JPH0633309B2 - Novel steviol glycoside, production method thereof and sweetener using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel steviol glycoside, a method for producing the same, and a sweetener using the same.

(Prior Art) In recent years, the use of artificial sweeteners such as saccharin, zultin, and cyclamate in general foods has been banned or regulated from the viewpoint of safety.

On the other hand, in recent years, the health effects of excessive sugar intake have begun to be a problem, and thus there is an eager need to develop a natural sweetener having less of these problems.

On the other hand, steviol glycosides obtained from Stevia, which is an Asteraceae plant native to Paraguay, South America, are a mixture of seven kinds such as stevioside C, D, E and dulcoside A, steviol bioside H.

Stevia sweeteners currently on the market are sold as a mixture of these. Among these steviol glycosides, a typical structural formula of stevioside is shown in (I) [Fig. 1 (a)].

Unlike sugar, stevioside is a low-calorie sweetener, and its sweetness is about 145 times higher, and it is attracting attention as a sweetener that replaces sugar.

However, the Stevia sweetener has bitterness and dislike,
Furthermore, it has a drawback that it has a long residual taste and has a tail. In order to improve these, many research reports and patent applications have been made on a method for improving the sweetness and sweetness of stevioside. Specifically, cyclodextrin glucanotransferase (hereinafter referred to as CGTase) produced by Bacillus megaterium is used for stevioside, and starch is used as a sugar donor for enzyme transfer to improve sweetness. The method of doing is also proposed. As described above, products in which the drawbacks of steviol glycosides have been improved have been produced, but they have not yet achieved satisfactory results.

The reason for this is that in the above-mentioned reaction, 1-glucose was added to the glucosyl group at position 13 or 19 of stevioside.
~ 3 molecules each of which is transferred to one side, or a mixture of both transferred to both sides is produced. Among these, those in which 1 to 3 molecules of glucose are transferred to the glucosyl group at the 13-position have improved sweetness and taste. However, from the glucosyl group at the 13th position,
It has been reported that the product in which a large amount of glucose has been transferred by the glucosyl group at the 19-position has reduced sweetness and taste.

Therefore, the present inventors found that stevioside's 19th CO
After blocking the glucosyl group esterified to OH (hereinafter referred to as the 19-position glucosyl group) with galactose, the 13-position
We proposed a method for selectively transferring glucose to a glucosyl group that is ether-bonded to OH (hereinafter referred to as the 13-position glucosyl group) (Japanese Patent Application No. 63-247371).

(Problems to be solved by the invention) In this method, in addition to those in which the glucosyl group at the 19-position is blocked with galactose, those in which the glucosyl group at the 13-position is blocked with galactose are considerably contained without being fractionated. Therefore, when CGTase was used for this and enzyme transfer was performed using starch as a sugar donor, the glucosyl group at the 19-position was blocked with galactose as expected.
1 to 3 molecules of glucose are transferred to the glucosyl group at the 13th position, and both the degree of sweetness and the sweetness are significantly improved. However, for those in which the glucosyl group at the 13th position is blocked with galactose, 1 is added to the glucosyl group at the 19th position. ~ 3 molecules of glucose are transferred, and this ratio reaches 10 to 15%, which is a cause of insufficient improvement in sweetness.

On the other hand, the present inventors previously proposed that β-D-fructofuranose is bound to the glucosyl group at the 19-position of rubusoside or stevioside to improve the sweetness quality (Japanese Patent Application No. -234675).

On the other hand, the structural formula of rebaudioside A is as shown in (II) [Fig. 1 (b)], and the content of rebaudioside A in steviaol glycosides contained in stevia is It is usually cultivated at around 15%. Rebaudioside A has a sweetness of 245 times, which is almost twice that of stevioside, and its sweetness is considerably better than that of stevioside. Therefore, research on improved cultivation methods has been conducted for a long time, and the quality of rebaudioside A is high. It has been improved to 50% and has been cultivated for 50 years.

However, such a high rebaudioside A content is not perfect in sweetness and sweetness similar to α-glucosyl stevioside, and rebaudioside A itself has a better sweetness than stevioside. Therefore, up to today, there have been no reports on the improvement of sweetness.

In view of the aforementioned Japanese Patent Application No. 1-234675, the present inventors predicted that the taste quality would be improved by causing β-D-fructofuranose to act also on rebaudioside A, As a result of earnest research on the enzymes used for this, Microbacterium sp. It was confirmed that a special β-fructofuranosyl transferase (hereinafter referred to as transferase) was produced from H-1 (Deposited Microorganisms Institute No. 11428). It was also discovered that β-D-fructofuranose is transferred to rebaudioside A by using this enzyme, and the taste quality is improved.

(Means for Solving Problems) The present invention has been completed based on the above findings,
The substance according to the present invention is a steviol glycoside having a structure in which β-D-fructofuranose is bonded at the 2-position to the 6-position of the glucosyl group at the 19-position of rebaudioside A, that is, β-fructofuranosyl. It is rebaudioside A.

