JPH067108A - Method for extracting and separating sweet substance of stevia rebaudiana bertoni - Google Patents

Abstract

PURPOSE:To obtain a high-purity stevia sweet substance by efficiently removing impurities such as colored substances and proteins from an extracted solution of leaves of Stevia rebaudiana Bertoni with an alcohol by a simple operation. CONSTITUTION:Leaves of Stevia rebaudiana Bertoni are extracted with a water- soluble alcohol such as methanol. The extracted solution is well mixed with water so as to give a volume ratio of the alcohol/water of 20/80 to 60/40. The mixed solution is separated by an ultrafilter having 20,000-150,000 fractional molecular weight and then by an ultrafilter. The filtrate is further separated by an ultrafilter having 1,000-10,000 fractional molecular weight to efficiently separate impurities such as colored substances and protein.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for extracting and separating stevia sweeteners, and more particularly to stevioside, rebaudioside A which are sweeteners contained in leaves of Stevia rebaudiana and chemicals related thereto. The present invention relates to a method for extracting and separating a stevia sweet substance, which comprises extracting a sweet substance having a structure (hereinafter referred to as stevia sweet substance) by alcohol and separating the substance.

[0002]

Stevia sweet substances are obtained by extracting stevia leaves with water or a solvent such as a water-soluble alcohol. However, this extract contains a large amount of various impurities such as saccharides, proteins, and inorganic elements having different molecular weights and polarities, in addition to the Stevia sweet substance. In addition, many of these substances include coloring substances. For example, when stevia leaves are extracted with water, the extract has a dark brown to blackish brown color, and when extracted with methanol, the extract has a dark green color. These impurities must be removed as much as possible to obtain a highly useful Stevia sweetener from Stevia leaves.

Many methods have already been proposed as methods for removing these impurities and obtaining a high-purity Stevia sweet substance. For example, after adding a coagulant to the Stevia leaf extract obtained by the above method to remove impurities in the extract by flocculation and precipitation, etc., the extract is passed through a porous synthetic adsorption resin. Then, the Stevia sweet substance adsorbed on the porous synthetic adsorption resin is eluted with a solvent, and the obtained eluate containing the Stevia sweet substance is treated with an ion exchange resin or the like for purification. Furthermore, after distilling off the solvent if necessary,
There is a method of crystallizing a stevia sweet substance by adding methanol, ethanol or the like.

However, while the above-mentioned method is excellent in the purification effect of the Stevia sweet substance, it has the following problems. 1) The pretreatment for removing the coagulating precipitate with the coagulant prior to bringing the extract into contact with the synthetic adsorption resin requires a large amount of the coagulating agent, which makes the handling of the coagulating precipitate after use complicated. 2) In the pretreatment prior to contacting the extract with the synthetic adsorbent resin, unless a lipid-soluble substance such as chlorophyll, protein, etc. is sufficiently removed from the extract, a large amount of organic solvent will be generated when the synthetic adsorbent resin is regenerated. It becomes necessary and the deterioration of this resin becomes faster.

On the other hand, the separation method using an ultrafiltration membrane 1) does not require a phase change at the time of separation, 2) can be separated at room temperature, 3) requires less energy, and 4) can simplify the process. In addition, it has been attracting attention as a means for separating and refining biochemical substances and food-related substances in recent years.

As a method for separating Stevia sweet substances using such an ultrafiltration membrane, Japanese Patent Laid-Open No. 51-52200 discloses that Stevia is extracted with water and the extract is treated by the ultrafiltration method. , A method for separating colored substances in an extract by a molecular sieving effect is disclosed.

However, since this method separates only by the size of the molecular weight, when a large amount of impurities having a molecular weight similar to that of the Stevia sweet substance is contained, the removal of the impurities is insufficient and the decolorization rate is high. There is a problem that is low. There is also a problem that the content of the stevia sweetening substance in the membrane permeate (filtrate) is low.

Further, in Japanese Patent Laid-Open Nos. 54-8768 and 8769, Stevia extract is ultrafiltered,
A method is disclosed in which slaked lime is added to the membrane-permeated liquid to adsorb and remove the coloring component. However, this method requires addition of slaked lime and separation operation in addition to ultrafiltration. Moreover, the removal of coloring components and other impurities is also insufficient.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems by efficiently removing impurities such as coloring substances and proteins by a simple operation, and providing a high-purity stevia sweetening substance. It is to propose a method for extracting and separating Stevia sweet substances that can be obtained.

