JPH0577675B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application 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 prohibited or regulated due to safety concerns. On the other hand, in recent years, the health effects of consuming too much sugar have begun to become a problem, and there is a strong desire to develop natural sweeteners that are less likely to cause such problems. On the other hand, stevioside obtained from Stevia, an Asteraceae plant native to Paraguay in South America, and Rosaceae, which grows wild in southern China, Guangxi, and Guangdong regions,
Rubusoside, which is obtained from the leaves of Rubus genus shrub, Atsuba genus, is a steviol glycoside as shown in the structural formulas () and () [Fig. 1 b, a], but unlike sugar, these are steviol glycosides. It is a sweetener with fewer calories and is about 114 to 145 times sweeter, so it is attracting attention as a sweetener that can replace sugar. However, the sweet taste of the steviol glycosides stevioside and rubusoside has a bitter and unpleasant taste, and furthermore, they have the disadvantage of having a long trailing residue, so it is difficult to glucosylate them with α- or β-glucosyltransferase. Although products that have improved these drawbacks have been produced, sufficient results have not yet been achieved. Regarding methods for improving the sweetness and sweetness quality of stevioside, there are many research reports and Japanese Patent Application Laid-Open No. 54-5070.
Numerous patent applications have been filed. Regarding rubusoside, a method has been proposed to improve the sweetness by adding cyclodextrin (Japanese Patent Laid-Open No. 71867/1983). Furthermore, the sweet taste quality is improved by transglucosyl group transfer to rubusoside using starch as a sugar donor using cyclodextrin glucanotransferase (hereinafter referred to as CGTase) produced by Bacillus megaterium. A method has also been proposed. In order to solve these drawbacks, the inventors conducted repeated research and filed Japanese Patent Application No. 150209/1983 and Japanese Patent Application No. 1/1999.
No. 56129 was filed. (Problems to be Solved by the Invention) However, the above-mentioned method of enzymatically transferring starch to rubusoside using CGTase as a sugar donor does not sufficiently improve taste quality, and the same is true for stevioside. . Furthermore, the content of the above-mentioned application filed by the inventors has improved the sweetness quality considerably, and although it has become mellower, it has not yet been perfected. The cause of these problems is that β-ether bonded to the 13-OH position of rubusoside in the above reaction.
-β that is ester-bonded to the glucosyl group (hereinafter referred to as glucosyl group at position 13) or COOH at position 19
-A mixture of 1 to 3 molecules of glucose is transferred to the glucosyl group (hereinafter referred to as the glucosyl group at position 19) to one side, and to both, but among these, the glucosyl group at position 13 is Those with 1 to 3 molecules of glucose transferred have sweetness,
Agric., Biol. ., Chem., 48(10)2483
~2488 reported. The same is true for stevioside.
1989 Agric., Biol., Chem., 53(6), 1603~
Reported in 1607. That is, since the transfer product obtained as a result of the above-mentioned reaction is a mixture of products with improved taste and products with deteriorated taste, the taste has not been sufficiently improved. According to Japanese Patent Application No. 1-56129 filed by the inventors
A method for selectively transferring galactose to the glucosyl group at position 13 has not yielded satisfactory results. On the other hand, the present inventors previously discovered that a special β-fructofuranosyltransferase is produced from Arthrobacter sp. As a result, each of the 19
This invention was completed by discovering that fructose is transferred to the glucosyl group at this position. (Means for Solving the Problems) The substance according to the present invention has β-D-fructofuranose at the 2-position of the β-glucosyl group that is ester-bonded to the carboxyl group at the 19-position of rubusoside or stevioside. is a steviol glycoside with a bonded structure, that is, β-fructofuranosyl rubusoside or β-fructofuranosyl stevioside. Specifically, the substance according to the present invention is represented by the structural formula ( ) ( ) [Figure 2 a, b]. The substance according to the present invention is prepared by adding Arthrobacter sp. It can be obtained by reacting with β-fructofuranosyltransferase produced by No. The steviol glycoside used in this reaction is not limited to purified rubusoside or stevioside, and may be, for example, an extract of sweet leaves or stevia, or an intermediate purified product obtained by further purification. As the sugar donor used in this reaction, sucrose, raffinose, stachyose, etc. are used. In this reaction system, the aqueous solution or suspension containing steviol glycosides and sugar donors has a concentration of rubusoside or stevioside of about 1 to 40% (W/
W), the concentration of sugar donor is approximately 1 to 50% (W/W), and the ratio of sugar donor to rubusoside or stevioside varies depending on the sugar donor used, but is in the range of 0.1 to 50 times. , preferably 1
~5 times the range. The β-fructofuranosyltransferase used in this reaction is the β-fructofuranosyltransferase produced by Arthrobacter sp. When acting on an aqueous solution or suspension containing the donor, it decomposes the sugar donor and transfers its fructose to the glucosyl group at position 19 of rubusoside or stevioside, producing β
-Any substance that produces fructofuranosyl rubusoside or β-fructofuranosyl stevioside can be used. The PH and temperature of the reaction solution are usually PH4-8, and the temperature is 20.
~70°C is suitable. The amount of enzyme activity to be used is closely related to the reaction time, and may be such that the reaction is completed in usually 5 to 120 hours, preferably 5 to 20 hours, but is not limited thereto. (Effect of the invention) By the method described above, the liquid obtained by the reaction is absorbed into an adsorption resin (product name: Diaion HP-20,
Mitsubishi Kasei Corporation) chromatography and high performance liquid chromatography for fractionation and fractionation, and the resulting fractions were subjected to structural analysis using nuclear magnetic resonance and methylation analysis. As a result, the structural formula (,) was obtained. β
- It was confirmed that it was fructofuranosyl rubusoside or β-fructofuranosyl stevioside. In addition, in the case of β-fructofuranosyl rubusoside, the sweetness of the reaction product obtained as described above is compared with the original rubusoside, and the molar ratio is
