JPH0453497B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel stevioside sweetener and a method for producing the same. Since the use of artificial sweeteners has been prohibited or regulated in recent years, stevioside has been increasingly used as a strong natural sweetener in diet sweeteners and the like. However, stevioside has a strong sweet taste as well as quite strong unpleasant tastes such as bitterness and astringency, so its range of use has been limited. To this end, various methods have been attempted to improve the taste. Typical examples include stevioside, pullulan, dextran,
A method for improving the taste of stevioside, characterized in that it is used in the coexistence of locust bean gum and one or more polysaccharides consisting of β-cyclodextrin (Japanese Unexamined Patent Publication No. 150358/1983), α-glucosyl stevioside A method for producing a sweetener (Japanese Patent Publication No. 57-18779), which comprises adding β-1,4-galactosyl sugar compound to an aqueous solution containing stevioside and β-1,4-galactosyl sugar compound.
Microorganisms having galactosyl transfer activity or β-
β by reacting with 1,4-galactosyltransferase
A method for improving the taste of a stevio sweetener characterized by producing and containing -1,4-galactosyl stevioside (Japanese Patent Application Laid-open No. 94367/1983) is known. However, the method described in has not been able to sufficiently improve the taste of stevioside. In addition, in the method described in and, α-glycosyl or β-galactosyl stevioside is produced from stevioside using α-glycosyl or β-galactosyltransferase, but in order to produce a high-quality sweetener, high purity is required. It is necessary to contain α-glycosyl or β-galactosyl stevioside. In other words, when using low-purity stevioside, even if α-glycosyl or β-galactosyl transglycosylation reaction is performed,
Because the impurities contained in the sweetener have a strong bitter taste, the overall taste is strongly bitter, and the sweetener is not a good quality sweetener. Therefore, in order to obtain a high-quality sweetener by the above method, it is necessary to considerably increase the purity of the stevioside used or to purify the obtained α-glycosyl or β-galactosyl stevioside. In fact, an invention for a purification method for α-glycosyl stevioside has been provided (Japanese Patent Application Laid-open No. 43659/1983). In view of these circumstances, the present inventors conducted intensive research on improving the sweet taste quality of stevioside, and as a result, they focused on the relationship between the structure of stevioside and the expression of sweetness. By clathrating it with cyclodextrin, we have invented a new stevioside sweetener that has a high-quality sweetness that could not be achieved by the conventional methods of ~. That is, the present invention is a sweetener comprising a stevioside clathrate with γ-cyclodextrin. In the present invention, steviosides are natural sweeteners extracted from Stevia rebaudiana BERTONI (hereinafter abbreviated as stevia), a plant belonging to the Asteraceae family, and are β-glycosyl glycosides whose aglycone is steviol. Stevioside (eg, stevioside, rebaudioside A and C, etc.) and stevioside related compounds having a steviol skeleton (eg, α-glycosyl stevioside, β-1,4-galactosyl stevioside, etc.), including enzyme-treated products thereof. Next, cyclodextrin (hereinafter abbreviated as CD) is a cyclic oligosaccharide homolog, and is a cyclic compound in which 6 to 8 D-glucoses are linked with α-1,4, and the number of D-glucoses in Afterwards,
When there are six, it is called α-CD, when there are seven, it is called β-CD, and when there are eight, it is called γ-CD. Among these CDs, only γ-CD forms a stable inclusion complex with steviosides.
It has been reported that the inclusion complex of CD is a type of intermolecular compound [Pharmacia, 4 , 315 (1968)], and it is thought that γ-CD, which has the largest cavity diameter, is compatible with steviosides in terms of molecular size. . The sweetener of the present invention is composed of a γ-CD clathrate of steviosides and γ-CD.
It can be obtained by inclusion of CD in the presence of water. Specifically, γ-CD is dissolved or suspended in warm water to a concentration of 5 to 50% (W/W), and steviosides are added to this at a concentration of 60 to 80% relative to γ-CD.