Specifically, the substance according to the present invention is added to an aqueous solution or suspension containing rebaudioside A and a sugar donor in Microbacterium sp.
No.) produced by acting a transferase. Its structural formula is represented by (III) [Fig. 2].
As rebaudioside A used in this reaction, rebaudioside A purified and separated is used, but it is not limited thereto.

As the sugar donor used in this reaction, sucrose, raffinose, stachyose and the like are used.

In this reaction system, the concentration of the aqueous solution or suspension containing rebaudioside A and the sugar donor is rebaudioside A
Is 1 to 40% (w / w), the sugar donor is about 1 to 50% (w / w), and the ratio of sugar donor to rebaudioside A varies depending on the sugar donor used, but ~ 50 times the range,
The range is preferably 1 to 5 times.

Suitable reaction conditions are usually pH 4 to 8 and temperature of 20 to 70 ° C. The amount of enzyme activity used is closely related to the reaction time, and it is usually 5 to 120 hours, preferably 5 to 20 hours, and the amount of enzyme activity may be such that the reaction is completed, but it is not limited thereto.

(Effect of the invention) The sweetness of the reaction product obtained as described above is 1.2 times in terms of molar ratio as compared with rebaudioside A which is the drug substance. Almost disappeared, the sweetness is well cut, and mellowness is added. Compared to conventional sugar transfer products, including those that we have developed and applied for patents,
It was confirmed that it was further improved. Therefore, the reaction solution of the transfer product thus obtained can be used as a sweetener as it is, but if necessary, the enzyme is inactivated and filtered, and then the solution is treated with an ion exchange resin such as H-form. It can be desalted using a strongly acidic cation exchange resin and an OH-type basic anion exchange resin to give a concentrated syrup-like sweetener, or the concentrated solution can be dried to give a powdered sweetener. Furthermore, the desalted reaction solution can be purified by a column chromatography method to separate and collect a transfer product, which can be used as a sweetener. Concentration, drying at this time,
The powdering may be performed by a known method.

The β-fructofuranosyl rebaudioside A obtained according to the present invention has a high degree of sweetness and a very good sweetness quality, so that low-calorie foods, drinks, so-called beauty foods, healthy foods, diets, etc. Suitable for sweetening food. It is also suitable for addition to oral quasi drugs for the prevention of dental caries such as mouthwashes, toothpastes, etc., and can be freely used in fields requiring sweetness, including other drugs.

(Examples) Hereinafter, the present invention will be specifically described with reference to Examples.

Example 1 (1) Preparation of enzyme Sucrose 0.5%, sodium nitrate 0.3%, dibasic potassium phosphate 0.1%
Microbacterium sp. Was added to an agar slant medium containing 1%, magnesium sulfate 0.05%, and magnesium chloride 0.02%. H-
Inoculated with 1 (Deposited by Microtechnology Research Institute, No. 11428), cultured at 30 ° C for 3 days, and then picked up 1 platinum loop and 1% sucrose, 0.05 yeast extract
%, Polypeptone 0.5%, Sodium Nitrate 0.3%, Dibasic Potassium Phosphate 0.1%, Magnesium Sulfate 0.02%, (pH7.2) inoculated in a liquid medium (60 ml medium / 500 ml shoulder flask), 30 The cells were cultivated with shaking at 0 ° C for 2 days. This was used as an inoculum, dispensed into a liquid medium having the same composition, and cultured with shaking at 30 ° C. for 2 days. After the completion of the culture, the culture solution was centrifuged to obtain a supernatant (crude enzyme solution). This solution contained 10 units of transferase in ml.

The activity measuring method is as follows. 5% sucrose solution (50 mM
Phosphate buffer solution pH6.5) Enzyme solution 200 appropriately diluted to 200μ1
After adding μl and allowing it to act at 40 ° C. for 10 minutes, the reaction solution is put into boiling water to inactivate the enzyme by heat, and glucose and fructose produced by the Somoginelson method are calculated as glucose. The enzyme activity was defined as 1 unit of the amount of enzyme that decomposes 1 μmol of sucrose per minute.

(2) Transfer reaction 97% rebaudioside A (Sample No. 1: Morita Chemical Co., Ltd.)
2.8 g and 20 g of sucrose were dissolved in a 20 mM citrate buffer solution (pH 5.7) to make 200 ml, and 50 units of the transferase prepared in the following (1) was added and reacted at 40 ° C. for 16 hours. The metastasis rate at this time was 82%. After that, the reaction solution in which the enzyme was inactivated by heating was adsorbed on an adsorption resin (trade name: Diaion HP-20: manufactured by Mitsubishi Kasei Co., Ltd.) and then eluted with 80% methanol to generate a rearrangement reaction with unreacted rebaudioside A. The mixture of substances was separated. Of this, 1/2 amount was distilled off from MeOH and freeze-dried to obtain 1.34 g of a mixture (Sample No. 2). Next, the above 1/2 amount was further chromatographed on a preparative column, and MeOH was distilled off and freeze-dried to obtain 0.92 g of a highly pure rearrangement reaction product (Sample No. 3).

