JPS59120073A - Sweetener and its preparation - Google Patents

Abstract

PURPOSE:To prepare a high-quality sweetener free from bitter taste, astringent taste and unpleasant after-taste, etc., by treating stevioside and/or rebaudioside- A with beta-glucosyl transferase using beta-glucosyl saccharide compound as a substrate. CONSTITUTION:Stevioside and/or rebaudioside-A are dissolved in water together with a beta-glucosyl saccharide compound (e.g. yeast glucan, laminarin, etc.). The aqueous solution is added with a beta-glucosyl transferase produced by microorganisms such as Flavobacterium dormitator, etc., and subjected to the enzymatic reaction at about 45-50 deg.C. The reaction product is purified e.g. by silica gel column chromatography, etc. to obtain the objective sweetener composed mainly of beta-glycosyl stevioside and/or beta-glycosyl rebaudioside-A.

Description

[Detailed Description of the Invention] [Summary and Background of the Invention] The present invention relates to a new modified natural product, more specifically, a novel modified natural β- The present invention relates to a method for producing a sweetener containing ri-1-nocosyl steviosides and β-1-nocosyl steviosides.

Stevia (5tevia Rebaudiana Be
It is a valuable perennial of the Asteraceae family that is native to Paraguay in South America, and its leaves contain diterpene-based glycoside sweet substances 1, mainly stevioside, which are extracted and purified. It is used as a sweetener as a stevia extract or a purified G-mastevia extract.

* As a glycoside sweet substance in Stevia leaves (Yo, a member of Stevioside, Lenoku Udeioside-A, C, 0.h,
The components of zulcoside A and zulcoside A are well known.

In general, stevioside is present in the internal body of Stevia leaves in terms of its content in terms of taste components, and is the main component of the sweetness component in the purified stevioside/stevia extract. Main island C, sweetness quality) Hibuki B is similar to sugar, and has a sweetness factor about 300 times higher than sugar, but on the other hand, it also has a bitter and astringent taste, so these unpleasant tastes are absorbed during the day. There are some remaining drawbacks.

Rebaudioside-A is the second-largest component after stevioside in terms of content, has a higher sweetness than stevioside, and is a high-quality substitute that does not have a bitter or astringent taste and does not leave these unpleasant tastes during the day. It is a flavor component.

Rebaudioside-C and dulcoside-A are both components that contain rhamnose, but the sweetness intensity is about 1 that of rebaudioside-A and stevioside, respectively.
It is only about /10, and the content is so small that it cannot be used as a sweetener. , Rebaudioside-〇, and E are both evaluated to be superior to stevioside in terms of meaty taste intensity and sweetness quality, but since they are trace components, they are difficult to be used as sweeteners. Below, for reference, the chemical structures of six types of Stevia flavor glycoside components that have been confirmed so far are shown.

-,,,J 4 "., Wro'-1" Dioside-A, which is found to be contained in stevia leaves, is superior to stevioside in both sweetness strength and quality, and also in terms of content. It is the second most abundant component after stevioside, and therefore plays an important role in the sweetness of stevia extracts and purified stevia extracts. It is.

Is it possible to extract this Rebaudioside-A from Stevia leaves, purify it, and then use it for legal purposes? (1) Purification costs will be high.

■ The stevioside fraction, which has the highest content, is obtained in large quantities as a by-product, and moreover, it has a stronger bitterness and astringency than the stevioside fraction, making it difficult to use as a sweetener.

Since such disadvantages/disadvantages arise, it becomes less meaningful to implement this method industrially.

Further, chemical synthesis of rebaudioside-A from stevioside has been studied, and it has been recognized that it is actually possible. However, -,1te,...,y synthesis requires a large number of reaction steps and is complicated.

■ The resulting product is considered a synthetic sweetener and cannot be used like a natural sweetener.

There are some drawbacks such as.

Therefore, the present inventor started research on synthesizing rebaudioside-A using enzymes using stevioside as a raw material. β using a compound as a substrate
-By acting glucosyltransferase, β-glycodyl stevioside and θ-glycodyl rebaudioside-A
They also found that these β-glycodyl derivatives have a very good quality of flavor, without bitterness, astringency, or unpleasant astringency.

