JPH0334990A - Sugar-transferred stevioside compound, production thereof and stevia-based sweetening using the same compound - Google Patents

Abstract

NEW MATERIAL:Compounds of the formula [R1 is alpha-G, alpha-G<4>-alpha-G, alpha-G<4>-alpha-G<4>-alpha-G (G is D-glucopyranosyl); R2 is H]. EXAMPLE:13-O-[alpha-D-glucopyranosyl-(1 4)-beta-D-glucopyranosyl-(1 2)-beta-D- glucopyranosyl]-19-O-[beta-D-glucopyranosyl]-steviol. USE:A sweetening excellent in quality of sweetness. PREPARATION:A transfer reaction between stevioside and an alpha- glucosylsaccharide compound is carried out enzymatically in the presence of cyclodextrin glucosyltransferase in an amount of <=5u/ml.

Description

[Detailed description of the invention]

(Industrial Field of Application) The present invention provides a novel su-hiori degree l'+! This invention relates to r-transferred compounds, their production methods, and novel stevia sweeteners using them. (Prior art) Stevia is a perennial plant belonging to the Asteraceae family from Paraguay in South America.
It contains flavor components and has been in increasing demand as a natural sweetener in recent years. Stehya U flavoring has a taste level of 100 to 300 times that of sugar, is stable to heat, and is suitable for food processing, while having the highest content of Stevia 1'' among flavorants. Stevioside has one problem in terms of taste quality, such as the onset of taste and taste other than taste is slower than that of sugar, and the sweetness is long-lasting. As a method for improving the t:f 1.115 of
No. 70, Japanese Patent Publication No. 61-28363-℃), although it has been made longer, it is still satisfactory, and the current situation is that it has not been revised to 1 (i'). As you can see, 1) Stevia 1-1 flavoring with improved taste quality is still not available, and 1-1 flavoring is not available yet. Therefore, the inventors of the present invention recognized that it was an important and urgent task to develop a Stevia vomit with improved taste quality, and began research. That is, the present invention aims to provide a novel stevioside transglycosylation compound, a method for producing the same, and a novel Stehya H flavoring agent using the same with improved taste quality. [Means for Solving the Problems] Based on this technical background of Stevia flavored rice, the present inventors aimed to develop a Stevia 1 seasoning that is safe and has excellent emitter quality. R fortune-telling, which has been intensively researched as a goal, a new theory (l; j), I! 1 metastatic substance
] ``14'' was found to be useful as a flavoring agent with excellent taste quality, and the present invention was completed. That is, the present invention is comprised of the following technical matters (1) to (3). (1) The following formula [wherein R1 is α-G, α-G4-α-G or α-G4
-α-G4α-G, R2 are both H (in the formula, G is D
- indicates glucopyranosyl). In addition, below III
is αG, R2 is H, 5-1a, ll+ is cx
-G'-rx-G, R2 or I4 substance is 5-2a,
R is α-G'-a-G'α-G, so the substance in which R2 is 11 is abbreviated as 5-3a]. (2) At least one stevioside glycosyltransfer substance among the three substances in (1) above is a total stevioside glycosyltransfer substance.
A stevia flavoring agent characterized by containing as an active ingredient a stevioside glycosyltransfer substance which is contained in a high concentration relative to an i-transfer substance. (3) The weight ratio of at least one stevioside sugar transfer substance among the three substances described in (+) above to all the stevioside sugar transfer substances is 70% or more. 2) The ``stevia'' flavoring agent listed. (4) By enzymatically transferring stevioside and α-glucosyl sugar compound in the presence of an clathrate compound using cyclode 1-s 1 to ring glucosyl 1-transferase, α-glucosyl sugar is transferred to Ro of stevioside. Stevia 1'' flavoring agent characterized in that the active ingredient is a stevioside glycosyltransfer substance in which compounds are linked through α-1,4 bonds. (5) Stevioside and α-glucosyl sugar compound;
The method for producing a stevioside' transglycosylation compound according to (1) above, characterized in that the transfer is carried out enzymatically in the presence of cyclotex I ring glucosyltransferase in an amount of 0/ml or less. The present invention will be explained in detail below. The present inventors have focused on the phenomenon that a compound obtained by enzymatically transferring an α-glucosyl sugar compound to stevioside has a slightly improved taste quality by 1" compared to stevioside. In other words, when an α-glucosyl sugar compound is enzymatically transferred to stevioside, stevioside is transferred to the 19th and 13th positions.
