JPH09107913A - Alpha-glucosylated stevia sweetener - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a stevia sweetener excellent in quality of sweetness. SOLUTION: This α-glucosylated stevia sweetener contains α- glucosylrebaudioside A in an amount of >=1.2 times based on the α- glucosylstevioside. This sweetener is obtained by extracting a plant body or a dried leaf of Stevia rebaudiana Bertoni containing a rebaudioside A in an amount of >=1.5 times based on the stevioside with a hydrous solvent, separating a sweetness ingredient from the resultant extract solution, carrying out the α-addition of glucose to the separated sweetness ingredient with a cyclodextrin glucosyltransferase. An α-1,4-glucosidase is reacted with the sweetener to afford the sweetener having a regulated added sugar chain.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art Stevia is a perennial chrysanthemum that originates 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. As the sweet component of stevia, 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, Zulcoside A, etc. are known. In the generally cultivated Stevia cultivar, stevioside is the main component of the sweetening ingredients, the content of rebaudioside A is about 3 to 4 of that of stevioside, and the content of rebaudioside C is slightly lower, Depending on the variety, there are various types such as those not containing rebaudioside A and C, and those containing rebaudioside C as a main component.

Among the tastes perceived by the tongue such as astringency and spiciness, the quality of sweetness is very delicate. 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 it has the drawback that the unpleasant taste such as bitterness remains 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 1.3 to 1.5 times that of stevioside, and its sweetness is mellow and does not leave an unpleasant taste similar to that of sugar, but slightly in stevia leaves. Only included,
There is also a drawback that it does not give a rich taste due to its sharp sweetness.

In order to improve the taste of these sweeteners, particularly α-obtained by adding α to glucose in order to improve bitterness of stevia extract containing stevioside as a main component.
A sweetener consisting mainly of glucosyl stevioside has been put into practical use (Japanese Patent Publication No. 57-18779). α-Glucosylation gives a rich sweet taste, but α-
Glycosyl stevioside has a unique aftertaste different from that of stevioside with a decrease in sweetness. In order to improve these, stevia extract containing stevioside, after adding α to glucose, there is a method of adjusting the sugar chain of glucose (Japanese Patent Laid-Open No. 2-163056), Stevia to further improve the taste quality. A method of isolating stevioside from the extract by recrystallization and α-adding glucose is also taken and is commercially available. However, although this method slightly improves the unique aftertaste, a mother liquor containing a sweetening component after the isolation of stevioside is produced, and this mother liquor cannot be used as a sweetener in terms of sweetness, and eventually, , Is a cause of increasing the production cost.

Isolation of rebaudioside A from stevia extract containing stevioside as a main component is impossible on an industrial scale. Even if rebaudioside A is isolated, rebaudioside A is recovered. Crystallization causes an increase in cost as the rate decreases. Therefore, it was difficult to industrialize α-glucosyl rebaudioside A obtained by α-adding glucose to rebaudioside A using cyclodextrin glucosyltransferase.

[0006] In addition, when glucose is α-added with an extract from a stevia plant or a dry leaf containing rebaudioside A in an amount not more than 1.5 times that of stevioside, α is added to the rebaudioside. The rate at which glucose is α-added to stevioside is higher than the rate at which glucose α-adds to side A. Therefore, in order to α-add glucose to rebaudioside A, excess enzyme, α-glucosyl glycation product, and reaction time are required. Furthermore, a large amount of glucose is added to stevioside due to the increase in reaction time, resulting in a decrease in sweetness. When trying to prevent this, a large amount of unreacted rebaudioside A remained, and eventually it was not possible to obtain a sweetener containing α-glucosyl rebaudioside A as a main component.

[0007]

SUMMARY OF THE INVENTION Therefore, the present inventors efficiently and inexpensively produce a natural sweetener having a sweetness similar to that of the above-mentioned sugar, a rich taste, and a high aftertaste and a high degree of sweetness. The present invention has been completed by extracting the sweetening component from a new variety of Stevia rebaudiana Bertney having an improved proportion of the sweetening component as a result of intensive studies.

