Novel steviol glycoside

Abstract

To provide a novel stevia sweetener component.SOLUTION: The component analysis of a novel steviol glycoside component and stevia extract and crystalline products containing them can identify the raw material of a sweetener containing RA as a main component, while making the quality control of sweeteners easy, thereby making the judgment of the appropriateness of the origin labelling of stevia variety and infringement easy. The invention relates to a novel steviol glycoside; a production of a novel steviol glycoside included in a Stevia Rebaudiana Bertoni variety which contains rebaudioside A in large quantities, and a production of a sweetener, food, pharmaceuticals, quasi drugs, and cosmetics containing them; an identification method of stevia variety; and an analytical method of the novel steviol glycoside.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to novel steviol glycosides, novel steviol glycosides contained in Stevia Rebaudiana Bertoni varieties rich in rebaudioside A, and the production of sweeteners, foods, pharmaceuticals, quasi-drugs, and cosmetics containing them, the identification of Stevia varieties, and a method for analyzing novel steviol glycosides.

Background Art

[0002] Stevia is a perennial plant of the Asteraceae family native to Paraguay, South America, and its scientific name is Stevia Rebaudiana Bertoni. Stevia contains components with a sweetness more than 300 times that of sugar, and is cultivated to extract this sweet component and use it as a natural sweetener. Known sweet components of Stevia include stevioside (C 38 H 60 O 18 ), rebaudioside A (C 44 H 70 O 23 ), rebaudioside C, D, E, dulcoside A, etc. In generally cultivated Stevia varieties, among the above sweet components, stevioside (hereinafter, ST) is the main component, and the content of rebaudioside A (hereinafter, RA) relative to 100 parts by weight of ST is about 40 parts by weight, and the content of rebaudioside C is slightly less than that, but there are various varieties, some of which have rebaudioside C as the main component. Under, ST) is the main component, and the content of rebaudioside A (hereinafter, RA) relative to 100 parts by weight of ST is about 40 parts by weight, and the content of rebaudioside C is slightly less than that, but there are various varieties depending on the variety, some of which have rebaudioside C as the main component.

[0003] Since ST has a sweetness 300 times that of sugar, it is widely used in the food industry as a natural sweetener, and its sweetness is relatively similar to that of sugar, but it is known that an unpleasant taste such as bitterness remains in the aftertaste when compared with RA. On the other hand, since RA has a good quality sweet taste and a sweetness 1.3 times to 1.5 times that of ST, generally, Stevia sweeteners with a higher RA content than ST The material is considered preferable. Therefore, the present inventors repeated cross-breeding and selection from conventional varieties to improve the varieties, obtained stevia varieties containing only a small amount of ST with respect to RA, and developed sweeteners from these varieties (see, for example, Patent Application 1 described later). However, among the tastes perceived by the tongue such as astringency, spiciness, and mellow taste, the mellow taste is very delicate. The delicate mellow taste does not depend only on the ratio of ST and RA, and since the mellow taste is improved when glucose is added to the chemical structures of various sweet components contained in stevia, methods for improving the mellow taste and richness by adding glucose to stevia sweet components have been developed (Patent Documents 2 and 3 described later).

[0004] Therefore, it is very important to analyze the unknown components contained in stevia even if the content is small. In particular, it is extremely important for taste quality control to identify the components with more glucose added than ST and to examine the glucose added to them. At the same time, since the taste quality is affected by the types of sweet components contained in the plant itself as a raw material, it is important to precisely grasp the contained sweet components in order to develop excellent stevia varieties and utilize them. Although the improvement of raw material plants will become more and more active in the future, it will be possible to grasp the variety improvement results in detail by specifically identifying the sweet components contained in the developed plants. On the other hand, for newly developed plant varieties, the present inventors have developed a variety determination method using genes (Patent Documents 4 and 5 described later), but in fact, there is no means for identifying the raw material plants for the sweeteners extracted and processed from these raw material plants or the products using them.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