Because it has D-glucose at the position, for example, cyclodextrin glucosyltransferase (E, C, 2,
4,1゜19) Enzymes such as "hereinafter referred to as CGTase",
When the transfer reaction of α-glucosyl sugar compounds is carried out, the reaction product becomes a complex mixture in which various α-glucosyl sugar compounds are transferred to the end of the bonding sugar at the 19th, 13th, and 13th positions. It is expected that such a mixture contains components with excellent taste quality (1↑) and components whose sweetness quality has not been improved. However, conventionally the individual lJ in this mixture,! ! It is difficult to divide the frequencies of I-Takumi-Kaimono, and in part, in JP-A-61-28363, α-1,4 monoglucopyranosyl stehiosite, α-14-diglucopyranosyl steviosite, α-14-diglucopyranosyl steviosite Although α-14-triglucopyranosylstehiozide has been molecularly identified, the relationship between its structure and sweetness has not been clarified, and its separation has not been sufficient. Therefore, the present inventors developed Stehioli'id! ! 17: It is important to isolate the individual components in the transfer material and clarify the relationship between their structure and 1+4' taste quality in order to produce Stechia sweetener with excellent taste quality. After coming to this realization, we conducted various studies on the isolation of the (ll11)J component in the stevioside transglycosylate and its sweet taste. As a result, the present inventors were able to isolate the individual components of stevioside glycosyltransferases, which had been considered difficult in the past, and also to elucidate the taste quality of stevioside. It, combined with glucose, maltose, and maltotriose, gives a taste quality of 1↑ that is equal to or better than Rebaudioside A, which is currently evaluated to have the best sweetness quality among stevia compounds. Also, 19-CO
It has been found that when O-glucose is transferred only to the 4th position (the 12th position in the formula of claim 1, hereinafter abbreviated as R2) and to both R and R2, there is no or very little improvement in sweet taste quality. Ta. Next, Table 1 shows the structures and taste qualities of a series of stevioside glycosyl transfer compounds, including the novel stevioside glycosyl transfer compounds of the present invention. (Leaving space below) Table 1 Structure of stehiozide saccharide inverting compounds and 1' taste multiple, -1
"Taste Stevioside S-1. a - 1h -2a -2b -2c -3a -3b -3c -3d m ; n O=0 1:0 0:1 2:O l;1 0:2 3;0 2;1 1:2 0;3 Sweetness multiple 60 80 33 05 36 36 17 46 50 21 0 * m: n Number of D-glucopyranosyl bound to R+(m) and 172(n)-1-1Taste multiple-B/A A; Each stevioside sugar 0.025W/W% of transition component
Sweetness of the solution B: Concentration (W/W)% of a sucrose solution that has the same sweetness as a 0.025W/W% solution of stevioside transglycosylation component. B; Equivalent to Rehaudiozide A C; Slightly superior to Stevioside but inferior to Rehaudioside FA D: Equivalent to Stevioside *Book** Compounds of 5-1a, 5-2a and 5-3a is as follows: 5-1a+ 13-0 [α-D-glucopyranosyl(1→4)-β-D-glucopyranosyl-(1→2)-β-D-glucopyrano1 zirco-], 9-0- [β-D-Glucobyranosylcostehiol 2a: 13-0-[α-mal l-syl β-D-glucopyranosyl β-D-glucopyranosyl] [β-D-glucopyranosyl] Biol 3a: 13-0- [α (1→4)-β (1→2)-β 1.9-0- [β Stevioside, that is, glucose, flu-1--su, flu-1-liose, etc. are added to α1,4 to 111 of stevioside. Stehiosite transglycosylates containing compounds bound alone or in combination;
Stevia 1 containing a large amount of sdehyde derivatives containing 4-bonded compounds alone or in combination
” The flavoring agent is conventional Stevia I11! J(1-->/l) (1-)2) 1.9-0 Sweetness quality is superior to the following It turned out that Next, the physical properties of each steviozite transglycosylation compound will be explained. Compounds 5-1a, 5-1b are 13-0 of steviocyto
-4 or 19 position of the terminal glucose on the sophorose side
Either one α-glucose is transferred to the 4th position of C00-glucose. (N, Kaneda et al.
al. Chem, Pharm, l1u11.25.2466
(1977) S-Ia is a colorless crystal with a melting point of 2]1 to 214
°C, and elemental analysis gave the molecular formula of C441170023. This results in compound 5-1a,
It was confirmed that 5-1b has one α-glucose transferred to steviocyto. Next, in C-NMR, S-1a was 95.6pp.
m is the anomeric carbon of 19COO glucose, 98.