That is, the present invention converts stevia sweetness components into β-glycodyl derivatives using enzymatic chemical reactions, eliminates the bitterness and astringency that are the drawbacks of stevia sweeteners, and improves the quality of sweetness without unpleasant astringency. It is made of wood.

The inventors have confirmed that stevioside is converted into β-glycodyl steviosides by the enzymatic reaction of the present invention, and that the reaction of the present invention also progresses to rebaudioside-A. did.

The enzymatic reaction in the present invention is not limited to highly purified stevioside and rebaudioside,
It may be a mixture of both, or it may be a stevia extract or a purified stevia extract. In short, as long as the raw material contains components such as stevioside and rebaudioside, these components can be converted into β-glycodyl derivatives to produce the sweetener according to the present invention.

The substrate used in the present invention is a β-glycosyl sugar compound. For example, in the case of β-glucosyltransferase produced by Flavobacterium dormitator, laminarin, pachyman, yeast glucan, lichenan, curdlan, etc. are used. It will be done.

Furthermore, the β-glucosyltransferase used in the present invention is
The enzyme does not necessarily need to be purified, and even the enzyme in the middle of purification, or even the culture sap, can sufficiently achieve the purpose.

The reaction of the present invention is carried out by allowing β-glucosyltransferase to act on water/solution containing a stevia glycoside sweet component and a β-glucosyl sugar compound. The condition or a condition close to it is selected. That is, temperature 30~60℃, PH4.5~
7,5 and the reaction time is preferably 2 to 100 hours.

In addition, the sweet component of stevia glycoside used in the reaction and β-
The ratio of the β-glucosyl sugar compound to the weight of the component of the Stevia package is preferably 0.25 to 0.25, although the ratio of the β-glucosyl sugar compound to the weight of the component of the Stevia package is not particularly limited.
A range of 100 is recommended. Furthermore, there is no particular restriction on the solid concentration of stevioside and β-glucosyl sugar compound in the reaction solution, but from the viewpoint of solubility, it is
It is better to set it to 0%.

The sweetness intensity is almost the same before and after carrying out the reaction according to the invention. However, the quality of the sweetness differs significantly before and after the reaction, and after the reaction, the bitterness and astringency seen before the reaction disappear, and moreover, the sweetness becomes smooth, mellow, and of high quality without any unpleasant residue.

As described above, the reaction solution containing the β-glycodyl derivative produced by the reaction of the present invention can be used as it is as a chemical solution, but if necessary, unreacted substrates are separated and ions are added. It can also be desalted using an exchange resin and concentrated into a liquid product. In addition, it can be dried and powdered to make a powder product, or it can be granulated,
It can also be a granule product.

The sweetener of the present invention obtained in this manner is particularly preferable as a sweetener for foods and medicines because it improves the taste defects peculiar to stevioside and provides a smooth, mellow, high-quality sweet taste.

The present invention will be explained below through experiments, but the technical scope of the present invention is not limited by these in any way.

[Production of sweetener of the present invention]

, Experiment 1 Production of sweetener 1-1 Preparation of enzyme Flavobacterium dortni.
tatorvar gl ucariol yt 1c
ae) with yeast 4%, KH21'040.3%,
FeSO4°71 (200.07% or Ranal sterilized Shita medium CPH8.7) 17 platinum loops were inoculated into 3.000rd (5000-volume Shi+-7y-mentor), 30
"C. Cultured with aeration for 72 hours. After culture, the culture solution was
3. Centrifuge for OOO'xG S to clear culture r
I got the liquid. The lachrymal fluid was concentrated to about 1/1 degree under reduced pressure at 30° C. and salted out with ammonium sulfate at 20-80% saturation. The salted out enzyme was collected by centrifugation at 10,000xG for 5 minutes and
．． ．． 01 iν1 Potassium phosphate' (K-Phos
phate) buffer, pH 5.0, and dialyzed overnight at 5°C.