The clathration reaction between γ-CD and steviosides is completed by stirring and mixing for 30 minutes to about 1 day, and the reaction solution is further dried by a commonly applicable drying method such as spray drying or freeze drying. By this, a sweetener containing a γ-CD clathrate of steviosides can be obtained. As a result, since γ-CD is expensive, it is also possible to use γ-CD of low purity. The content of γ-CD clathrates of steviosides in the sweetener thus obtained is 50 to 100% on a solid basis. Sweeteners obtained in this way can be produced by purifying steviosides using commonly known saccharide purification methods, such as gel filtration, ion exchange resin, adsorption resin methods, etc., or combinations thereof, depending on the purpose. It is possible to produce sweeteners with high content. In addition, the sweetener of the present invention, which is composed of a γ-CD clathrate of steviosides, can be obtained by allowing cyclodextrin glycosyltransferase to act on a starch suspension or its hydrolysis solution, and then adding steviosides thereto. , can be produced by continuing an enzymatic reaction. In this case, steviosides are included in γ-CD to form an inclusion compound, which changes the equilibrium relationship of CD production.
Since the process shifts to the CD production side, a sufficient amount of γ-CD is produced to include all the added steviosides. The strain name of cyclodextrin glycosyltransferase (hereinafter abbreviated as CGTase) used in this method is α-1,4-glucan-4-
Glycosyltransferase (EC, 2.4.1.19)
It is an enzyme that produces CDs from starch or its hydrolyzate, and the producing bacteria include Bacillus.
macerans, Klebsiella pneumoniae, Bacillus
circulans, Bacillus megaterium, Bacillus
Examples include Bacillus ohbensis sp., Micrococcus luteus, etc. Among them, Bacillus ohbensis sp.
-124285) is preferably used. The starch or its hydrolyzate used in the above method includes, for example, potato starch, sweet potato starch, corn starch, etc., and dextrins obtained by hydrolyzing these using liquefied α-amylase or acid. Starch or its liquefied form α
-Amylase-treated products are preferably used. The enzymatic reaction in the above method consists of two steps, the first step being normal CD production,
Add 5 to 20 units/starch (g) of CGTase (JP-A-49-124285) to 5 to 20% (W/V) of liquefied starch, and react at pH 6 to 9 and 40 to 70°C for about 10 to 24 hours. let 1 unit of CGTase here means PH6.5,
0.2 ml of enzyme solution appropriately diluted with 2.5 ml of 0.05% (W/V) soluble starch solution containing 0.05 M phosphate buffer.
Add 2ml of 0.6N and react for 10 minutes at 37℃.
Add HCl to stop the reaction, then 0.01% (W/
V) Add 1 ml of iodine potassium iodide solution to develop color, and then measure the absorbance at 660 nm.
It represents the amount of enzyme that causes the absorbance to disappear. In the second step, 10 to 40% (w/w) of steviosides to the starch used in the first step were added to the above reaction solution, and the conditions were the same as in the first step (pH 6 to 9,
Continue the reaction at 40-70 °C for approximately 15-40 hours. The two-step reaction thus yields a sweetener containing 20 to 66% of steviosides gamma-CD clathrates on a solids basis. γ-CD of steviosides in this reaction product
Since the components other than the clathrate are mostly maltodextrins such as maltose having α-1,4 bonds, it is possible to concentrate the reaction solution itself and use it as a sweetener, and it is usually possible to do so. By drying and pulverizing the sweetener using a suitable drying method, a high-quality sweetener can be produced as it is, and the production cost can be significantly reduced. Naturally, the sweetener obtained by the above method is processed by gel filtration or purification using ion exchange resin, etc., depending on the purpose of use, to obtain the γ-CD inclusion of steviosides. It can be made into a sweetener with a high body content. Next, the sweetener of the present invention is composed of a γ-CD clathrate of steviosides,
Also, the excellentness of the sweetener of the present invention as a sweetener will be explained with reference to experimental examples. Experimental example 1 Stevioside and CD inclusion experiment Precisely weigh 3.0 g of pure stevioside (Tama Biochemical) into several containers, and add 0 to 5 g of β-CD to each container.
In addition, purified water was added to make a total volume of 100 ml. Similarly, add γ-CD to 3.0g of stevioside from 0 to 8.
g was added, and purified water was added to each to make 100 ml. Each of the above mixed solutions of stevioside and CD was shaken at room temperature for 24 hours, the stevioside concentration in the solution portion was measured, and solubility phase diagrams of stevioside and β-CD and stevioside and γ-CD were created.