130-glucose anomeric carbon in lppm, 10
The anomeric carbon of the terminal glucose of 13-0 sobolose is at 6.6 ppm and 1.03. The anomeric carbon of α-glucose transferred to lppm was observed. Also S-
1114J, the anomeric carbon of 19-Coo glucose at 95.4 ppm, the anomeric carbon of 13-0-glucose at 97.8 ppm, and the anomeric carbon of 13-Coo glucose at 06.5 ppm.
The anomeric carbon of the terminal glucose of 0-sophorose, or 1
The anomeric carbon of α-glucose transferred to 0.02.8 ppm was observed. However, in "C-N)IR, which of compounds 5-Ia, S-1, and b has α-glucose at the 4-position of the terminal glucose on the 13-0 sophorose side of steviocyto, or at the 4-position of 19-COO-glucose. It could not be determined whether the tumor had metastasized or not (Table 2).
A similar C-NMR analysis was conducted in JP-A No. 61-28363, but the binding site of α-glucose was not identified in this analysis. Therefore, the cauldron method (K, Ohtani et al. Te
trahedronLett,, 25.4531 (
1,984)) was tried. In the cauldron method, the 19-Coo-glucosei rule of steviocyto is used to cleave conistert glycosides, and the cleaved sugars are methylated at the cleavage site to become methyl glycosides. The 19th position Memethi glue resting silica gel glue layer Chroma I obtained by this method
・Confirmed by graphography (deployment medium: Ri 1 J Horin-methanol water, (in: 1), compound 5-1a,
The structure of S-1,b was determined. Methyl glucoside from S-1a, 5II
) Methyl glucoside was obtained from each. (Table 6) As a result, compounds S-1a and S, in which one molecule of α-glucose was transferred to steviocytosite using cyclodate strrine transferase
-1b are respectively 34a as the structure is shown in Table 1
is the one in which α-glucose is transferred to the 4th position of the terminal 5ii glucose on the 13-0-sophorose side of steviocyto, and s-ib is the one in which α-glucose is transferred to the 4th position of the 19-COO-glucose in steviocyto. It was decided that there was. Compounds S-2a, S-2b, and S-2c have α-mal I.
- one glucose has been transferred, or one α-glucose has been transferred.
One of them has been transferred one after another. (N.Kane
daeL al. cllcm. Pharm. I! u11
．． , 25, 2466 (1977)) S-2a is a colorless crystal with a melting point of 222-225'C, and elemental analysis gave it the molecular formula of CSOllBQO2B. S-2b
It was a colorless solid with a melting point of 220-223°C, and elemental analysis gave it a molecular formula of C5o111100□,l. S
-2c is a colorless crystal with a melting point of 224-227°C, and the molecular formula of CSOolloo□8 was determined by elemental analysis. This results in compounds S-2a, S-2b, S
It was confirmed that -2c is a product in which one α-mal I·-su or one α-glucose was transferred to steviocyto'. Next, in 13C-N phylum R, S-2a is 95.7pp
m is the anomeric carbon of 19COO-glucose, 97.
(The anomeric carbon of 130-glucose in ippm is 1
The anomeric carbon of the terminal glucose of 130-sophorose is 1, 02.5, 102.7 ppm at 06.7 ppm.
, a transferred anomeric carbon of α-glucose was observed. S-2b has 19COO-glucose anomeric carbon at 95.2 ppm and 130-glucose at 97.3 ppm.
The anomeric carbon of glucose is 106.7 ppm13
-O The anomeric carbon of the terminal glucose of soporose is]
The anomeric carbon of α-glucose transferred at 0.02.7 and 1.02.8 ppm was observed. Also S-2c↓J:
The anomeric carbon of 1,9-COO-glucose is at 95.5 ppm, the anomeric carbon of 13-0-glucose at 97.8 ppm, and the anomeric carbon of the terminal glucose of 13-0-sophorose at 106.5 ppm6, +02.7 , α- transferred to 103.1 ppm
An anomeric carbon of glucose was observed. However, +3
In CNMR, compounds S-2a, S-2b.
Which of S-2c is the 4th position of the terminal glucose on the 13-0-sophoDose side of steviocyto, or 1.9-Coo
- It could not be determined whether one α-maltose or one α-glucose was transferred to the 4-position of glucose. (Table 2) Therefore, the Ohtani method (K. Ohtani et al.
trahedronfelt. , 25. 4537(
1984)) was attempted. Compounds S-2a, S-2 obtained by cleaving only the ester glycoside moiety using the Otani method
When the 19-position methyl glycoside of each of b and S-2c was confirmed by silica gel thin layer chromatography (developing solvent: chloroform-methanol-water, 6:4:1), methyl glucoside was found from compound S-2a. , S-2
Methyl glucoside was obtained from b, and methylfur 1-trioside was obtained from S-2c. (Table 6
) As a result, compounds S-2a and S, which are two molecules of α-glucose transferred to steviocytosite using sifurobukis-1-phosphorus-glucosy-1 full transferase, were obtained.