The dialysate (enzyme 1) was equilibrated with Q, QIM IJ potassium phosphate buffer, p+-15,0. Passed through a SE-Sephadex C-50 column to remove the adsorbed enzyme.
, olra potassium phosphate buffer, Pt (5,Q).
n) Concentrate using a PM-10 membrane filter, and the concentrated solution is biogel (13io Gel)-P.
The gel was filtered through a 150 column. When the purity of the gel-filtered enzyme was tested using electrodynamics, a homogeneous sample could not be obtained, so SP-Sephadex (SeFI
The enzyme was concentrated by rechromatography and roughy using a Hadex C-50 column, and an enzyme preparation homogeneous in disk electrophoresis was obtained. (Enzyme 2) The final injection of Enzyme 2 is 9-, and its protein and C
The a degree was 1.1 Jnf/rri as a result of measuring the absorbance of 280 samples using bovine serum albumin as a standard. Furthermore, the activity of enzyme 2 was 132 units/ml as the activity of β-1,3-glucanase. Here, 1 unit of activity means 0.5 m! of a laminarin solution (5 my/m!, ) dissolved in Q, l + J sodium succinate buffer, )') I 5.8, !
Add 0.5 d of appropriately diluted enzyme solution to bring the total volume to 1
After reacting at 40°C for 10 minutes, Somogyi (
Somogyi) reagent 1- is added to stop the reaction,
The resulting reducing sugar is converted to Somogyi Ne/l/77 (Som
ogyi-Ne1son) method, 10
It refers to the amount of enzyme that produces 1 μmole of reducing sugar (as glucose) per minute.

[Molecular weight]

22.000 (BioGel P-150 Kell
25.000 (SO8 Denki Eido) 28
．． 000 (Ultracentrifuge only L S 20W = 3.46
) The partial specific volume value was set to 0.75 cc/liter.

[Amino acid composition]

Molecular weight determined by ultracentrifugation 28. As a result of calculating the number of amino acids using OOO, it was found that it was made up of 234g1 of amino acids (Table 19゜). Also, from this composition, the partial specific volume value was determined to be 0.711 cc, /g', and this The molecular weight according to the ultracentrifugation method described above is approximately 24,
It becomes 000.

(Left below) Table 1 [Substrate specificity] Table 2 shows the results of investigating the specificity of enzyme 2 using various substrates.
Shown below.

Table 2 shows that this enzyme acts only on substrates having β-1,3 bonds, such as laminarin, pachyman, yeast glucan, and lichenan, and produces reducing sugars.

From this result, it is concluded that the enzyme is β-1,3-dalcanase.

[Optimum reaction conditions]

The optimal reaction conditions for Enzyme 2 are shown below.

Optimum temperature 45-50℃ Optimum pH 5.5-65 Temperature stability Stable up to 50℃, H stability μH 6.0-9.0 [Glucose transfer reaction] P- Nito I:'7E=/Le β-D-Gelcondo (P-Nit
ro'pheny I-β-D-glucosid
e) and laminarin at 0.0. Dissolve 1 μL of enzyme 2 in OIM IJ potassium acid buffer, PH60.
(11μ2) and reacted at 50°C for 20 minutes. After the reaction, 50 μt of the reaction solution was spotted on Toyo F paper height 50, and
-Butanol:Ethanol:Water-5: It was developed in a descending method using a developing solution of 2:2. Paper E was air-dried, and the spot position of the reaction product was confirmed using an ultraviolet lamp.

FIG. 1 shows chromatograms of a solution treated in the same manner as above without adding an enzyme and a solution reacted with the addition of an enzyme.

From the drawing, it can be seen that unreacted P is present in the solution in which the enzyme is added and reacted.
In addition to -nitrophenyl-β-D-curcoside, numerous spots of reaction products produced by sugar transfer can be seen.

The results show that this enzyme has glycosyltransfer activity.

1, this sugar transfer occurs due to the substrate specificity of the enzyme.
It is concluded that - binding causes sugar transfer.

1-3 Production of sweetener Highly purified stevioside 12 is added to 0.01 M phosphorus until spots and peaks of sweet components of stevia-based glycosides other than stevioside are no longer detected in thin-layer chromatograms and high-performance liquid chromatograms. The mixture was heated and dissolved in 50 ml of acid potassium buffer, pH 6.0, and then laminarin 12 was added and dissolved.