(3) Structural analysis Sample No. 3 fractionated / isolated by the above method was treated with lithium iodide, 2,6 lutidine and a methanol reagent to selectively decompose the ester bond at position 19 by β -D-
It was confirmed that fructofuranose was transferred to the glucosyl group at position 19. Next, β was determined by ¹ H, ¹³ C-NMR analysis.
It was confirmed that one molecule of -D-fructofuranose was bound at the 2-position, and further its methylation analysis (complete methylation → acid hydrolysis → reduction → acetylation → gas chromatograph) revealed that β-D- It was confirmed that fructofuranose was bonded to the 6-position of the glucosyl group.

From the above results, as shown in structural formula (III) [FIG. 2], β-D-fructofuranose is located at the 2-position at the 6-position of the glucosyl group ester-bonded to COOH at the 19-position of rebaudioside A. The structure was determined to be β-fructofuranosyl rebaudioside A bound by.

The ¹³ C-NMR chart at this time is shown in FIG.

Test Example Regarding the samples No. 2 and No. 3 obtained in Example 1, glucose transfer stevioside (manufactured by Toyo Sugar Co., Ltd.) (sample No. 4), which is currently on the market, is the highest among currently commercially available synthetic products. A sensory test was performed using aspartame (trade name: manufactured by Ajinomoto Co., Inc.), stevioside, and rebaudioside A (sample No. 1), which are said to have the degree of sweetness and the quality of sweetness, as standard products.

(1) Sweetness Test Preparation of Aqueous Solution of Specimen Based on the literature values already reported, the aqueous solutions shown in Table 1 were prepared so that each sweetness was approximately 3 to 6% in terms of sucrose.

Sucrose aqueous solution The following 6 types of sucrose aqueous solutions were prepared.

3.5,4.0,4.5,5.0,5.5,6.0 (%) Test method The sucrose aqueous solutions were arranged in order from the lowest concentration, and 10 panel members were selected to have the same sweetness as the test aqueous solution. The sweetness based on the test is shown in Table 2.

As described above, β-fructofuranosyl rebaudioside A (high-purity product) is about a molar ratio conversion as compared with the standard product.
It was 1.2 times.

(2) Sweetness test 5% for each sample from the sweetness test result of (1)
A sweetness aqueous solution equivalent to the sucrose aqueous solution was prepared, and the sweetness qualities (bitterness, residual taste, mellowness) were examined.

First of all, for the standard 3 points, the 3 items of bitterness, residual taste and mellowness were scored by a scoring method (Table 3) using a panel of 20 people, and then the sample (No. 2, No.
3) For 2 points, a panel of 12 people was used and similarly scored. The results are shown in Table 4.

As described above, β-fructofuranosyl rebaudioside A has bitterness, residual taste, and mellowness of all items, but is slightly inferior to aspartame, but rebaudioside A is said to have very good sweetness, Glucose transfer was improved to a better quality than stevioside.

[Brief description of drawings]

1 (a) and 1 (b) are structural formulas of stevioside and levidioside A which is a raw material of the present invention, and FIG. 2 is β-fructofuranosyl rebaudioside A which is a substance according to the present invention. Structural formula, Fig. 3 shows ¹³ C of sample No3
-NMR chart, measurement conditions are equipment: JEOL JEN GX-40
0 (100MHz), solvent: Pyridin-d5, internal standard: Tetrametyls
ilan (TMS).

Claims (3)

[Claims] 1. A steviol glycoside having a structure in which β-D-fructofuranose is bonded at the 2-position to the 6-position of a β-glucosyl group which is ester-bonded to COOH at the 19-position of rebaudioside A. 2. An aqueous solution or suspension containing rebaudioside A and a β-fructosyl sugar compound is added to Microbacterium sp. H-1 (Department of Microbiology Research Institute, No. 11428)
COOH at position 19 of rebaudioside A characterized by acting on β-fructofuranosyl transferase produced by
At the 6-position of the β-glucosyl group that is ester-bonded to β-D
-A method for producing a steviol glycoside having a structure in which fructofuranose is bound at the 2-position. 3. An aqueous solution or suspension containing rebaudioside A and a β-fructosyl sugar compound is added to Microbacterium sp. Of the β-glucosyl group that is ester-bonded to COOH at position 19 of rebaudioside A obtained by reacting β-fructofuranosyl transferase produced by H-1 A sweetener comprising a steviol glycoside having a structure in which β-D-fructofuranose is bound to the 6-position at the 2-position.