As shown in Figure 2, S-2b and S-2c have α-maltose transferred to the 4-position of the terminal glucose on the 13-0-sobolose side of steviocyto, and S-2b has the structure shown in Figure 2. One α-glucose is transferred to the 4th position of the terminal glucose on the 13-0 sobolose side of steviocyto and the 4th position of 19-COO-glucose, and S-2c is transferred to the 4th position of the 19-COO-glucose of steviocyto. It was determined that one alpha-mallease had been transferred. Compounds S-3a, S-3b, S-3c, S-3d
is the 4-position of the terminal glucose on the 13-0-sophorose side of steviocyto, or the 4-position of the 1.9-COO-glucose.
One α-maltotriose slucose is transferred to each position. (N. Kaneda et al. Chem. Phar
m. [lull. + 25 + 246 (i (1977)
) S-3a is white 45) rice, and elemental analysis shows that C
s I, II 9. (The molecular formula of 11J was obtained. S-3b is outstanding), and elemental analysis revealed that CS
The molecular formula of 61190033 was given as 8. 5-3c is a white powder, and elemental analysis gave it a molecular formula of C36+190033. 5-3
d is a white powder, and elemental analysis shows that it is C56+19゜0.
33 molecular formulas were given. This allows compound 5-
3a, S3b, 5-3c, 5-3d are the 4th position of the terminal glucose on the 13-0-sophorose side of steviocyto,
Or 1.9-Co. It was confirmed that either one α-maltotriose or one α-maltose and one α-glucose were transferred to the 4-position of glucose. Next, in "C-NMR, 5-3a is 95.6 ppm
The anomeric carbon of 19COO-glucose is 97.8
13 glucose anomeric carbon in ppm, 106.5
The anomeric carbon of terminal glucose of 13-0 sophos 11-s was observed at ppm, and the anomeric carbon of transferred α-glucose was observed at 102.5.102.7 (2C) ppm. S-3b has the anomeric carbon of 19-COO-glucose at 95.5 ppm, the anomeric carbon of 13-glucose at 98.0 ppm, and the anomeric carbon of 13-O- at 106.7 ppm.
The anomeric carbon of the terminal glucose of soporose is 102
．． The transferred anomeric carbon of α-glucose was observed at 3.103.1 (2C) ppm. 5-3c is 9 95.5 ppm to 1.9-COO-glu=r-sno7
/ ? - carbon carbon, 13-glucose anomeric carbon at 97.9 ppm, 13-0-sophorose terminal glucose anomeric carbon at 106.8 ppm, 1.02.6
．． ]03.0 (2C) ppm, the transferred anomeric carbon of α-glucose was observed. In addition, 5-36 has 95.4 ppm of 19-Coo-glue:
The anomeric carbon of 1-su is 97.8 ppm, and the anomeric carbon of 13-curcose is 106.5 ppm of 13-0.
-The anomeric carbon of the terminal glucose of sophorose is 10
2.6.102.9(2C)ppmG, transferred α-
An anomeric carbon of glucose was observed. However, “C
-In NMII, compound S3a, 5-31+, 5
-3(:, Which of 5-3d is 13-0 of steviocyto
- 4th position of the terminal glunis of sophorose, 4) Skull 1
．． It could not be determined whether one α-maltotriose or one α-mal l·-s and one α glun 1-s were transferred to the 4-position of 9-Coo-glucose. (
Table 2) Therefore, the Otani method (K, Ohtani et al, Te
trahedronl, ett,, 25.4537 (
1,984,)) was attempted. In the Otani method, only the Nisder glycoside moiety was removed using 9J 1lIi, and the 19-position methyl glycosides of each of compounds 5-3a, 5-3h, 5-3c, and 5-3d were prepared using silica gel 7! As confirmed by J-layer chromatography (developing solvent: chloroform-methanol-water, 6:,'+:i), methyl glucoside was found from compound 5-3a, methyl maltoside was found from 5-3b, and 5-3c Methylmaltotriozyde 1' was obtained from 5-3d, and methylmaltoside oxide was obtained from 5-3d. (Table 6) As a result, compounds 5-3a, 5-3b, 5-3 in which three molecules of α-glucose were transferred to steviocytosite using cyclodextrin glucosyltransferase
As shown in Table 1, 5-3a and 5-3d are those in which α-maltotriose is transferred to the 4-position of the terminal glucose on the 13-o-sophorose side of steviocyto, and 5-3d is the structure shown in Table 1. 3b is α-maltose at the 4th position of the terminal glucose on the 13-0-sophorose side of steviocyto, and α-glucose is 1 at the 4th position of 19-Coo-glucose.
