KR101724569B1 - New method for producing rubusoside - Google Patents

Abstract

The present invention relates to a process for preparing lucsoside from stevioside, comprising the step of treating viscose or naringin or an agase or both, a composition for degrading glucose of stevioside containing biscotrim, naringinase or both as active ingredients , Stevioside and viscose, naringin and / or a complex containing both, and a food composition, pharmaceutical composition or cosmetic containing the complex. In the present invention, an enzyme capable of producing rubusoid is extracted from a commercially available enzyme with stevioside as a substrate.

Description

New method for producing rubusoside < RTI ID = 0.0 >

The present invention relates to a process for preparing lucsoside from stevioside, comprising the step of treating viscose or naringin or an agase or both, a composition for degrading glucose of stevioside containing biscotrim, naringinase or both as active ingredients , Stevioside and viscose, naringin and / or a complex containing both, and a food composition, pharmaceutical composition or cosmetic containing the complex.

In recent years, the use of artificial sweeteners such as cyclamate, saccharin and its salts has been banned or restricted in terms of stability. In contrast, sugar intake has been avoided due to health problems There is an urgent need to develop natural sweeteners that can replace them.

At this point, stevioside is a low-calorie sweetener compared to sugar, and its sweetness is about 200-300 times higher than sugar, so its price is rapidly rising. Stevioside is a sweetener extracted from Stevia rebaudiana BERTONI, a perennial herbaceous chrysanthemum which originated in South America Paraguay, and is composed of stevioside, rebaudioside A, B, C, E and so on. Rubus suavissium S. LEE is a kind of tea that has been used in China and Japan for a long time in the name of "spike". It is a kind of tea which is called polyphenol (tannin, flavonoid) and Rubusoside Contains a large amount of glycosides, Rubusoside has about 150-200 times the sweetness of sugar, non-calorie, anti-cavitation and clean feeling (cool taste) is a sweetness material. The isosteiovol skeleton is made of uglycon beta-glucosyl glycoside.

The market for stevioside has started to be formed in accordance with the government's regulations on the use of saccharine since 1990, and it is now mainly used as a saccharine substitute sweetener and a low calorie sweetener. Stevioside is a non-calorie diet sweetener that is not absorbed in vivo. In addition, it is known that it is suitable for use in various processed foods because it does not change the sweetness, and it is non-bioavailable for pickled foods because it forms a precipitate in 5-18% saline solution. However, aftertaste is long and has a disadvantage of bitterness and discomfort in addition to sweetness. Therefore, there is a problem that the usage and the usage limit are generated, and it is necessary to improve the quality of the stevioside.

(1) a method of adding one or more natural saccharide sweeteners such as sugar, glucose, and fructose, (2) a method of combining with a salt of amino acid or amino acid, (3) a method of adding cyclodextrin (JP-A-60-98957), and the like. However, the above method has a disadvantage in that a considerably large amount of additives must be added and stevioside is lost as a low-calorie sweetener. Another method for improving quality was to make a "rebaudioside A" analogue, which was an excellent model of sweetness and sweetness, utilizing biotransformation enzyme technology. The only commercially available enzyme-treated stevioside with a total of 1-12 glucose added randomly to the 13-OH or 19-OH site of stevioside using commercially available CGTase. However, CGTase can not selectively add only one glucose, and if added glucose is completely decomposed by intestinal microorganisms and converted to an energy source, it has the disadvantage that the number of calories increases by the number of hydrolyzed glucose .

Until now, the only enzyme that degrades stevioside is beta-glucosidase derived from Clavibacter and is an enzyme with high degradability of glucosyl ether. Glucosyl ether can be obtained by saponification of stevioside.

Therefore, the present inventors have found that the use of stevioside, which is a relatively low-cost material compared to rubusoidal, is an economical method that provides a refreshing feeling, and rubusoidal, which is a material having excellent skin irritation and moisturizing effect, And to mass-produce them through biotechnology.

The main object of the present invention is to provide a process for preparing one or more glucose-free steviosides, including the step of treating the stevioside with Viscozyme enzyme, Naringinase or Naringinase enzyme or both.