It was confirmed that the taste was 1.5 times that of the original product, and the taste was significantly less bitter and mellow compared to the original product. Furthermore, the sweetness level of β-fructofuranosyl stevioside is 1.2 times the molar ratio compared to the original stevioside, and in terms of sweetness in particular, there is almost no bitterness and the sweetness is sharp, and compared to conventional sugar transfer products, It was confirmed that the bitterness, residual taste, and mellowness were much improved, and even better than Rebaudioside-A. Therefore, the reaction solution of each transfer product obtained in this way can be used as a sweetener as it is, but if necessary, after deactivating the enzyme and filtering, the solution is treated with an ion exchange resin. For example, it can be desalted using an H-type strongly acidic cation exchange resin and an OH-type basic anion exchange resin, and then concentrated to form a syrupy sweetener, or this concentrated liquid can be dried to form a powdery sweetener. You can also. Furthermore, the desalted reaction solution can be purified by column chromatography, and the transferred purified product can be separated and collected to be used as a sweetener. At this time, concentration, drying, and powdering may be carried out by known methods. Since the β-fructofuranosyl rubusoside and β-fructofuranosyl stevioside obtained by this invention have a high degree of sweetness and a very good sweetness quality, they can be used as low-calorie foods and drinks, low-calorie luxury foods, etc. It is suitable for sweetening so-called beauty foods, health foods, and diet foods. It is also suitable for addition to oral quasi-drugs for preventing tooth decay, such as gargles and toothpastes, and can be freely used in fields that require sweetness, including other pharmaceuticals. (Example) Hereinafter, the present invention will be specifically explained with reference to Examples. Example 1 (1) Preparation of enzyme Arthrobacter S.B. K-1 (Bacterium No. 10736) was inoculated on an ordinary agar slant culture medium, and after culturing at 37°C for 2 days, one platinum loopful was taken and 1.2% Yeast extract, 0.8% polypeptone, 4% soluble starch, 0.4%
A liquid medium (60 ml medium/500 ml shoulder flask) consisting of (NH ₄ ) ₂ HPO ₄ and 0.1% MgSO ₄ .7H ₂ O (PH 7.0) was inoculated and cultured with shaking at 37° C. for 2 days. This was used as an inoculum, dispensed into a liquid medium having the same composition, and cultured with shaking at 37°C for 2 days. After the culture was completed, the culture solution was centrifuged to obtain a supernatant (crude enzyme solution). This solution contained 30 units of β-fructofuranosidase per ml. The enzyme activity measurement method is as follows. Enzyme solution appropriately diluted to 200μ in 20% sucrose solution containing 40% xylose (50mM phosphate buffer PH6.5)
After adding 200μ and allowing it to react at 40℃ for 10 minutes, a portion of the reaction solution was poured into boiling water to heat inactivate the enzyme.The amount of glucose and fructose released was determined using the F kit, and fructose was determined from the amount of glucose. Subtract the amount to calculate the amount of fructose transferred. One unit is the amount of enzyme that transfers 1 μmol of fructosyl group per minute. (2) Transfer reaction Dried sweet leaves were crushed, warm water was added for extraction, a filter aid was added, and after thorough stirring, the liquid was filtered to obtain a clear liquid. Furthermore, after adsorption with an adsorption resin (trade name: Diaion HP-20 manufactured by Mitsubishi Kasei Corporation), it was recrystallized to prepare rubusoside (sample No. 1) with a purity of 97%. After dissolving 9 g of rubusoside and 191.5 g of sucrose prepared in the above method in 50 mM phosphate buffer (PH6.5) to make 280 ml, (1)
β-Fructofuranosidase prepared at 2500
unit was added and reacted at 50°C for 16 hours. After the reaction, a solution in which the enzyme was heated and inactivated was adsorbed on an adsorption resin, and then eluted with 80% methanol to form a mixture of unreacted rubusoside and rearrangement reaction product (sample no.
2) was fractionated. This rearrangement reaction product was further chromatographically fractionated using a preparative column to obtain a highly pure rearrangement reaction product (sample No. 3). (3) Structural analysis Sample No. 3, which was fractionated and isolated using the method described above, was subjected to β It was confirmed that -D-fructofuranose was transferred to the glucosyl group at position 19. Next, ¹ H, ¹³ C-NMR analysis confirmed that one molecule of β-D-fructofuranose was bound at the 2-position, and further methylation analysis (complete methylation → acid hydrolysis → reduction → acetyl It was confirmed from chemical analysis (gas chromatography) that the β-D-fructofuranose was bonded to the 6-position of the glucosyl group. From the above results, as shown in the structural formula (), β-Fructofuranosyl rubusoside has β-D-fructofuranose bonded at the 2-position to the 6-position of the glucosyl group that is ester-bonded to COOH at the 19-position of rubusoside. The sides and structure have been decided. The ¹³ C-NMR chart at this time is shown in Figure 3. Example 2 (1) Transfer reaction 9 g of 97% pure stevioside (Sample No. 4 - manufactured by Maruzen Kasei Co., Ltd.), 153.2 g of sucrose, and 2013 units of β-fructofuranosidase prepared in (1) of Example 1. The reaction was otherwise carried out under the same conditions as in Example 1 (2). This reaction solution was purified and fractionated by the method of Example 1 (2),
A mixture of unreacted stevioside and the rearranged product (Sample No. 5) and a highly purified rearranged product (Sample No. 6) were obtained. (2) Structural analysis As a result of structural analysis of the above-mentioned fractionated and isolated sample No. 6 in the same manner as in Example 1 (3), glucosyl ester bonds to COOH at position 19 of stevioside as shown in the structural formula (). The structure was determined to be β-fructofuranosyl stevioside bound to the 6-position of the group at the 2-position of the β-D-fructofuranosheath. The ¹³ C-NMR chart at this time is shown in Figure 4. (Test Example) Samples No. 2, No. 3, and No. obtained in Examples 1 and 2.
5, No. 6 was subjected to a sensory test using aspartame, rebaudioside-A, rubusoside (sample No. 1), and stevioside (sample No. 4) as standard products. (1) Sweetness Test Preparation of Aqueous Solutions of Samples Aqueous solutions shown in Table 1 were prepared so that each sweetness level was approximately 3 to 6% in terms of sucrose based on previously reported literature values.