[0008]

SUMMARY OF THE INVENTION The inventors have endeavored to produce an extract containing rebaudioside A as a main component, which does not require the removal of stevioside. A sweetener containing 1.2 times or more of Dioside A was completed. This sweetener was prepared by extracting Stevia rebaudiana Bertney plant or dried leaves containing 1.5 times or more of rebaudioside A with respect to stevioside with water or a water-containing solvent, and separating it from the obtained extract. Glucose is α-added to the sweetening component using cyclodextrin glucosyltransferase. Further, α-1,4-glucosidase is allowed to act on the above-mentioned sweetener to obtain a sweetener having an added sugar chain regulated.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION A new variety of Stevia rebaudiana Bertney containing 1.5 times or more of rebaudioside A relative to stevioside (JP-A-60-160).
No. 823, JP-A-63-173531),
These plants or dried leaves were extracted with water or a water-containing solvent, and the sweet component (rebaudio extract) separated from the resulting extract (hereinafter referred to as Stevia extract) was subjected to α-addition of glucose using cyclodextrin glucosyltransferase. Then, a sweetener containing 1.2-fold or more of α-glucosyl rebaudioside A with respect to α-glucosyl stevioside is obtained. Furthermore, sweetness containing 1.2 times or more of α-glucosyl rebaudioside A with respect to α-glucosyl stevioside in which an additional sugar chain obtained by reacting α-glucosylated product with α-1.4-glucosidase is adjusted. Get paid.

The breeding process of a new Stevia cultivar for obtaining the rebaudio extract used in the present invention is as follows. However, the breeding process is not limited to this breeding process, and is 1.5 times or more that of stevioside. Any breeding method and cultivation method may be used so long as Stevia containing Rebaudioside A is obtained.

From October to December 1979, a Stevia native cultivar S having a rebaudioside A content of 6/10 of that of stevioside was used in the Niimi plant of Morita Chemical Industry Co., Ltd. in Ashimi, Niimi City, Okayama Prefecture. Artificially bred in Japan and sown the obtained seeds at the nursery greenhouse in 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 sweetener content was investigated in the beginning of the month, and seedlings containing 1: 1 or more of rebaudioside A relative to stevioside were selected and designated as SF1.

[0012] SF1 was propagated in cuttings, 10 to 12 of the same year
The seeds were artificially bred in a greenhouse in the month, and the obtained seeds were sown in a nursery greenhouse in February 1981, and the seedlings germinated and grown in early May of the same year were transplanted to the field, and in early August of the same year. The sweetness ingredient content was investigated, and seedlings containing rebaudioside A at a ratio of 1: 1.5 or more relative to stevioside were selected and designated as SF2.

SF2 was similarly cut and propagated in cuttings.
~ December artificially crossed in a greenhouse, sow the resulting seeds in a nursery vinyl house in February 1981, and transplant the seedlings germinated and grown in early May of the same year to the field, and in August of the same year. The sweetness ingredient content was investigated at the beginning of the period, and seedlings containing rebaudioside A in an amount of 1: 2.56 or more relative to stevioside were selected and designated as SF3.

100 seedlings obtained from each of SF2 and SF3 were proliferated by cuttings, and the content of sweetening components in the dried leaves was examined. As a result, dried leaves A (stevioside 3.6) were obtained.
%, Rebaudioside A 5.6%, rebaudioside C 1.1%), dried leaves B (stevioside 2.7%, rebaudioside A 7.1%, rebaudioside C 1.1)
%), 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 cation exchange resin and anion exchange resin to remove ionic impurities as 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 and elution with a hydrophilic solvent to concentrate the eluent, followed by 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 can be obtained by α-adding glucose to a rebaudio extract with cyclodextrin glucosyl transferase. Further, α-glucosyl rebaudioside A having an added sugar chain regulated can be obtained by allowing α-1.4 glucosidase to act on α-glucosyl rebaudioside A obtained above.