[0010]

The present invention is the following method for extracting and separating a stevia sweet substance. (1) A method for extracting and separating a stevia sweet substance, which comprises adding water to an alcohol extract obtained by extracting stevia leaves with alcohol, mixing the mixture, and separating the mixture with an ultrafiltration membrane. (2) The method according to (1) above, wherein the alcohol is methanol. (3) Volume ratio of alcohol / water is 20/80 to 60/4
The method according to (1) or (2) above, wherein water is added so that the amount becomes 0. (4) The method according to any one of (1) to (3) above, wherein separation is carried out by an ultrafiltration membrane having a molecular weight cut off of 20,000 to 150,000. (5) Separation by an ultrafiltration membrane having a molecular weight cutoff of 20,000 to 150,000, and then separating the filtrate by an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 10,000. The method described in either.

In the method of the present invention, stevia leaves are first extracted with alcohol. Examples of alcohols include water-soluble alcohols such as methanol and ethanol.
Of these, methanol is preferred. The ratio of alcohol to stevia leaf is 10 to 30 ml volume part, preferably 10 to 20 ml volume part of alcohol to 1 g weight part of dried stevia leaf.

The extraction temperature is preferably room temperature to 80 ° C. However, in the case of methanol or ethanol, the temperature is from room temperature to the boiling point. By extracting under such conditions, stevia sweetening substances (stevioside, rebaudioside A and sweetening substances having a chemical structure similar to these)
An alcohol extract containing is obtained. This extract has a dark green color because it contains chlorophyll.

Next, water is added to the above alcohol extract and mixed well. The amount of water added is such that the alcohol / water volume ratio is 20/80 to 60/40, preferably 25/75 to 50.
It becomes / 50. When water is added to the dark green alcohol extract in such a ratio, it is easily dissolved in alcohol, and the solubility of fat-soluble substances such as chlorophyll and other impurities which are difficult to dissolve in water is lowered, and these are precipitated, It becomes an emulsion. When this emulsion-like mixed liquid is separated by an ultrafiltration membrane, precipitates and high-molecular-weight impurities are removed, and a colorless or pale yellow transparent liquid is obtained in the filtrate part (permeation part).

The ultrafiltration can be carried out by a generally adopted method, but it is preferably carried out by the following method. First, the emulsion-like mixed solution was cut to a molecular weight cutoff of 2
It is filtered with an ultrafiltration membrane of 10,000 to 150,000, preferably 50,000 to 150,000. As a result, solid particulate impurities and dissolved high molecular weight impurities in the mixed liquid remain in the concentrating portion (impermeable portion) and are removed. Then, the filtrate is fractionated to have a molecular weight of 1
It is filtered with an ultrafiltration membrane of 000 to 10,000, preferably 1,000 to 5,000. As a result, among the high molecular weight substances compared to the stevia sweet substance, high molecular weight impurities which were not removed by the ultrafiltration membrane of 50,000 to 150,000 are removed in the concentration part.

Most of the coloring substances and other impurities can be removed by filtration through an ultrafiltration membrane having a molecular weight cut off of 20,000 to 150,000, and a colorless or pale yellow filtrate is obtained. Although filtration with an ultrafiltration membrane having a molecular weight of up to 10,000 can be omitted, further filtration with an ultrafiltration membrane having a molecular weight cut-off of 1,000 to 10,000 can provide a stevia sweetener having a higher purity.

In the present invention, the cutoff molecular weight means that the rejection rate is 90% when a standard substance such as dextran having an average cutoff value of the cutoff molecular weight is filtered through a membrane.

By performing the extraction and separation as described above, a colorless or pale yellow transparent separation liquid having a high content of the stevia sweetening substance can be obtained from the stevia leaf. After distilling off the solvent from this separated liquid, a high-purity stevia sweet substance can be obtained by a method such as drying under reduced pressure.

[0018]

As described above, according to the present invention, water is added to the alcohol extract of Stevia leaf and mixed, and the mixture is separated by an ultrafiltration membrane. Impurities such as substances and proteins can be efficiently removed by a simple operation, and a high-purity stevia sweet substance can be obtained. After separation with an ultrafiltration membrane having a molecular weight cutoff of 20,000 to 150,000, the filtrate was filtered to a molecular weight cutoff of 10
By separating with an ultrafiltration membrane of 0 to 10,000, a higher-purity stevia sweet substance can be obtained.