Another object of the present invention is to provide a composition for degrading glucose of stevioside containing a biscuit enzyme, a naringinase or an enzyme or both as an active ingredient.

It is still another object of the present invention to provide a method for improving the sweetness of a stevioside-containing plant or food, which comprises treating a stevioside-containing plant or food with a biscozyme enzyme or a naringinase enzyme.

Still another object of the present invention is to provide a stevioside; And a complex containing a biscozyme enzyme, a naringinase enzyme, or both.

In one embodiment, the present invention provides a method for preparing one or more glucose-free steviosides, comprising the step of treating the stevioside with a biscozyme enzyme, a naringinase or an enzyme, or both.

It is preferable that the at least one glucose-eliminated stevioside of the present invention is selected from the group consisting of rubusoidal, steviol monoglucoside, and steviol.

In another aspect, the present invention provides a composition for degrading glucose of stevioside, which comprises a biscozyme enzyme, a naringinase or an enzyme, or both as active ingredients.

In another aspect, the present invention provides a method for improving the sweetness of a stevioside-containing plant or food, which comprises treating a stevioside-containing plant or food with a biscozyme enzyme or a naringinase enzyme.

In the present invention, the treatment of a viscose enzyme or a nitinergic or an enzyme may be performed by treating these enzymes themselves; It is possible to treat microorganisms producing biscozyme enzyme or naringinase enzyme.

All of the enzymes having beta-glucosidase activity do not form rubusoidal with the stevioside as the substrate.

The present inventors have found that a buffer solution is added to a stevioside suspension suspended in physiological saline and various enzymes such as Tricoderma logibrachiatum cellulase, Amano Corporation Visozyme, Aspergillus niger naringinase, A. niger hemicellulase, A. niger hersperdinase, Amano Corporation Ultraflo, A. niger cellulase, Amano Corporation celluclast, by the addition of beta-glucosidase Almond) for reaction at each optimum temperature, The reaction product was subjected to thin layer chromatography or high performance liquid chromatography to confirm whether or not lucusoid was produced. As a result, it was confirmed that Viscozyme and Naringinase could form lucusoside.

Therefore, biscozymes and naringinases and azelaases were selected as enzymes for selective hydrolysis of glucose at the 13-OH position of stevioside, and the present invention uses these enzymes to prepare rubusoidal and steviol monoglucoside . (Fig. 1).

"Stevioside" is a sweet substance contained in leaves of stevia, a perennial plant of Asteraceae. Stevia sweeteners have excellent sweetness several hundred times as much as sugar but are characterized by a slow rise in sweetness compared with sugar, a characteristic that the so-called rise in sweetness is slow, a characteristic in which the sweetness remains relatively long as an aftertaste, Feature. In addition, stevia side, which is abundantly contained in the above-mentioned stevia sweetener, has defects accompanied by unpleasant taste and bitter taste unique to aftertaste, and it is clearly felt uncomfortable when used in a coffee drink or a soft drink. Accordingly, the present invention seeks to produce rubusoidal with improved taste from stevioside, which is a relatively inexpensive material.

[Chemical Formula 1]

 The above formula (1) represents stevioside, and is represented by a schematic diagram in which two molecules of glucose and one molecule of glucose are respectively connected to steviol.

On the other hand, "Rubusoside" can be obtained with a yield of 5% or more in the leaf of Rubus suavissium S. LEE, which is a multi-component raw material of Rosaceae, and can be obtained from non-calorie, It has a diminishing (cool taste) sweetness material. As a substitute for sugar, it can provide good supplementary effects to people who have diseases such as obesity, diabetes, cardiovascular disease, kidney. In addition, rubusoide is a di-terfan glycoside which is excellent in skin irritation mitigating action, and contains natural moisturizing factors and amino acids, and is excellent in moisturizing effect, so that it can be applied as a cosmetic material.

(2)

The above formula (2) represents rubusoside, and is represented by a schematic diagram in which one molecule of glucose and one molecule of glucose are connected to steviol, respectively.

"Viscose" is a glucosease produced from Aspergillus fungi. In the present invention, the viscose is Aspergillus aculeatus is preferably used. In some cases, microbes capable of producing and secreting viscose can be used to prepare rubusoidal from stevioside. At this time, the treatment condition of the viscose is not particularly limited, but it is preferable to treat the viscose with stevioside under the conditions of pH 4 to 5 and 40 to 50 ° C to prepare rubusoid.