The results are shown in FIG. The solubility phase diagram in Fig. 1 was reproduced by Higuchi et al.
and KAConnors, Adv.Anal.Chem.Inst., 4 ,
117 (1965)]. That is, from FIG. 1, it is recognized that γ-CD and stevioside show a Bs type solubility phase diagram, and β-CD and stevioside show an A _L type solubility phase diagram. Also, when we calculate the apparent stability constant K' from Fig. 1, we find that γ-CD (11,250M ^-1 ) > β-CD (259M ^-1 ), and γ-CD has a large K' and is similar to the Bs type. It was revealed that it forms a very strong clathrate with stevioside. In addition, in the case of γ-CD, the solubility phase diagram of the Bs type is shown, so from the analysis of its plateau region and the analysis of the inactive crystals obtained by the arrow (↓) in Figure 1, γ- It was demonstrated that CD and stevioside form an inclusion complex in a molar ratio (1:1). Experimental Example 2 Comparison of inclusion complex of stevioside with γ-CD and mixture of stevioside and γ-CD γ-CD [RINGDEX-C, Sanraku Ocean Co., Ltd.]
Add 200 ml of pure water to 100 g of Stevioside (manufactured by Tama Seikagaku Co., Ltd.), mix well in advance, and prepare 62 g of pure stevioside (manufactured by Tama Biochemical Co., Ltd.).
was further added and kneaded well for 3 hours at room temperature. An increase in viscosity was observed during kneading, suggesting that an inclusion phenomenon was occurring. After kneading, 160 g of white powder (clathrate) was obtained by freeze-drying. Separately, mix well 100 g of γ-CD and 62 g of pure stevioside, and mix stevioside and γ-CD.
A mechanical mixture of 162 g was prepared. γ− of stevioside prepared as above
Physical properties of inclusion complexes by CD, stevioside and γ
Table 1 shows the results of comparing the physical properties of mechanical mixtures of -CD.

[Table] Clathrates obtained in this experiment, stevioside and γ-
The infrared absorption spectra (IR) of the mechanical mixture of CD and isosteviol, which is the stevioside skeleton, measured by the KBr tablet method are shown in Figures 2 and 3.
As shown in Fig. 4 and Fig. 4. IR of the inclusion body obtained in this experimental example (Fig. 2),
IR of mechanical mixture of stevioside and γ-CD
(Figure 3) The difference between 1600~
It has an absorption area of 1800 cm ^-1 . It can be easily determined from the IR of isosteviol in Figure 4 that this region is an absorption band specific to the steviol skeleton.
A clear change in absorption peak was observed between the clathrate and the mixture, which proves that γ-CD is included in the stevioside skeleton. Furthermore, the IR of the sweetener obtained in Example 2 below is 5th.
As shown in the figure, a change was similarly observed in the absorption band derived from the stevioside skeleton, which proves that the stevioside skeleton is included. Experimental example 3 Steviosides and γ- of steviosides
Taste test of clathrates using CD Panel selection Five aqueous solutions of 2%, 2.5%, 3%, 3.5%, and 4.0% sucrose are arranged at random and ranked from sweetest to sweetest. make them do We selected 20 people whose Spearman's rank correlation coefficient (Rs) between sucrose concentration and sweetness order was 0.9 or higher. Preparation of sweetener Commercially available γ-CD [RINGDEX manufactured by Sanraku Ocean Co., Ltd.
-C, purity 99%] was dissolved in 40 ml of boiling water, then 16 g of Stevitux-80 [manufactured by Tama Seikagaku Co., Ltd., total content of stevioside, rebaudioside A and C, 82.1%] was added, and a stirrer was added. The mixture was left to cool overnight while stirring. When this solution was freeze-dried and pulverized, 35 g of white powder (referred to as the product of the present invention) was obtained. Taste test The sample solution is 8% in advance to make the sweetness almost the same.