[0006] Traditionally, stevia sweeteners have been standardized by analyzing five components, including ST, RA, rebaudioside A, dulcoside A, and steviol biosides, but no knowledge was available about other unknown components. Even if the sweet components are known, there is no way to confirm their presence because the analytical method has not been established. However, recently, the JECFA standard has been set for seven steviol glycosides, and the importance of confirming the sweet components contained in stevia, clarifying unknown components, and understanding the subtle effects of these components on taste has been recognized. The objective of the present invention is to determine the structure of minor sweetening components contained in Stevia varieties and to determine their taste effects on Stevia sweeteners. A further object of the present invention is to provide a means for identifying the stevia plant used as a raw material for stevia sweeteners and products using the same. [Means for solving the problem]

[0007] The present inventors have searched for novel steviol glycosides contained in stevia varieties with RA as the main component, and have found 10 novel steviol glycosides that may have a subtle effect on taste quality. Furthermore, they have found that the contents of these components vary between varieties, and that certain components are present only in stevia varieties with RA as the main component, and have confirmed that these can be used as markers for the plant-derived sweetener, thereby completing the present invention. Effect of the Invention

[0008] The steviol glycosides of the present invention have a structure in which more glucose is added than ST or RA, and by including these, a stevia sweetener with excellent full-bodied taste is provided. In addition, by confirming the steviol glycoside X of the present invention in the form of an extract or crystals, the origin of the raw materials used can be inferred, and by analyzing the final product, it is possible to determine whether the raw plant material infringes on patents or other rights. For example, the present invention provides the following: (Item 1) Below formula [ka] (In the formula, R1 and R2 are hydrogen atoms or TIFF2025096594000002.tif174152 It represents the glycan of Steviol glycosides I to X, represented by the following formula: (Item 2) An extract of Stevia rebaudiana Bertoni, a plant of the Asteraceae family, containing rebaudioside A as its main component, and containing steviol glycoside X. (Item 3) A method for producing high-purity rebaudioside A from the extract described in item 2. (Item 4) A method for producing a food product, comprising adding the extract according to item 2 in an amount of 1% or less. (Item 5) 4. A method for producing a food, comprising adding 1% or less of high-purity rebaudioside A obtained by the method according to claim 3. (Item 6) How to identify stevia varieties by steviol glycoside X. (Item 7) Analytical method for steviol glycosides I-X. [Brief description of the drawings]

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] The stevia varieties referred to as the main RA component in the present invention are varieties containing more RA than ST, and the varieties described in Patent Application No. 2001-200944, Japanese Patent Application No. 2007-506004, etc. The extract obtained from the dried leaves has a higher RA content than the ST content and contains rebaudioside D (R-D), steviol glycosides III, V, VI, VII, X, and components with more glucose added than ST and RA, whereby a sweetener with excellent kokumi taste can be obtained. Furthermore, by recrystallizing them, a high-purity RA sweetener containing extremely small amounts of ST and steviol glycoside X can be efficiently obtained.

[0011] The first aspect of the present invention is the following formula

Chemical formula

[0012] A second aspect of the present invention is an extract containing steviol glycoside X (rebaudioside O), which is obtained by extracting the plant body of Stevia rebaudiana Bertoni, which has rebaudioside A as a main component, or its dried leaves with water or a hydrous solvent.

[0013] A third aspect of the present invention is a method for obtaining high-purity rebaudioside A containing steviol glycoside X (rebaudioside O) by recrystallization or the like from the extract of the above aspect.

[0014] A fourth aspect of the present invention is a method for producing a food by adding 1% or less of the extract obtained by the method of the second aspect above to the food.

[0015] A fifth aspect of the present invention is a method for producing a food by adding 1% or less of the high-purity rebaudioside A obtained by the third aspect above to the food.

[0016] Steviol glycoside X does not exist in the extract obtained from the raw material variety having ST as the main component, but the extract obtained from the raw material variety having RA as the main component contains glycoside X. Therefore, it is possible to determine whether the raw material variety has ST or RA as the main component. That is, since steviol glycoside X does not exist in the extract obtained from the variety having ST as the main component by removing ST by crystallization and the extract having RA as the main component, or the high-purity product obtained by recrystallization from them, it is possible to confirm the raw material variety. A sixth aspect of the present invention is a method for identifying stevia varieties by steviol glycoside X.