[Table] Sucrose aqueous solution The following six types of sucrose aqueous solutions were prepared. 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 (%) Test method Aqueous sucrose solutions were arranged in descending order of concentration, and 10 panel members were asked to choose one with the same sweetness as the sample aqueous solution. The sweetness level based on the test is shown in Table 2.

[Table] As shown above, β-fructofuranosyl rubusoside is approximately 1.5 times that of the standard product in terms of molar ratio.
The sweetness of β-fructofuranosyl stevioside was about 1.2 times that of the standard product on a molar basis. (2) Sweet taste quality test Based on the results of the sweetness test in (1), prepare a sweet aqueous solution equivalent to a 5% sucrose aqueous solution for each sample, and test their sweet taste qualities (bitterness, residual taste, mellowness). I did it. First, regarding the three standard products (rubusoside, which has poor taste, is excluded due to testing circumstances), bitterness, residual taste,
The three items of mellowness were scored using the rating method (Table 3) using a panel of 20 people, and then using these ratings as a reference, the test products (No. 2, No. 3, No. 5, No. 6) The four points were scored in the same way using a panel of 12 people. The results are shown in Table 4.

【table】

[Table] As shown above, the improvement in taste quality was insufficient for β-fructofuranosyl rubusoside, but β-fructofuranosyl stevioside was superior to aspartame in terms of bitterness, residual taste, and mellowness. Although slightly inferior, the sweetness quality was significantly improved to that of Rebaudioside-A, which is said to have very good sweetness.

[Brief explanation of drawings]

Figures 1a and b show the structural formulas of rubusoside and stevioside, which are the raw materials of the present invention, and Figures 2a and b show the structural formulas of β-fructofuranosyl rubusoside and β-fructofuranoside, which are the raw materials of the present invention. The structural formula of cilstebioside, Figure 3 is sample No.
^3.13C -NMR chart, measurement conditions are equipment:
JEOL JMN GX-400 (100MHz), solvent:
Pyridin−d5, internal standard: Tetramethylsilan
(TMS), Figure 4 is a ^13C -NMR chart of sample No. 6, measurement conditions are equipment: JEOL JMN GX-400
(100MHz), solvent: Pyridin-d5, internal standard:
Tetramethylsilan (TMS).

Claims (1)

[Scope of 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 that is ester-bonded to the carboxyl group at the 19-position of rubusoside or stevioside. 2 Add β-fructofuranosyltransferase produced by Arthrobacter sp. K-1 (Fiber Deposit Bacteria Deposit No. 10736) to an aqueous solution or suspension containing rubusoside or stevioside and a β-fructosyl sugar compound. 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 that is ester-bonded to the carboxyl group at the 19-position of rubusoside or stevioside. manufacturing method. 3 Add β-fructofuranosyltransferase produced by Arthrobacter sp. A sweet taste consisting of a steviol glycoside with a structure in which β-D-fructofuranose is bonded at the 2-position to the 6-position of the β-glucosyl group that is ester-bonded to the carboxyl group at the 19-position of rubusoside or stevioside obtained by the reaction. fee.