The concentration of the sample solution, which is said to have the same sweetness as the sucrose solution, was determined. In other words, Stevitx-80 is
0.064% (W/V) aqueous solution, the product of the present invention is 0.14%
(W/V) was prepared as an aqueous solution. The sweetness of this test solution was then tested by the 20 panel members mentioned above. The results are shown in Table 2.

[Table] From Table 2, it was found that the product of the present invention and Stevicus-80 had the same degree of sweetness. Since the total contents of stevioside, rebaudioside A and C in both test solutions were almost the same, it was found that inclusion of steviosides with γ-CD did not reduce sweetness. In addition, the product of the present invention has significantly reduced bitterness and is a sweetener with excellent sweetness. Experimental Example 4 Heating Test Selection of Panel Members Ten panel members were selected using the method described in Experimental Example 3. Heating method Stevitux-80 and α-G Sweet [Toyo Seito
Co., Ltd., steviol glycoside content 83%] and the white powder prepared in Experimental Example 3 (referred to as the product of the present invention).
The sweetness of the three types of samples was measured at a sugar concentration of 8% (W/V).
A corresponding aqueous solution was prepared, 50 ml each was placed in a Nessler tube, and heated uncovered on a water bath at 95-100°C for 4 hours. After that, water was added in the amount equal to the amount of water that had evaporated, and the 10 panel members mentioned above tested the sweetness and bitterness of each sample before and after heating. The results are shown in Table 3.

[Table] As seen from Table 3, Stevitx-
All respondents answered that 80 had a low sweetness level and felt a strong bitterness. In addition, those who answered that there was no change in sweetness or bitterness with α-G sweet
There were 2 out of 10 people. On the other hand, the product of the present invention has a sweetness level of 10
Six of the respondents agreed that there was no change in bitterness. From this, it can be seen that this sweetener clearly has superior stability against heating compared to other stevioside products that are clathrates of steviosides with γ-CD. From the results of Experimental Examples 1 to 4 above, it is clear that sweeteners consisting of clathrates formed by clathrating steviosides and γ-CD in the presence of water are conventional steviosides or steviosides and other clathrates. Unlike a simple mixture with polysaccharides, it has a mellow sweetness without unpleasant tastes such as bitterness or acridness, its taste does not change when heated, and its solubility increases greatly, etc. It is recognized that it has excellent properties as a sweetener. The novel stevioside sweetener with improved taste of the present invention can be used as a sweetener as it is, but if necessary, other sweeteners may be added or fillers, salting agents, and acidulants may be added. It can be used in combination with etc. Other sweeteners used include sugar, glycyrrhizin, saccharin, dihydrochalcone, sweet tea extract, phyllodultin, aspartame, maltose, isomerized sugar, glucose, starch syrup, etc., and fillers include dextrin, starch, etc. , lactose, guar gum, tamarind, methylcellulose, gum arabic, etc. as salting agents.
For example, sodium chloride, potassium chloride, etc. are used as acidulants, such as d-tartaric acid, citric acid, dl
-Organic acids such as malic acid, succinic acid, and fumaric acid and their salts. The novel stevioside sweetener with improved taste of the present invention harmonizes well with various taste improvers, such as substances with umami, sour, salty, astringent, and pungent tastes, so that it improves the sweetness of various beverages and favorite foods. It has processing suitability that makes it suitable for attaching. Furthermore, it is difficult to serve as a nutritional source for oral bacteria related to tooth decay.
It is effective in sweetening beverages, luxury foods, etc. for the purpose of preventing tooth decay. The sweetness factor of the novel stevioside sweetener of the present invention is influenced by the ratio of γ-CD to steviosides, but since the sweetness factor is higher than that of ordinary sugar, a small amount added is sufficient to achieve the purpose. Since the amount of decomposition and absorption in the body is small, it can be used to sweeten low-calorie foods and beverages for diabetics and obese people, as well as low-calorie luxury foods, beauty foods, health foods, and diet foods. I can do it. Next, examples of the present invention will be shown, but the present invention is not limited thereto. Example 1 Commercially available γ-CD [manufactured by Sanraku Ocean Co., Ltd.]
RINGDEX-C, purity 99%] Add 200ml of water to 120g, mix well, and add Stevitx-80 to this.