5-3c is steviocyto 1.
One α-glucose and one α-maltose have been transferred to the 4th position of the terminal glucose on the 3-0-sophorose side, and 5-3d is the terminal glucose [“ 1.9-COO-
It was determined that α-maltotriose was transferred to the 4th position of crucose. Stevioside' IJ7j transfer product produced by the method described in JP-A No. 5,1-5070 and JP-A No. 61-28363, in which an α-glucosyl sugar compound is transferred to commercially available stevioside. 1 The composition of the transition product is
The results of analysis using the method developed by the present inventors are shown in Table 2. (Leaving space below) 2 Table 2 Analysis of each transglycosylated compound in commercially available transglycosylated stevia sweeteners g/
100g Stevia sweetener Commercial product A Commercial product B Commercial product C Commercial product D3.37
8,22 3.44 1.462.0
1 7.08 1.96 1.984
．． 77 2.51 3.45 2.7
93.24 15.32 4.18
1.383.57 2.62 2.43
1.2B2.84 2.41.
2.473.80 4.26 3.
06 1.354.98 7.36
4.08 1.222.70
2.08 1. ．． 323 As a result, among the transglycosylation products, the proportion of compounds transferred only to "R" with excellent taste was about 20 to 45%, and the proportion of transglycosylation products with no improved taste was about 80 to 55. %, and it was assumed that this was the reason why the sweetness quality was not sufficiently improved. Therefore, the present inventors conducted research on the relationship between the proportion of stevioside glycosyltransferases transferred only to R1 and the taste quality. The results are shown in Table 3. (Leaving space below) 4 Table 3 Percentage of stevioside glycosylated compounds transferred only to R and sweet taste *Percentage of compounds transferred only to R1 among glycosylated substances. **The amount of stevioside compound was kept constant. A: Better than Rehaudeiosai FA B: Rebaudeiozai; Equivalent to 'A C: Rehaudeiozai F
Inferior to A, but slightly higher fC than stevioside 5 D: Equivalent to stevioside As shown in Table 2, the stevia sweetener has a proportion of 70% or more of the stevioside sugar transfer product transferred to R1 compared to conventional products. It was found that the taste quality of Stevioside R1 was far superior.Next, R1 of Stevioside contains glucose, maltose,
Stevioside transglycosylates containing compounds in which maltotriose etc. are bonded with α-1,4 bonds, singly or in combination, and glucose, maltose, mal-I-trios etc. in α-1 R of stevioside , a method for efficiently producing a stevia flavoring agent containing a large amount of stevioside transglycosylates containing compounds bound by 4-bonds, either singly or in combination, will be described. As for the method of using chemical synthesis method in combination, after transferring an α-glucosyl sugar compound to F1G using CGTase, 19-glucosyl sugar compound is transferred to Stevio-ruvioside, which is obtained by deglucosylating the glucose at position 19 of Stevio rubioside. Stevia 4 contains a compound in which glucose, maltose, malto-l-liose, etc. are bonded to R5 of stevioside through an α-1,4 bond by glucosylating the carboxyl group at the position. ”. Flavors can be manufactured. (Reference; Journal of the Chemical Society of Japan 1981. (5), P726-735) As an enzymatic method, stevioside and α-glucosyl sugar compound are
When transferring with Tase, by adding various clathrate compounds, compounds in which glucose, maltose, malto]-liose, etc. are bonded to R1 of stevioside through an α-1,4 bond can be used alone or It is possible to efficiently produce a stevia sweetener containing a large amount of mixed stevioside glue. Examples of clathrate compounds include cyclodextrin and zylic bisdesmocytes. In particular, cyclodextrin itself is more preferred since it serves as a donor for the transglycosylation reaction by COTase. Furthermore, in the enzymatic method, by adjusting the concentration of cc'rase used in the transfer reaction to 5 u/mF or less, Sla, 5-2a, 5-2a, which has excellent sweetness,
Only 5-3a could be produced in high yield. This is because the addition reaction at position 13 of steviol proceeds specifically by selecting the above enzyme concentration. This method is
Stevioside 1% and soluble starch or T- as substrate
Cyclodextrin alone or in mixture 1.00-2.