The cyclodextrin glucosyltransferase used in the present invention is Bacillus macerans.
( Bacillus macerans ), Bacillus megaterium ( Baci
llusmegaterium ), Bacillus stearothermophilus
( Bacillus stearothermophilus ) and the like originated in Bacillus. α-1.4 for glucosidase
1.4 Glucosyl sugar compounds include α-amylase that randomly cleaves sugar chains, β-amylase that cleaves maltose units from its non-reducing end, and glucoamylase that cleaves glucose units from its non-reducing end. The transfer reaction between cyclodextrin glucosyltransferase and α-1.4 glucosidase is carried out at pH 4 to 7, preferably pH.
5 to 6, the reaction temperature is 20 to 80 ° C., preferably 40 to 70
The temperature is good, and the amount of 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 by 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 sweetener also has the same sweetness even if an additional sugar chain is adjusted. There was little residual taste and the sweetness was excellent. Further, the sweetener of the present invention is sweeter than the case of producing α-glucosyl rebaudioside A using rebaudioside A obtained by crystallization from stevia extract instead of rebaudio extract. This is advantageous in that the crystallization cost required for isolating rebaudioside A and the increase in production cost due to the decrease in recovery rate can be avoided.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1 Rebaudio Extract A 40 g of dried Stevia leaf A (Stebioside 3.6%, Rebaudioside A 5.6%, Rebaudioside C 1.1%) was sweetened with 10 to 20 times the amount of water. Extraction was carried out several times until no longer possible, and the extract was packed with 200 ml of a cation exchange resin (Amberlite IR-120B) and an anion exchange resin (Amberlite IRA-94) 2.
Pass it through a column filled with 00 ml, and pass the liquid through a synthetic adsorption resin.
(Diaion HP-20) It is passed through a column filled with 200 ml to adsorb the sweet component, washed thoroughly with water, and then washed with methanol 40
Elute with 0 ml. The eluent is concentrated under reduced pressure and dried to give a pale yellow powder. The analysis results are shown in the table below.

Example 2 Rebaudio Extract B Instead of the dried Stevia leaf A in Example 1, dried leaf B
(Stebioside 2.7%, rebaudioside A7.1
%, Rebaudioside C 1.1%) was used. The results are shown in the table below.

Example 3 Rebaudio Extract C Instead of Stevia dry leaf A in Example 1, dry leaf C
(Stebioside 1.0%, rebaudioside A9.1
%, Rebaudioside C 0.9%) was used. The results are shown in the table below.

[0022] ST: Stevioside RA: Rebaudioside

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

Each reaction solution was divided into two equal parts, the reaction solution was filtered to remove suspended matter, and the filtrate was passed through a column packed with 100 ml of synthetic adsorption resin Diaion HP-20 and thoroughly washed with water. Then elute with 300 ml of methanol. The eluent is a cation exchange resin (Amberlite IR-120
B), anion exchange resin (Amberlite IRA-94)
After desalting and decoloring through a column packed with, white powder obtained by concentrating and drying under reduced pressure was designated as α-glucosylated sweeteners Aa, Ba and Ca.

Example 5 Addition sugar chain regulation
1.0% of solids was added to Nagase & Co., Ltd. and reacted at a temperature of 50 ° C. for 5 hours. After the reaction, the enzyme was inactivated by heating at 95 ° C for 30 minutes. Each reaction solution is filtered to remove suspended matter, and then each filtrate is used as a synthetic adsorption resin DIAION HP.
-20 is passed through a column packed with 100 ml, washed thoroughly with water, and then eluted with 300 ml of methanol. The eluent can be passed through a column packed with a cation exchange resin (Amberlite IR-120B) and an anion exchange resin (Amberlite IRA-94) for desalting and decolorization, and then concentrated and dried under reduced pressure. The obtained white powders were designated as sugar chains controlling α-glucosylated sweeteners Ab, Bb and Cb.

Α-Glucosylated sweeteners Aa, Ba and Ca obtained in Example 4 and three types of sugar chain-regulating α-adducts obtained in Example 5 in which sugar chains were further adjusted The yields of the glycosylated 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

Aa and A-obtained in Examples 4 and 5
b, Bb, Cb were subjected to thin layer chromatography. Measurement conditions Thin layer plate HPTLC Fertingplatten kieselgel 60F 254 (manufactured by MELK) Developing solvent Chloroform: Methanol: Water = 20: 30: 8 Coloring agent 50% sulfuric acid The thin layer plate is Shimadzu Chromatoscanner 91.
Type 0 (manufactured by Shimadzu Corporation, reflective zigzag scanning method),
It was read at a measurement wavelength of 350 nm. A-a, A-b, B
Figures 1 to 4 show the thin-layer chromatograms of -b and Cb read by a chromatographic scanner.