100g of was added and kneaded for 2 hours in an automatic mortar. When the obtained slurry was freeze-dried and crushed,
215 g of white powder was obtained. The content of γ-CD inclusion complexes of steviosides contained in this white powder sweetener was 90.0%. As a result of a taste test, the thus obtained sweetener exhibited a high quality sweetness with a significantly reduced bitterness compared to the raw material Stevicus-80. In addition, the sweetness level was the same or slightly lower than that of the steviosides contained therein. Example 2 83.3 g of potato starch was well suspended in 800 ml of water, and starch liquefaction enzyme neospitase [Nagase Sangyo Co., Ltd.]
After adding 42 mg of [Made in Japan] and heating to 80°C with stirring to obtain a liquefied starch solution, it was sterilized at 120°C for 10 minutes and cooled to 50°C. 833 units of Bacillus ohbensis were added to this liquefied starch solution.
Add the derived CGTase (Japanese Patent Application Laid-Open No. 49-124285),
The total amount of liquid was 900ml. At this time, the pH of the liquid was 6.8. This liquid was kept at 50°C to start the first stage reaction. After 20 hours of reaction, Stevitux-50 (manufactured by Tama Biochemical Co., Ltd., total content of stevioside, rebaudioside A and C 50) was added as a stevioside.
%]30g dissolved in 100ml of warm water was added, and the second stage reaction was continued at 0°C for 24 hours. After the reaction was completed, the mixture was heated at 100° C. for 10 minutes, filtered, and dried to obtain 110 g of a white powder sweetener. The content of γ-CD inclusion complexes of steviosides contained in this powdered sweetener was 34%. In a taste test, this powder had a sweetness level that was equal to or slightly lower than that of the steviosides it contains.
It was a high-quality sweetener with almost no bitterness. Next, some application examples of the sweetener of the present invention will be described. Application example 1 Application to coffee Next, a coffee drink was prototyped by mixing. Commercially available instant powdered coffee [Ajinomoto General Foods, Inc.]
Trademark: Maxim] 13.5g Sweetener obtained in Example 1 1.3g Hot water 970ml Also, as a comparative example, in place of the sweetener obtained in Example 1 in the above formulation, 0.22 g of Stevitux-80 was added.
A prototype was made with the addition of g. As a result of a sensory test conducted by 12 panel members using a two-point preference method, the sweetener obtained in Example 1 was found to have significantly improved bitterness and sarcastic taste compared to Stevitux-80. Application example 2 Application to mackerel miso In a pot, put 4 mackerel fillets (280g), 200ml of dashi soup,
Add 50ml of sake and 3.5g of the sweetener obtained in Example 2,
Bring to a boil, then add pork and simmer over a simmering heat. Next, 30 g of miso was added to the broth, and then boiled, and 3 ml of soy sauce was added to make a trial mackerel simmered in miso. Further, as a comparative example, miso-stewed mackerel was prepared in the same manner as above except that 0.45 g of Stevicus-80 was added instead of the sweetener obtained in Example 2. The results of a sensory test conducted by 15 panel members showed that the mackerel simmered in miso using the sweetener obtained in Example 2 was superior to the comparative product in terms of sweetness and flavor with no bitterness. Ta.

[Brief explanation of the drawing]

Figure 1 is the solubility phase diagram of stevioside and cyclodextrin, Figure 2 is the infrared absorption spectrum of the inclusion complex of stevioside with γ-CD, and Figure 3 is the mechanical mixture of stevioside and γ-CD. FIG. 4 is an infrared absorption spectrum of isosteviol, and FIG. 5 is an infrared absorption spectrum of the sweetener obtained in Example 2.

Claims (1)

[Scope of Claims] 1. A sweetener comprising a stevioside clathrate with γ-cyclodextrin. 2. A method for producing a sweetener comprising a stevioside clathrate with γ-cyclodextrin, which comprises clathrating steviosides and γ-cyclodextrin in the presence of water. 3. Inclusion of steviosides with γ-cyclodextrin, which is characterized by allowing cyclodextrin glucosyltransferase to act on a starch suspension or its hydrolysis solution, then adding steviosides thereto, and continuing the enzymatic reaction. A method for producing a sweetener made from body parts.