This is carried out by adding 0.5 u of Leik I'te, 1-strine synthase (manufactured by Amano Pharmaceutical Co., Ltd., trade name: Contizyme) to a mixture with 61% and treating. As the above-mentioned enzyme, any cyclodextrin synthase other than CGTase can be used as long as it specifically promotes the addition reaction at the 13th position of steviol. The enzymatic reaction in the present invention may be either a hatch type reaction or a continuous type reaction using an immobilized enzyme. Also, the reaction time is
If it lasts too long, by-products other than the target substance will be produced and the taste quality will deteriorate, so it is appropriate to leave it within 32 hours. The pH may be in any range as long as the enzymatic reaction can proceed, and is preferably 3.0 to 8.0. 8 Below, the present invention will be specifically explained with reference to Experimental Examples and Examples. [Experiment example] 1) Transglycosylation reaction on stevioside Stevioside (hereinafter abbreviated as S) extracted from stevia leaves 1.0.7 g
and 13.6 g of soluble starch and 320 iR of water. IM Acetate Huffy-(pH=5.6) 3.16
mfl and CC, Tase (Hacilus stearothermophilus production 1400 units/ml) 0.41 ml
was added and incubated at 40'C for 16 hours to carry out the reaction. The enzyme was inactivated by boiling for 15 minutes. (Figure 1
) 2) Separation of transglycosylation products After filtering the reaction mixture obtained in 1), porous resin column chromatography (DIAION IIP-20
), water, 40% methanol, 65% methanol
Elution was performed sequentially with 80% methanol, methanol, and acetone. 7.14 g of the 65% methanol fraction was subjected to silica gel column chromatography (solvent: chloroform methanol-water, 13:7:l), fr-1 to fr-8
was obtained in the yield shown in FIG. Further 9 silica gel column chromatography loloform-methanol-water, fr-2, -4, -6, fr-2; 13:
7:1 fr4; 10:5:1. fr-6;
6:4:1) respectively. 1.7 g of the fraction with 1 molecule of α-glucose transferred to S (Konni top), 1.13 h of the fraction with 2 molecules transferred (S-2), and 1 g of the fraction with 3 molecules transferred (Ko-1). Each yield was .38 g. Next, high performance liquid chromatography (column: TSK
Separation was performed using a SL00S-120T). Loss 1 (
(Solvent: 70% methanol), the interlocking is different from (Solvent: 62.5% methanol) 7a, , S-2b
, From the previous article, Difference 1 (solvent: 59% methanol),
Seiko, S. and S. were isolated with the yields shown in Figure 1. 3) Confirmation of structure The structure was determined by elemental analysis, 'H-1nlR,'C-NMR, and analysis of the reaction product using a lithium iodide reagent.In addition, the appearance, specific rotation, and The melting point was measured. Each analysis method is described below. H-NMR: JI Mi OL-GX400 conductive nuclear magnetic resonance apparatus (manufactured by JEOL Ltd.) 0 Deuterated pyridine solution, 400 MHz, 30°C'C Ni IJMR: JEOL-FX100 nuclear magnetic resonance apparatus (
(manufactured by JEOL Ltd.) Deuterated pyridine solution, 100 MHz, 30'C Elemental analysis: Yanagiki C, I (, N coder MT-3 type (Yanagimoto Seisakusho) Specific optical rotation: Union Automatisoku Decital Volarimeter Pl+-101 Melting point: Elemental analysis (Table 3) using Dancro Yanagiki melting point measuring device Mono-α-14-
1111 tW was determined to be a glucosyl transfer product. Furthermore, when 5-1a is reacted with a lithium iodide reagent, methyl glucoside is obtained, and 5-1b is methylmaltoside 1, so 5-1a is 130-
[α-D-glucopyranosyl-(1→4)β-D-glucopyranosyl-(1→2)-βD-glucopyranosyl)-19-0-[β-D-glucopyranosyl]-steviol, S-1, b is 13 -○1 [β-D-glucopyranosyl-(1,->2)-βD-
glucopyranosyl)-1,9-0-(α-I]glucopyranosyl-(1→4)-β-D-glucopyranosyl)-
It turned out to be stehiol (Figure 2). '11
-NMR and "C-NMI?" analysis (Table 34) confirmed that the difference was a di-α-1,4 glucosyl transfer product. Also, the reaction with lithium iodide reagent 5-2a is 13-○-[α-maltosyl-(1
→4)-β-D glucopyranosyl-(1→2)-β-D
-curcopyranosyl]-1,9-0-(β-D-glucopyranosyl)-steviol, the difference is 13-0-(αD
-Glucopyranosyl(1→4)-β-I)-Glucopyranosyl-(1→2)-β-D-glucopyranosyl)-
11-0-(α-1]-glucopyranosyl-(1→4)
-β-D-glucopyranosyl]steviol, the difference is 1
．． 3-0-(β-D-glucopyranosyl-(1-)2)
-β-D-glucopyrano2syl]-19-0-[α-maltosyl-(1→4)-β
-D-glucopyranosyl]-steviol (Figure 2). 'II-NMR and 13C-N
MI? According to the analysis (Tables 4 and 5), L amount, continuous use 5-3c
, 5-3d was confirmed to be a tri-α-1,4-glucosyl transfer product. Furthermore, when a reaction with a lithium iodide reagent is carried out, methyl glucoside, methyl maltoside, methyl maltotriside, and maltotetrozide are obtained, respectively.