The Cb obtained in Example 5 was also 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 UV210 nm C-b high performance liquid chromatogram is shown in FIG.

[0029]

[Table 3] Analytical values A-a A-b 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 next to “G” indicates the number of glucose added.

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 in Example 5 in which an additional sugar chain was further adjusted. In order to compare the sweetness of the sweetened sweeteners Ab, Bb, and Cb, a sweetness test was conducted by a two-point comparison method by five panelists who were excellent in judging the taste of the Stevia sweetener.

Comparison of A with Aa and Ab A bitter taste with a mild sweetness and a residual taste with astringency A (1.16mg / ml) 3 1 2 3 A-a (1.44mg / ml) 2 4 3 2 Has bitterness, mellow sweetness, has residual taste, has astringency A (1.16mg / ml) 4 1 2 3 A-b (1.44mg / ml) 1 4 3 2

Comparison of B with B-a and B-b Bitterness Mild sweetness Residue with astringency B (1.10 mg / ml) 3 1 2 3 B-a (1.44 mg / ml) 2 4 3 2 Has bitterness, mellow sweetness, has a residual taste, has astringency B (1.10mg / ml) 4 1 2 3 Bb (1.44mg / ml) 1 4 3 2

Comparison of C with Ca and Cb There is bitterness There is a mellow sweetness There is a residual taste There is astringency C (1.00mg / ml) 3 1 2 3 C-a (1.44mg / ml) 2 4 3 2 There is bitterness, mellow sweetness, there is a residual taste, there is astringency C (1.00mg / ml) 3 2 3 3 C-b (1.44mg / ml) 2 3 2 2 2 Each of A to C compared to A to C It was found that a and b were excellent in taste.

Test Example 2 In order to compare the sweetness qualities of the invented product and α-glucosyl stevioside (commercially available product), a sweetness test was carried out by a two-point comparison method using a panel excellent in taste test of 5 persons.

Those with good quality sweetness People with residual taste Aa (1.44 mg / ml) 3 2 Commercial product (1.80 mg / ml) 2 3 Good quality sweetness Someone who has assumed Ab (1.44mg / ml) 3 2 Commercial product (1.80mg / ml) 2 3

Those with good quality sweetness Those with residual taste Ba (1.44mg / ml) 4 2 Commercial product (1.80mg / ml) 1 3 Good quality sweetness Person who said to be B-b (1.44mg / ml) 4 1 Commercial product (1.80mg / ml) 14

Those with good quality sweetness Those with residual taste Ca (1.44mg / ml) 4 1 Commercial product (1.80mg / ml) 1 4 Good quality sweetness With residual taste A person who has Cb (1.44mg / ml) 51 Commercial product (1.80mg / ml) 0 4 The present invention is α-where glucose is added to stevioside.
It was found that the taste was superior to that of glucosyl stevioside.

[Brief description of the drawings]

FIG. 1 is a diagram in which a thin layer chromatogram of Aa is read by a chromatographic scanner.

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

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

FIG. 4 is a diagram in which a thin layer chromatogram of Cb is read by a chromatographic scanner.

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

Claims (4)

[Claims] 1. A sweetener containing 1.2-fold or more of α-glucosyl rebaudioside A with respect to α-glucosyl stevioside. 2. A plant or dried leaves of Stevia rebaudiana Bertney containing 1.5 times or more of rebaudioside A relative to stevioside is extracted with water or a water-containing solvent, and separated from the obtained extract. A sweetener containing 1.2 times or more of α-glucosyl rebaudioside A relative to α-glucosyl stevioside, which is obtained by α-adding glucose to the thus-sweetened component using cyclodextrin glucosyltransferase. 3. The sweetener according to claim 1 or 2 with α-
A sweetener obtained by reacting 1,4-glucosidase with an added sugar chain adjusted. 4. A plant or dried leaves of Stevia rebaudiana Bertney containing 1.5 times or more of rebaudioside A to stevioside are extracted with water or a water-containing solvent, and the resulting extract is sweetened. Separate the ingredients,
A method for producing a sweetener containing 1.2 times or more of α-glucosyl rebaudioside A relative to α-glucosyl stevioside, which is obtained by α-adding glucose to this sweetening ingredient using cyclodextrin glucosyl transferase.