"Narin or Agase" is a long nalgin hydrolytic enzyme that clears the bond between laminase and glutinase of Narin Long to remove bitter taste. The naringin or azeotropic agent is Penicillium decumbens . In some cases, rubusoid can be prepared from stevioside using a microorganism capable of producing and secreting the naringin or an agase. At this time, the treating conditions of the naringin and the azine are not particularly limited. Preferably, the treating conditions are pH 4.5 to 5.5 and 35 to 50 ° C, more preferably pH 5.0 to 5.4 and 37 ° C, To prepare rubusoidal.

In yet another aspect, the present invention provides a stevioside; And a complex containing a biscozyme enzyme, a naringinase enzyme, or both.

Here, the stevioside may be stevioside itself or a mixture thereof, or a stevioside-containing plant or food. In addition, the complex of the present invention may be in a form in which stevioside is formed with a decomposition product of stevioside by an action of a biscozyme enzyme, a naringinase, an enzyme enzyme or both.

The complex of the present invention may be obtained by cleaving glucose from stevioside by a biscozyme enzyme or a naringinase enzyme, and then removing the biscozyme enzyme or the naringinase enzyme.

The degradation product of the above-mentioned stevioside may contain lubucoside, steviol monoglucoside, or both.

In another aspect, the present invention provides a sweetener composition comprising the complex.

Suitable uses for the sweetener compositions provided herein are well known to those skilled in the art. For example, the sweetener composition may be used in place of conventional sweeteners in a sweetener composition such as feed, food, beverage, medicine, tobacco, functional foods, oral care products /

The sweetener composition may be present in the sweetener composition in an amount sufficient to impart the desired sweetness to the sweetener composition.

In yet another aspect, the present invention provides a food composition comprising the complex as a sweetener.

The term "food composition" is intended to encompass all types of food compositions including, but not limited to, protective hydrocolloids (eg gums, proteins, modified starches), binders, film-forming agents, encapsulating / encapsulating materials, wall / sheath materials, matrix compounds, coatings, emulsifiers, , Solubilizing agents (oils, fats, waxes, lecithins, etc.), adsorbents, carriers, fillers, auxiliary compounds, dispersants, wetting agents, processing aids (solvents), fluidizing agents, taste- -Forming agents, antioxidants and antimicrobial agents.

In addition, multivitamins and mineral supplements may be added to the food composition of the present invention to obtain the proper amount of essential nutrients that are missing in some diets. Multivitamins and mineral supplements can also be useful for the prevention of disease, and protection against nutritional loss and deficiency due to life patterns.

In another aspect, the present invention provides a dental caries prevention or food composition containing the complex.

The complex according to the invention may be in any form suitable for administration to the body, in particular any form usual for oral administration, for example food or feed (additives / supplements), food or feed pre-mixture, fortified food or Solid forms such as feeds, tablets, pills, granules, dragees, capsules and inhaled formulations such as powders and tablets; Or liquid, such as solutions, emulsions or suspensions, for example, beverages, pastes and oily suspensions. The paste may be incorporated into a hard or soft shell capsule whereby the capsule may be a matrix of, for example, (fish, pig, poultry, cattle) gelatin, plant protein or lignin sulfonate. Examples of other application forms are transdermal, parenteral or injection dosage forms. The dietary and pharmaceutical compositions may be in the form of controlled (delayed) release formulations.

The pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable excipient, diluent or adjuvant. For example, standard techniques disclosed in Remington ' s Pharmaceutical Sciences, 20th edition Williams & Wilkins, PA, USA can be used in the formulation. For oral administration, tablets and capsules containing suitable binders such as gelatin or polyvinylpyrrolidone, suitable fillers such as lactose or starch, suitable lubricants such as magnesium stearate, and optional additional additives, Is preferably used.

In another aspect, the present invention provides cosmetics containing the complexes described above. In the present invention, cosmetics characterized by exhibiting a moisturizing effect are preferable.