Maltotriosyl-(1→4)-β-D-glucopyranosyl-(1→2)β-D-glucopyranosyl]-19-
○-[β-D glucopyranosyl]-steviol, 5-
3b is 130-[α-maltosyl-(l→4)-β-D
Glucopyranosyl-(1→2)-β-D-curcobyranosyl: l-1,9-0-[α-D-glucopyranosyl-(]→4)-β-D-glucopyranosyl] steviol, V is 13-C1- [α-D-glucopyranosyl-(1→4)-β-I]-glucopyranosyl=(1→2
)-β-D-glucopyranosyl]3 1.9-0-(α-Mal I syl-(■→4)-β-D
glucopyranosyl]-steviol, S-3cj 13
0-[β-D-glucopyranosyl-(1→2)β-D-
Curcopyranosyl:]-1,9-○-[α-maltotriosyl-(1-)4. )-β-1)-glucopyranosyl]-steviol (Figure 2). Also, 'II-NMI?' of each metastasis? Speck l-le 3rd to 5th
In the figure, 5-1a, 5-3a's 'C-NMR
The spectra are shown in Figures 6-8G. Table 6 shows the appearance, specific rotation, and melting point of each transition product. (Leaving space below) 4 Table 4 Elemental analysis value of each sugar transfer product CaaH7oOzs As H2O C3GHII002+1 20 CsOH800ze 120 C56) 190033 H20 5 6 7 8 9 4 Table 7 Appearance, specific rotation, melting point of each sugar transfer product Colorless crystal Colorless crystalColorless crystalColorless crystalColorless crystalWhite crystalWhite crystalWhite crystalWhite crystalWhite crystal+12.7°16.1°138.1°-1-32,6°+1.2.0°+54.5°-1-48 , 6゜-1-93,6' +68.6゜211~214 217~220 222~225 220~223 224~227 (Leaving space below) 1 Table 8 1 frame of silica gel thin layer of Otani method reaction product I. Migration rate by graph * Migration rate Distance traveled by solute / 1-city separation between origin and solvent tip

[Coloform: methanol: water = 6:4:1 color development Sulfuric acid color development 120°C 4) Calamity and H゛Taste overtones 1 and 11 Taste quality 4) Test of taste multiple and sweetness quality for each transition product obtained I did the word 1. The sweetness factor was determined by a sensory test using the limits method, and the concentration of a sucrose solution with the same taste level as the sample (equivalent stimulation PSE) was determined. The results were shown in Figure 2. (Example) Example 1 Stehiozide 50■ and T-cyclodextrin 10
Dissolve 1 mg in Karyu water and heat at 40°C130°C13
After incubation for an hour, COTase (Hacilus stearothermophilus production 50 unit/mf
l> to 0.1mff1. IM acetate Hanoff y-(p
H5,6) Add 0.011mR and heat to 40°C11
The reaction was carried out for 6 hours. After completion of the reaction 3: The enzyme was inactivated by leaving it on a boiling water bath for 15 minutes. The reaction products were then analyzed as previously described and the results are presented in Table 9.
Shown below. In addition, 1 core of unrefined Stehio'su'ite was collected. The Suichihia l1llA,ll obtained in this way had an extremely excellent taste quality. Table 9 Transglycans Weight (mg) S-1a 12.67S-1b
],, ]6 8S-2a 8.31 S-2b 2.56 S-2c ],, 26S-3a
3.57S-3b 1
．． ．． 21S-3c i, 46 S-3d 0.03G total 32.