[0019]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples. Example 1 16.0 g of crushed Stevia dried leaves was charged into a 300 ml round-bottomed flask equipped with a stirrer and a cooling tube at the upper part, 240 ml of methanol was added, and the mixture was stirred at 60 ° C. for 4 hours. By this operation, a dark green extract containing a stevia sweet substance was obtained. The extract and stevia leaf were filtered through TOYO5B filter paper, and the stevia leaf remaining on the filter paper was washed with 10 ml of methanol and similarly filtered through TOYO5B filter paper. By filtration twice, 202 ml of a dark green extract (hereinafter sometimes referred to as solution A) was obtained.

90 ml of this solution A was poured into a flask with a stopper, methanol and water were added in the procedure shown in FIG. 1, and ultrafiltration was carried out. First, methanol and water were added to 90 ml of the extract so that the volume ratio of methanol / water was 50/50 and the solid concentration in the mixture was 0.5%, and they were mixed well. This mixed solution (hereinafter sometimes referred to as solution B) became cloudy in green and had an emulsion form.

Then, the emulsion-like mixed solution was subjected to ultrafiltration membrane separation by a membrane separation test device "CarboSep Micro Module" manufactured by Sumitomo Heavy Industries Envirotech Co., Ltd. Ultrafiltration membrane separation was performed using a ceramic inorganic membrane having a molecular weight cut off of 150,000 to obtain 280 ml of filtrate and 48 ml of concentrated liquid. The filtrate was pale yellow and transparent. Of the 280 ml of the filtrate, 40 ml (hereinafter sometimes referred to as liquid C) is subjected to analysis, and 120 ml of the polyether sulfone having a molecular weight cut-off of 3000 is obtained using an ultrafiltration system "Miniset" manufactured by Fuji Filter Industry Co., Ltd. Separation was carried out using an ultrafiltration membrane to obtain 80 ml of a filtrate (hereinafter sometimes referred to as liquid D) and 27 ml of a concentrated liquid. The filtrate was colorless or pale yellow and transparent.

The liquids A, C and D obtained by extraction with methanol and separation with an ultrafiltration membrane were dried under reduced pressure after distilling off the solvent, and the weight of solids was measured. After that, the following analysis was performed with each device and conditions.

[Quantitative Analysis of Stevioside and Rebaudioside A] Apparatus: Hitachi, Ltd., L-6000 High Performance Liquid Chromatograph Column: MERCK, Licrosofa 100NH ₂
(5 μm) Temperature: 40 ° C. Mobile phase: acetonitrile / water (80/20), 1.0 m
1 / min Detection wavelength: 210 nm The content rates of stevioside and rebaudioside A were calculated by the following formula.

[Equation 1]

[Decolorization Rate] Each of the solid substances obtained from the liquids A, C and D by the above method was dissolved in methanol to prepare a methanol solution having a solid concentration of 0.4%, and the solution was prepared. The absorbance of was measured. Hereinafter, these absorbances are referred to as the absorbances of solution A, solution C, and solution D, respectively. Equipment: Shimadzu Corporation, self-recording spectrophotometer UV-21
00 Absorbance measurement wavelength: 430 nm Control solution: methanol Here, the decolorization rate was calculated from the following formula using the absorbance measured by the above method.

[Equation 2]

[Quantitative Analysis of Protein] The protein was analyzed by the Bradford method using a reagent manufactured by Bio-Rad. The protein removal rate was calculated by the following formula.

[Equation 3]

[Rejection Rate] The concentration of stevioside or rebaudioside A (C ₀ ) in the liquid to be subjected to the membrane separation and the concentration of stevioside or rebaudioside A (C ₁ ) in the filtrate were measured and calculated by the following formula. Rejection rate = (C ₀ −C ₁ ) / C ₀ × 100 (%)

The membrane separation conditions and the membrane separation results are shown in Table 1.

[Table 1]

As shown in Table 1, stevia leaves were extracted with methanol into a dark green extract to add water to form an emulsion, which was then filtered through an ultrafiltration membrane having a cut-off molecular weight of 150,000. A decolorization rate of 96.5% was obtained. For the purpose of decolorization, only filtration with this ultrafiltration membrane having a cut-off molecular weight of 150,000 is sufficient.

