0.0 RAG5 3.6 0 4.1 0 7.5 0.4 STG6 2.0 0 2.3 0 0.1 0 RAG6 2.4 0 5.3 0 3.2 0 STG7 1.5 0 0.8 0 0.0 0 RAG7 1.8 0 1.2 0 1.5 0 STG8 0.5 0 0.2 0 0.0 0 RAG8 0.6 0 0.5 0 0.5 0 "G" indicates glucose, and the number following "G" indicates the number of added glucose.

Test Example 1 Rebaudioside extract A obtained in Examples 1 to 3,
B and C, and their α-glucosylated sweeteners Aa, Ba and Ca obtained in Example 4, and α-glucosyl obtained by further controlling the added sugar chain obtained in Example 5 In order to compare the sweetness of the sweetened sweeteners Ab, Bb and Cb, a sweetness test was performed by a two-point comparison method by five panelists who were excellent in judging the taste quality of the stevia sweetener.

Comparison between A and A-a, Ab: Bitterness: Mellow sweetness: Tastefulness: A (1.16 mg / ml) 3 12 3 A-a (1.44 mg / ml) 2 4 3 2 Bitterness Sweet mellowness There is a bitterness Astringency A (1.16mg / ml) 4 1 2 3 A-b (1.44mg / ml) 1 4 3 2

Comparison of B with Ba, Bb Bitterness Mellow sweetness Savory taste Astringency B (1.10 mg / ml) 3 12 3 Ba (1.44 mg / ml) 2 4 3 2 There is bitterness. Sweetness is mellow. There is a bitterness. There is astringency. B (1.10 mg / ml) 4 12 3 B-b (1.44 mg / ml) 14 3 2

Comparison of C with Ca and Cb Bitterness is mellow Sweet taste is astringent C (1.00 mg / ml) 3 12 3 C-a (1.44 mg / ml) 2 4 3 2 There is bitterness. The sweetness is mellow. There is a bitterness. There is astringency. C (1.00 mg / ml) 3 2 3 3 C-b (1.44 mg / ml) 2 3 2 2 Each of A to C as compared with A to C a and b were found to be excellent in taste.

Test Example 2 In order to compare the sweetness quality of the invention product and α-glucosyl stevioside (commercially available product), a sweetness test was conducted by a two-point comparison method using panelists excellent in taste tests of five persons.

A-a (1.44 mg / ml) 32 Commercial product (1.80 mg / ml) 23 A person who considers to be a good-quality sweet A-b (1.44 mg / ml) 32 Commercial product (1.80 mg / ml) 23

B-a (1.44 mg / ml) 42 Commercial product (1.80 mg / ml) 13 A person who considered good quality sweetness Speaking person B-b (1.44mg / ml) 41 Commercial product (1.80mg / ml) 14

Those who say that they have good sweetness Those who say that they have a residual taste C-a (1.44 mg / ml) 41 Commercial products (1.80 mg / ml) 14 Those who say that they have a good sweetness C-b (1.44 mg / ml) 51 Commercial product (1.80 mg / ml) 04 The present invention provides α-
It was found that taste was superior to glucosyl stevioside.

[Brief description of the drawings]

FIG. 1 is a diagram in which a thin-layer chromatogram of Aa is read by a chromatoscanner.

FIG. 2 is a diagram in which a thin-layer chromatogram of Ab is read by a chromatoscanner.

FIG. 3 is a diagram in which a thin layer chromatogram of Bb is read by a chromatoscanner.

FIG. 4 is a diagram in which a thin-layer chromatogram of Cb is read by a chromatoscanner.

FIG. 5 is a high performance liquid chromatogram of Cb.

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-62-25949 (JP, A) JP-A-60-160823 (JP, A) JP-A-63-173531 (JP, A) JP-A-3-3 83558 (JP, A) JP-A-3-262458 (JP, A) JP-A-2-163056 (JP, A) (58) Fields studied (Int. Cl. ⁷ , DB name) A23L 1/236 A23L 1 /twenty two

Claims (2)

(57) [Claims] 1. A stevia sweetener comprising stevioside, rebaudioside A, α-glucosyl stevioside and α-glucosyl rebaudioside A, wherein the α-glucosylated product is 80% or more, and α-glucosyl rebaudio. A stevia sweetener in which Side A is present at least 1.2 times that of α-glucosyl stevioside and the number of added sugars of α-glucosylated product is at most 4 or less, and is at least 1.5 times that of stevioside. A plant or dried leaf of Stevia rebaudiana Bertney containing Baudioside A is extracted with water or a water-containing solvent, and glucose is α-added to the sweet component separated from the resulting extract using cyclodextrin glucosyltransferase. Then, glucoamylase is allowed to act to increase the number of added sugars to 4 or less. Sweeteners and adjusting to. 2. Stevioside, Rebaudioside A,
In a stevia sweetener comprising α-glucosyl stevioside and α-glucosyl rebaudioside A, α
-Stevia sweetness in which the glucosylated product is 80% or more, α-glucosyl rebaudioside A is 1.2 times or more as large as α-glucosyl stevioside, and the number of added sugars of the α-glucosylated product is 4 or less at most. A method for producing a raw material, comprising extracting a plant or dried leaf of Stevia rebaudiana Bertney containing rebaudioside A at least 1.5 times the amount of stevioside with water or a water-containing solvent, and obtaining the resulting extract. A method comprising adding α to glucose using cyclodextrin glucosyltransferase to a sweet component separated from a liquid, and then adjusting the number of added sugars to at most 4 by using glucoamylase.