Add 0.5 rnl of enzyme 2 to this solution and heat at 50℃ for 1 hour.
I reacted at night. The reaction solution was kept in boiling water for 10 minutes to inactivate the enzyme and obtain a liquid sweetener. (Sample 1) The thin layer chromatogram of Sample 1 is shown in FIG.

For N layer chromatography, HPTLC silica gel 60 thin solvent 700 form:methanol)v:water=30:20:4
After developing and air drying, 20% sulfuric acid was sprayed. It was heated to develop color.

From the figures, it is clear that novel reaction products are obtained from stevioside by the enzymatic reaction of the present invention.

It is also seen that rebaudioside-A is produced by this reaction, albeit in a small amount. This is considered to be reasonable considering that the enzyme used in the present invention is β-],3-curcanase which has glycosyltransfer activity.

Experiment 2 Purification of reaction product Sample 1 obtained in Experiment 1-3 was purified using synthetic adsorption resin HP-5020.
After passing 0 fnl through a column packed with SVI to adsorb unreacted stevioside and reaction products, the column was thoroughly washed with water to remove carbohydrates and salts.

Next, the adsorbate was eluted using a mixed solvent of acetone:water=1=1, and the eluate was concentrated. Dry to about 1.3
You will get 2 powdered sweeteners. (Sample 2) 12 of sample 2 was subjected to silica gel column chromatography (Wako gel c-
2 (10, φ2.j x 95 cm, unfolded 'f8
tiX hachloroform: methanol: water = 80
:20:4) to obtain 200 ml of sample 3.

The thin layer chromatogram of sample 3 is shown in FIG.

From the drawing, it can be seen that sample 8 is highly purified. It can also be seen that it is a different substance from rebaudioside-A.

On the other hand, from FIG. 2, it is clear that reaction products other than sample 3 are present, but they could not be fractionated, probably because they were small in quantity.

Experiment 3 Structure Determination of Sample 3 Sample 3 is a substance different from rebaudioside-A, and is considered to be a glycoside in which one glucose residue is bound to stevioside from the thin layer chromatogram.

On the other hand, the substrate specificity of the enzyme 20 used in this reaction was β-1
, 8 - Since the sugar transfer side to stevioside acts specifically on the bond, as shown in the figure below, sample 8
Since it is known from the spot position of the thin layer chromatogram that it is different from Rebaudioside-A, it is clear that it is not ■.

Therefore, in order to confirm whether the condition is ``■'' or ``■'', the residual glucone bonded to c16- after alkali hydrolysis was chemically cut and thin-layer macromathography was performed. If ■, Nuteviol biozide (Ste
A spot should be generated at the position (violbioside), and a spot should be generated at another position if it is ■. The results are shown in FIG.

From the drawing, it can be seen that the spot position of the alkaline hydrolyzate of Sample 3 is very close to the position of Rebaudioside-B, but slightly shifted downward. In other words, it is considered to be a new substance different from rebaudioside-B.

From this result, it is considered that the chemical structure of Sample 3 is that Curcone 1 residue is β-1,3-bonded to the position of ■ of the ■■■ transfer site of stevioside.

FIG. 5 shows the carbon-13 nuclear magnetic resonance spectrum of Sample 3. Samples were measured for pulse FT-width in pyridine at a concentration of 100 ml/ml at room temperature.

The chemical shift is 123.6185.21 for each of the central signals of the three 1-replet signals of pyridine di.
It was expressed as 8 ppm as 49.8 ppm. As a result, the chemical shift of C-20 of stevioside is 15,9
ppm (literature value 15.4' ppm), C-19
So 17'f, 8 ppm (literature value 177, 0 ppm
) was obtained, so the method for determining the chemical shift was considered to be correct.

From the carbon-13 nuclear magnetic resonance Nubek l-/shikara shown in the figure, β-D bound to C-18-OH was present at 98.1 ppm.
-Anomeric carbon of glucone, 96.2 ppmK
(, -19 varnish 1, anomeric carbon of chilled crucose,
It can be seen that each signal of the anomeric carbon of the β-sophorone moiety is detected at 105.4 ppm. Also,
Comparing the carbon-13 nuclear magnetic resonance vectors of sample 3 and rebaudioside-A, sample 3 has 84.6 ppm and 8
5.6ppm.