Further, when this filtrate was separated by an ultrafiltration membrane having a molecular weight cut off of 3000, a decolorization rate of 96.5% was obtained. In addition, the contents of stevioside and rebaudioside A in the filtrate solids were increased by 26% and 28%, respectively, as compared with those in the mixed solution, and the protein removal ratio was 6%.
It was 2.1%.

Example 2 90 ml of the solution A obtained in Example 1 was poured into a flask equipped with a stopper, water was added according to the procedure shown in FIG. 2, and ultrafiltration was performed. First, 90 ml of extract has a methanol / water volume ratio of 25/75, and the solid concentration in the mixture is 0.4.
Water was added to 8% and mixed well. This mixed liquid (hereinafter sometimes referred to as E liquid) was cloudy in green and had an emulsion form. Then, in the same manner as in Example 1, filtered through a ceramic inorganic membrane having a molecular weight cutoff of 150,000,
301 ml of filtrate and 45 ml of concentrate were obtained. The filtrate was pale yellow and transparent. Of 301 ml of the filtrate, 40 ml (hereinafter sometimes referred to as F liquid) was subjected to analysis, and 130 ml was subjected to a molecular weight cut-off of 3 by the same method as in Example 1.
Separation was carried out using an ultrafiltration membrane made of polyethersulfone of 000 to obtain 90 ml of a filtrate (hereinafter sometimes referred to as G liquid) and 27 ml of a concentrated liquid. The filtrate was colorless or pale yellow and transparent.

The liquids F and G obtained by extraction with methanol and separation by an ultrafiltration membrane were dried under reduced pressure after distilling off the solvent, and the weight of the solids was measured. After that, the same analysis as in Example 1 was performed. Table 2 shows the membrane separation conditions and the membrane separation results.

[0032]

[Table 2]

As shown in Table 2, stevia leaves were extracted with methanol and water was added to the dark green extract to form an emulsion, which was then decolorized by filtration through an ultrafiltration membrane with a cut-off molecular weight of 150,000. A pale yellow transparent filtrate with a rate of 92.4% was obtained.

Further, when this filtrate was separated by an ultrafiltration membrane having a molecular weight cut off of 3000, the decolorization rate was 93.5% and the protein removal rate was 76.0%. The contents of stevioside and rebaudioside A in the filtrate solids were increased by 17% and 14%, respectively, compared with those in the mixed solution.

Comparative Example 1 10.0 g of crushed dried Stevia leaf was placed in a round bottom flask having a capacity of 300 ml equipped with a stirrer and a cooling tube at the upper part, 150 ml of methanol was added, and the mixture was stirred at 60 ° C. for 4 hours. By this operation, a dark green extract containing a stevia sweet substance was obtained. The extract and stevia leaf were filtered through TOYO5B filter paper, and the stevia leaf from which the extract was separated was washed with 10 ml of methanol,
Similarly, it was filtered with TOYO5B filter paper. Two filtrations yielded 175 ml of a dark green extract.

75 ml of this extract was poured into a flask with a stopper, and 70 m of methanol was added without adding water.
1 was added. This methanol mixed solution was filtered through a ceramic inorganic membrane having a molecular weight cut off of 150,000 in the same manner as in Example 1 to obtain 113 ml of a filtrate and 31 ml of a concentrate. The filtrate was dark green and could not be decolorized by an ultrafiltration membrane.

Comparative Example 2 10.0 g of crushed dried Stevia leaf was charged into a round bottom flask having a capacity of 300 ml equipped with a stirrer and a cooling part on the upper part, 200 ml of water was added, and the mixture was stirred at 80 ° C. for 4 hours. By this operation, a brownish brown extract containing the stevia sweet substance was obtained. This extract and Stevia leaf are TOY
It is filtered with O5B filter paper, and the stevia leaf remaining on the filter paper is washed with 10 ml of water.
It filtered with the filter paper of. By twice filtration, 190 ml of a brownish brown extract (hereinafter sometimes referred to as solution J) was obtained.