When used in the form of cosmetics, one or two or more kinds of moisturizing agents selected from glycerol, propylene glycol, sorbitol, ethylene glycol, diethylene glycol, polyethylene glycol, polypropylene glycol and 1,3-butylene glycol, Can be used.

By using the viscose and / or the naringin or the azela, the quality of the taste can be improved from the cheap stevioside, and mass production of rubusoidal which can be applied to foods, cosmetics, medicines and the like can be mass-produced. The rubusoidal or steviol monoglucoside produced by the production method of the present invention can be used as a sweetener for general food, diet food, medicines, quasi-drugs and the like. For example, various kinds of pickled foods such as various kinds of liquor such as soy sauce, miso, powdered soup, various soft drink such as coffee, carbonated drink, lactic acid bacterium drink, and milk drink, various liquor such as shochu, fruit wine, Ice-cream, sherbet, and the like, and the like.

FIG. 1 is a graph showing the results of selecting rubusoside-producing enzymes using stevioside as a substrate using commercially available β-glucosidases.
Fig. 2 is a graph showing the production yield of rubusoidal from different pH conditions.
Figure 3 is a graph showing the results of stevioside reaction with Viscozyme or Naringinase for the production of lubusoidal and steviol glucoside in g units.
FIG. 4 is a graph showing ¹ H-NMR, ¹³ C-NMR, HMQC, HMBC, HH COZY and DEPT spectra of rubusoidalide prepared and purified in the present invention.
FIG. 5 is a graph showing ¹ H-NMR, ¹³ C-NMR, HMQC, HMBC, HH COZY and DEPT spectra of steviol monoglucoside prepared and purified in the present invention.

Example 1. Selection of lucoside-forming enzyme from various enzyme sources

In order to select the enzyme producing rubusoside, 2 g of stevioside was suspended in 10 ml of physiological saline, and the appropriate amount of the suspension was taken and added to 20 mM sodium acetate buffer (pH 5.2) and sodium phosphate buffer Solution (pH 7.0) was added. Herein, various enzymes ( Tricoderma logibrachiatum cellulase, Amano strain Visozyme, Aspergillus niger naringinase, A. niger hemicellulase, A. niger hersperdinase, Amano strain Ultraflo, A. niger cellulase, Amano strain celluclast, Almond beta-glucosidase ) Were added and reacted at the optimum temperature. After 24 hours, 10 ml of each reaction product was taken and examined for the production of rubusoidal by thin layer chromatography or high performance liquid chromatography. Most of these enzymes did not act as catalysts on stevioside, but biscozyme and naringinase were found to be lucoside forming enzyme. (Fig. 1)

Example 2. Establishment of Optimum Reaction Conditions for the Selected Enzymes

The optimum condition of the produced enzyme was confirmed at various temperature and pH conditions using the viscose selected in Example 1 above. In order to investigate the optimum pH and pH stability, it was found that the residual activity of Biscotrozine (32 mM, pH 3-11) and enzyme after mixing well in a refrigerator for 24 hours was examined. It was confirmed that it was stable at pH 3.5-pH 7.5, and the optimum reaction pH was confirmed at pH 4.0. The optimum temperature and temperature stability showed that biscozyme was abruptly unstable at 60 ℃ or higher and the optimal reaction temperature was 40 ~ 50 ℃. In addition, naringin and azela were confirmed to be stable at pH 3.5-pH 7.5, and the optimum reaction pH was confirmed at pH 5.2. The optimum temperature and temperature stability showed that Naringin and Agase were abruptly unstable at 55 ℃ or higher and the optimum reaction temperature was 35 ~ 50 ℃.

Example 3. Establishment of Optimum Conditions for Producing Loubushoids Generated

And 0.2 ml of a sample containing 5 mg of stevioside 50 mM Bricton Robinson buffer (pH 3.0 to pH 11.0) was reacted at 45 ° C for 16 hours. The results of analysis by HPLC of pH 4.0, 5.0, 6.0 and 8.0 are shown in FIG.

FIG. 2 shows the production yield of lucusoside from different pH conditions. As shown in FIG. 2, the yield of biscotrim is tended to increase under pH-acidic condition, And showed a small side production.