105.7 ppm, 106.2 ppm, 1
Each signal of 0.06.8 ppm was detected. Of this signal, 105.7 ppm, 106.2 p
The signals at 106.8 ppm and 106.8 ppm cannot be assigned because they are located very close to each other, but it is thought that one of these signals is a signal of the anomeric carbon of β-D-glucose transferred in the reaction according to the present invention. . In addition, 84.6 ppm, 85.6 ppm1.3-bonds, 2,3゜4 of the introduced sugar.
This is thought to be due to the individual signals being shifted.

Considering these results in total, the chemical structure of sample 3 is considered to be a new substance in which a β-1,3-glucosyl bond is introduced into the G-2 sugar residue of stevioside.

Experiment 4 Sensory test 4-1 Comparison of sweetness level Powdered sweetener according to the present invention obtained in Experiment 2 (Sample 2)
The sweetness level was compared using stevioside as a control.

As test liquids, sample 2 and stevioside were added at 1 volume each.
A sample of 0.00 mm was taken, water was added thereto, the sample was closed with 200 mm, and the sample was subjected to an examination.

The sensory test panel was conducted by five inspectors who were excellent in evaluating sweetness.

As a result, 1 person said that sample 2 had a strong sweetness, and 4 people said that stevioside had a strong sweetness, and the sweetness of sample 2 was considered to be weaker than that of stevioside. This was thought to be because the reaction of the present invention resulted in β-1,3-bonding of D-glucose in an amount equal to or more than the same mole to stevioside, and the molecular weight of the reaction product became larger than that of stevioside.

Therefore, in order to equalize the sweetness level, the concentration of sample 2 was 0.05.
%, and the concentration of the control product Nutebioside was 0.04.
%, O, Oa%, and each solution prepared to have a concentration of 0.02% was compared. The results are shown in Table 8.

Margin Table 3 Below: Comparison of Sweetness Levels From the results shown in Table 3, it can be seen that the sweetness level of the 0.05% solution of Sample 2 is equal to the sweetness level of the approximately 0.035% solution of stevioside.

4-2 Comparison of sweetness quality In order to make the test solutions uniform in sweetness, sample 2 was prepared at a concentration of 0.05%, and the control product stevioside was prepared at a concentration of 0.035% and used for the test. The results were as follows: Stevioside had a good sweet taste. 0 people had a good sweet taste. Sample 2 had a good sweet taste. 5 people unanimously concluded that Sample 2 had a good sweet taste.

The reason why the sweetness quality is good is that the control product stevioside has a unique strong bitter taste and exhibits unpleasant residue, whereas Sample 2 does not have a bitter taste and does not exhibit unpleasant residue. It was said to have a mild and sweet taste.

These experimental results show that the sweetener according to the present invention is a smooth, supple, and high-quality sweetener that does not exhibit the bitter taste or unpleasant residue peculiar to stevioside. this is,
It is concluded that this is due to the conversion of stevioside into β-1,3-glycosyl nutebiosides by the reaction of the present invention.

[Example of the present invention]

Next, first and second embodiments of the present invention will be described.

Example 1 Rebaudioside was determined by thin layer chromatography analysis.
Pure rebaudioside-Alf that has been highly purified to the point that no nutevia-based sweet components other than A can be detected.
1M potassium phosphate buffer, pH 6.0, 50++d!
Further, yeast glucan 12 and 0.5-enzyme 2 were added, and the mixture was reacted at 50° C. overnight. The reaction solution was kept in boiling water for 10 minutes to inactivate the enzyme by heating.
Unreacted yeast glucan was removed by centrifugation at 10,000×G for 10 minutes to obtain a liquid sweetener.

FIG. 6 shows thin layer chromatograms before and after this reaction.

From the drawings, it can be seen that rebaudioside is produced in the reaction according to the present invention.
It can be seen that sugar transfer occurs in A as well.