The concentration of solids in the extract was 0.5.
Water was added so that the amount became 10% and mixed well (hereinafter sometimes referred to as K solution). 300 ml of this K liquid is used in Example 1
In the same manner as above, using an ultrafiltration system "Miniset", separation was carried out using a polyethersulfone ultrafiltration membrane with a molecular weight cut-off of 10,000, and 248 ml of filtrate (hereinafter sometimes referred to as L liquid) and 51 ml of concentrated liquid were collected. Obtained. The filtrate was light yellow to brown and transparent.

Liquids J and L obtained by extraction with water and separation by an ultrafiltration membrane were dried under reduced pressure after removing the solvent, and the weight of the solid was measured. Then, it analyzed by the method similar to Example 1. However, an aqueous solution having a decolorization rate of 0.4% solid concentration was prepared, and water was used as a control solution in Example 1.
It was obtained in the same way as. The membrane separation conditions and the membrane separation results are shown in Table 3.

[0040]

[Table 3]

As shown in Table 3, a brown-brown extract obtained by extracting Stevia leaves with water was filtered through an ultrafiltration membrane having a cut-off molecular weight of 10,000 to obtain a pale yellow to brown transparent filtrate. However, the decolorization rate was 73.0%, which was lower than the decolorization rate of Example 1 or Example 2. In addition, the contents of stevioside and rebaudioside A in the filtrate solids did not increase compared with those in the mixed solution, and were smaller than those in the examples.

Comparative Example 3 10.0 g of crushed dried Stevia leaf was charged into a round bottom flask having a capacity of 300 ml equipped with a stirrer and a cooling tube on the upper part, 200 ml of water was added, and the mixture was stirred at 80 ° C. for 4 hours. By this operation, a brownish brown extract containing the stevia sweet substance was obtained. This extract and Stevia leaf are TOY
It is filtered with O5B filter paper, and the stevia leaf remaining on the filter paper is washed with 10 ml of water.
It filtered with the filter paper of. By twice filtration, 180 ml of a brownish brown extract was obtained.

40 ml of this extract (hereinafter sometimes referred to as M liquid) was subjected to analysis, and 75 ml of methanol was added to 75 ml so that the solid content concentration in the mixture was 0.9%, and they were mixed well. . A brown or gray precipitate was formed in the lower layer of this mixed solution (hereinafter, sometimes referred to as N solution).
The mixture containing this precipitate was treated with a centrifuge to remove the precipitate, and 140 ml of a supernatant was obtained. This supernatant liquid was yellow to brown and transparent.

110 ml of 140 ml of the supernatant was treated with a ceramic inorganic membrane having a molecular weight cut off of 20,000 using the ultrafiltration membrane tester "Carbosep Micro Module" as in Example 1, and the filtrate 72 ml (hereinafter , P solution) and 37 ml of concentrated solution were obtained. The filtrate was light yellow to brown and transparent.

The M liquid, N liquid and P liquid obtained by water extraction and separation with an ultrafiltration membrane were dried under reduced pressure after distilling off the solvent, and the weight of the solid was measured. Then, it analyzed by the method similar to Example 1. Table 4 shows the membrane separation conditions and the membrane separation results.

[0046]

[Table 4]

As shown in Table 4, stevia leaves were extracted with water, and the mixture containing methanol was filtered through an ultrafiltration membrane having a molecular weight cut off of 20,000 to obtain a pale yellow to brown transparent filtrate. Although it was obtained, the decolorization rate was 74.0%, which was lower than the decolorization rate of Example 1 or Example 2. In addition, the contents of stevioside and rebaudioside A in the filtrate solids did not increase compared with those in the mixed solution, and were smaller than those in the examples.

[Brief description of drawings]

FIG. 1 is a flowchart showing a separation method according to a first embodiment.

FIG. 2 is a flowchart showing a separation method according to a second embodiment.

Claims (5)

[Claims] 1. A method for extracting and separating a stevia sweet substance, which comprises adding water to an alcohol extract obtained by extracting stevia leaves with alcohol and mixing the mixture, and separating the mixture with an ultrafiltration membrane. 2. The alcohol is methanol.
The method described. 3. The alcohol / water volume ratio is 20/80 to.
Water is added so that it may become 60/40.
The method described. 4. The method according to claim 1, wherein the separation is carried out by an ultrafiltration membrane having a molecular weight cut off of 20,000 to 150,000. 5. The method according to claim 1, wherein after separating with an ultrafiltration membrane having a molecular weight cutoff of 20,000 to 150,000, the filtrate is separated with an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 10,000. The method described in crab.