In order to investigate the effect of stevioside added at the initial addition, 1-6% stevioside was added to the reaction mixture.

As a result of investigation of the effect of temperature, it was confirmed that the formation of rubusoidal was abruptly decreased at a temperature of 50 ° C or higher, similar to the result of the optimum reaction temperature of the enzyme.

Example 4. Purification of the resulting lubusoidal and steviol monoglucosides

100 g stevioside and 20 mM sodium acetate buffer solution (pH 4.0) and 20000 U of viscose and naringin agar were added at pH 3.0-5.2, which is the optimum condition for producing lubusoidal, and reacted at 45 ° C for 2 weeks. As a result of the reaction, the enzyme reaction solution which was transparent at the beginning of the reaction was analyzed by HPLC after thoroughly stirring the reaction solution having a light color, Respectively.

FIG. 3 shows the result of stevioside reaction with Visocozyme or Naringinase, which is used for the production of lubusoidal and steviol glucoside in g units. A and B show the result of treating Narininase and Viscozyme, respectively, and C, D and E are Stevioside, Rubusosoide, and Steviol. As shown in FIG. 3, it was confirmed that the reaction product was the main product, rubusoide, and the second most abundant steviol monoglucoside, as in the preliminary experiment. The reaction solution was passed through an AW 90 resin to remove residual proteins and salts. Separation and purification were carried out using Diaion HP-20 or C ₁₈ column chromatography using an ethanol concentration gradient (0-80%) to finally obtain rubusoidal 24 g and steviol monoglucoside (20 g).

The molecular weight and molecular formula of the compound obtained in Example 3 were determined using LC / MS in order to grasp the structures of the obtained lubusoidal and steviol monoglucoside, respectively. Further, ¹ H NMR, ¹³ C NMR, HOMO-COZY, 1H-Detected heteronuclear multiple-quantum coherence (HMQC), and Heteronuclear Multiple-Coherence (HMBC) through a nuclear magnetic resonance (NMR) Bond Coherence) and DEPT (Distortionless Enhancement by Polarization) spectra. The structure of the reaction product obtained in the above experiment was analyzed by ¹³ C-NMR spectroscopy. The analytical sample was prepared by dissolving 40 mg of each of the sample luuxoside and steviol monoglucoside in D ₂ O, which was separated and purified in the above experiment.

FIG. 4 and FIG. 5 show ¹ H-NMR, ¹³ C-NMR, HMQC, HMBC, HH COZY and DEPT spectra of the lubusoidal and steviol monoglucosides prepared and purified in the present invention, NMR analysis of the product revealed that the product produced from the stevioside of viscose was rubusoidal and steviol monoglucoside.

Claims (22)

A method for selectively producing lucuboside, comprising treating a stevioside with a viscoszyme.
The method according to claim 1,
Wherein the step of treating the enzyme further comprises treating a naringinase or an enzyme (Naringinase).
delete delete The method according to claim 1,
Wherein the viscose enzyme selectively hydrolyzes glucose at the 13-OH position of stevioside.
The method according to claim 1,
Wherein the viscose enzyme is derived from Aspergillus aculeatus .
3. The method of claim 2,
Wherein the naringinase enzyme is derived from Penicillium decumbens .
3. The method of claim 2,
Treating the viscoszyme or the nitinase or the enzyme may be performed by treating these enzymes themselves; Wherein the microorganism expresses a biscozyme enzyme or a naringinase enzyme.
The method according to claim 1,
Wherein the enzymatic reaction with the biscozyme enzyme is carried out at a pH of 4 to 5 and a temperature of 40 to 50 degrees.
3. The method of claim 2,
Wherein the enzymatic reaction by the naringinase enzyme is carried out at a pH of 4.5 to 5.5 and a temperature of 35 to 50 degrees.
A composition for selective preparation of rubusoidal from stevioside, comprising as an active ingredient a biscottic enzyme.
A method for improving the quality of a food containing steviosides, characterized in that it comprises treating the stevioside-containing food with a biscozyme enzyme, characterized in that the method selectively produces lucusoside.
delete 12. The method of claim 11,
Wherein the composition further comprises a naringinase or an enzyme.
delete delete delete delete delete delete delete delete

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