In addition, when we conducted a sensory test on the solutions before and after the reaction, we found that there was no difference in the degree of sweetness before and after the reaction, but the quality of the sweetness was slightly better in the solution after the reaction than in the solution before the reaction. Admitted.

From this, in the method according to the present invention, the reaction also progresses to rebaudioside-A, producing β-1,3-glycosyl rebaudioside-A, and as a result, the taste quality is further improved. I understand that.

In other words, the sweetener produced by the method according to the present invention has significantly improved sweetness ′α compared to the sweetener that does not carry out the present invention (particularly in the case of stevioside, since the characteristic bitterness and unpleasant residual taste are eliminated) , the effects of the present invention are remarkable. On the other hand, although rebaudioside-A originally has good sweetness, it can be further improved by implementing the present invention.

), Therefore, we believe that the present invention is particularly preferable as a sweetener for foods and medicines, and can be usefully used.

Example 2 Commercially available purified stevia extract (product name: Stevia 5F-AB
, manufactured by Ikeda Toka Kogyo Co., Ltd.) 2? 0. oIM Potassium phosphate buffer 5o- dissolved in Pachyman 22. 2 ml of Enzyme 1 was added and reacted at 50°C overnight. The reaction solution was kept in boiling water for 10 minutes to inactivate the enzyme by heating, and unreacted pachyman and insoluble matter were removed at 10.000 x G.
It was removed by centrifugation for 10 minutes, and the liquid was obtained.

FIG. 7 shows a range chromatogram before and after this reaction.

From the drawings, it can be seen that various reaction products are produced by the reaction of the present invention. That is, since the enzyme used in the present invention reacts with both stevioside and rebaudioside-A, it is thought that spots of such various reaction products appeared. □ On the other hand, when we conducted a sensory test on the solution before and after the reaction, we found that the liquid sweetener of the present invention has a smooth and mellow taste that does not exhibit the bitterness, astringency, or unpleasant residual taste seen in the sweetness before the reaction. It had a good quality sweetness. In addition, the H' taste level before and after the reaction is as follows:
I couldn't see any difference.

[Brief explanation of drawings]

The figure shows P-nitrophenyl-β-D-ri/L. Penose chromatograms of a mixed solution of coside and laminarin treated with enzyme 2 (1) and a control product (21) without the same enzyme. Figure 2 shows a mixed solution of stevioside and laminarin. Sample 1 (3) obtained by treating enzyme 2 with other standard controls (1° steviol bioside, 2
Stevioside, 4: Rebaudioside-A, 5: Rebaudioside-B. 6: Glucose), Figure 3 is the thin layer chromatogram of Sample 3 (2) and other standard controls (1: Stevioside, 3 Rebaudioside-A), Figure 4 is the sample Alkaline hydrolyzate (2) of 3 and other standard reference products (l: mixture of steviolbioside and rebaudioside-B1, stevioside and rebaudioside-A, 3: rebaudioside B14
: Thin layer chromatogram of Stevio-Rubioside),
Figure 5 shows the carbon and nuclear magnetic resonance spectra of Sample 3, Figure 6 shows the spectra of rebaudicide A and yeast glucan treated with Enzyme 2 (3), and other standard controls (1:
Thin layer chromatogram of Valadioside-A and Yeast Glucan, 2: Yeast Glucan + Enzyme 2.4 Nigelcose, 5: Rebaudioside-A), Figure 7 shows commercially available purified Stevia extract, Bakiman and ζ This enzyme 1 is applied (
3) and other standard controls (1: paxin and enzyme 1,
2: Stevia extract purified product and Pachyman, 4: Stevioside, 5: Rebaudioside-A16: Glucose).

Claims (3)

[Claims] (1) -4,t containing β-glycosylstehioside and/or make glycosyl rebaudioside-AM
- A sweetener characterized by: (2) Claim No. 11118i containing stevioside and/or rebaudioside-8!
lν sweetener. (3) Using stevioside and/or rebaudioside-A with a θ-glucosyl sugar compound as a substrate, θ-
1. A method for producing a sweetener comprising θ-glycodyl stevioside Mineji and/or β-glycodyl rebaudioside-A Yi produced by the action of curcosyltransferase.