756 1 Hi rate (%) 38.7 5.1 25.4 7.8 3.8 10.9 3.7 4.5 0.1 100.0 4 Example 2 4150 mg of stewage osa and 50 mg of soluble starch, After dissolving 70 mg of zyclic hisdesmoside in 1 ml of grass distilled water and pre-incubating with IIO'C for 13 hours, CGTase (50 uni
t/mQ, ) to 0.1 mL IM acetate y-(
pH5,6) Add 0.011 mfl and heat at 40°C1
The reaction was carried out for 6 hours. After the reaction was completed, the mixture was left on a boiling water bath for 15 minutes to inactivate the enzyme. The reaction products were then analyzed as previously described,
The results are shown in Table 10. Incidentally, unreacted stehiozite was collected. The Stechia sweetener thus obtained is a sweet sugar transfer product -1a -1b -2a -2b -2c -3a -3b -3c -3d Total Table 10 Weight (mg) 7.54 0.35 4.2] 0゜16 0.26 3.76 0.076 0.062 0.15 16.568 Ratio (%) 45.5 2.1 25.4 0.9 1.6 22.7 0.5 0.4 0.9 100.0 Example 3 Reaction products obtained in Example 1 and Example 2 +4]
The component that had transferred to the 3rd place was separated and the quality of the emetic substance was evaluated, and it was found to be very good. (See Table 1 + t0 cell example 4 6 Stevioside was added with a single substrate of soluble starch or T-cyclodextrin and a mixture of both at pH 5.
, 6 in 0.01M acetate buffer, cyclodextrin synthase (manufactured by Tenshu Pharmaceutical Co., Ltd., trade name Contilime 60).
Table 11 Stevioside transglycosylation reaction conditions Sample 7 Soluble starch 1.61 The content of stevioside and 1!L 21+, !4$1+ metabolites was quantified using HP+-C) under the following conditions.
Items that have been completed are listed as "Other". (Table 12) HP
LC analysis conditions column: YMG-PACK R-ODS-54,6X
250mm8 Medium = 57% methanol Speed: 1.0ml/min Output: 210nm Degree: SO'C Water (margins below) 9 Table 12 Components of each sample (% by weight) What is water splitting table (%)? (S-1a+S-2a)/(S-1a
+5-1b+5-2a+5-2b+S-2c) 100
Shows the fraction. In addition, 5-3a, 5-3b, 5-3c and 5-3d
Each component of sample 7 cannot be quantified under the two-legged condition. From the results of 8.15.16, the effective concentration of cyclodextrin glucosyltransferase is 5 u/m, l! In the following case, the transglycosylation reaction is performed at R3 of sophorose at position 13 of stevioside with an efficiency of 70% or more. These three or more sugar compounds are cut back by enzymes such as β-amylase to increase the content of 5-1a and 5-2a, which have better sweetness and a higher sweetness ratio, and further improve the sweetness. I can do it. The obtained samples 14 and 18 were examined for their sweetness multiple and quality. The sweetness factor was determined by a sensory test using the limits method, and the concentration of a sucrose solution having the same sweetness as the sample (equivalent stimulation PSE) was determined. The sweetness multiple was 150 for sample 14 and 136 for sample 18. The sweetness quality was equivalent to that of a 4% sucrose solution and was examined by 10 sensory testers who were excellent in sweetness, and 8 of them agreed that Sample 14 had a sweetness quality that was closer to that of sucrose. [Effects of the Invention] The stevia sweetener of the present invention is a novel stevia sweetener with excellent sweetness, and its usefulness is remarkable compared to conventional products. Furthermore, the present invention provides an efficient method for producing a stereotransfer compound with excellent sweetness.

[Brief explanation of drawings]

Figure 1 shows the separation method and yield of stevioside glycosylated products, and Figures 2 to 4 show 'H-N' of each stevioside glycosyltransferred product.
+'IR spectra are shown, and FIGS. 5 to 7 are diagrams showing 13C-NMR spectra of stevioside transglycosylates.

Claims (1)

[Claims] 1. The following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R_1 is α-G, α-G^4-α-G, or α-
G^4-α-G^4-α-G, R_2 both represent H (in the formula, G represents D-glucopyranosyl)] A stevioside transglycosylation compound represented by the following. 2. Stevia, wherein at least one stevioside glycosyl transfer substance among the three substances according to claim 1 contains as an active ingredient a stevioside glycosyl transfer substance that is contained in a high concentration relative to all stevioside glycosyl transfer substances. sweetener. 3. The stevia sweetness according to claim 1, wherein the weight of at least one stevioside glycosyltransfer substance among the three substances according to claim 1 is 70% or more by weight relative to all the stevioside glycosyl transfer substances. fee. 4. The 4-terminal glucose position of 13-0-sophorose of stevioside (Claim 1 R_1 position in the paragraph description formula, hereafter R_1
A stevia sweetener characterized in that the active ingredient is a stevioside transglycosylate in which an α-glucosyl sugar compound is bonded to a stevia (abbreviated as (abbreviated)) through an α-1,4 linkage. 5. 5u of stevioside and α-glucosyl sugar compound
2. The method for producing a stevioside glycosyltransfer compound according to claim 1, characterized in that the enzymatic transfer is carried out in the presence of cyclodextrin glucosyltransferase at an amount of less than /ml.