[0017] The seventh aspect of the present invention is a method for analyzing steviol glycosides I to X by high performance liquid chromatography (hereinafter referred to as HPLC).

[0018] To achieve these objectives, the inventors searched for the sweet components contained in varieties having RA as the main component, as well as varieties such as Japanese Patent Application No. 2001-200944 and Japanese Patent Application No. 2007-506004, found novel sweet components, and determined their chemical structures. Furthermore, it was confirmed that these components are useful as sweeteners, and a method for identifying varieties and an analytical method using these components were completed.

[0019] For the confirmation of novel components, extraction is carried out from the dried leaves of the variety having RA as the main component in Example 1 (hereinafter referred to as Variety A), Japanese Patent Application No. 2001-200944 (hereinafter referred to as Variety B), and Japanese Patent Application No. 2007-506004 (hereinafter referred to as Variety C) with water or a water-containing organic solvent. Next, the extract is concentrated as it is, or, if necessary, ionic impurities are removed with a cation exchange resin, an anion exchange resin, activated carbon, etc., the sweet components are adsorbed onto an adsorption resin and eluted with a hydrophilic solvent, and if necessary, the eluate is treated again with a cation exchange resin, an anion exchange resin, activated carbon, etc., and the eluate is concentrated and dried to obtain an extract, or confirmation can also be made with an extract obtained by appropriately performing conventional purification means such as decolorization.

[0020] The novel steviol glycosides of the RA-C extract obtained in Example 1(1) below are described in Example 5 Therefore, by separating and mass-analyzing with a high performance liquid chromatography mass spectrometer, the structure of each glycoside [Chemical formula] (In the formula, R1 and R2 each represent a hydrogen atom or the above sugar chain) was determined. Steviol glycoside I (glucoside B) has a structure with a molecular weight of 788 that is confirmed around a retention time (hereinafter referred to as R.T.) of 13 minutes in the chromatogram of high performance liquid chromatography in Fig. 1 It is a glycoside. Steviol glycoside II (Rebaudioside G) is a glycoside having a structure with a molecular weight of 804, which is confirmed around R.T. 15 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. It is. Steviol glycoside III (Rebaudioside I) is a glycoside having a structure with a molecular weight of 1128, which is confirmed around R.T. 28 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. It is. Steviol glycoside IV (Rebaudioside H) is a glycoside having a structure with a molecular weight of 1112, which is confirmed around R.T. 29 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. Steviol glycoside V (Rebaudioside L) is a glycoside having a structure with a molecular weight of 1128, which is confirmed around R.T. 34 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. Steviol glycoside VI (Rebaudioside K) is a glycoside having a structure with a molecular weight of 1112, which is confirmed around R.T. 34 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. Steviol glycoside VII (Rebaudioside J) is a glycoside having a structure with a molecular weight of 1112, which is confirmed around R.T. 35 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. It is. Steviol glycoside VIII (Rebaudioside M) is a glycoside having a structure with a molecular weight of 1290, which is confirmed overlapping with Rebaudioside D around R.T. 35 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. Steviol glycoside IX (Rebaudioside N) is a glycoside having a structure with a molecular weight of 1274, which is confirmed around R.T. 43 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. Steviol glycoside X (Rebaudioside O) is a glycoside having a structure with a molecular weight of 1436, which is confirmed around R.T. 51 minutes in the chromatogram of high performance liquid chromatography in Fig. 1. However, as is well known to those skilled in the art, the above R.T. varies due to gradient elution analysis.

[0021] As described above, the presence or absence of steviol glycoside X in the final product provides important information for identifying the raw material variety. At the same time, since these novel steviol glycosides can be confirmed by high performance liquid chromatography, comprehensive taste quality control and / or quality control can be achieved by grasping the presence of these sweet components even when used as sweeteners.

[0022] The obtained extract and its crystals can be used as sweeteners in candies, jellies, beverages, powdered beverages, instant noodles, jams, frozen desserts, chewing gums, Japanese confectioneries, health foods, chocolates, tabletop sweeteners, baked confectioneries, snacks, fresh foods, lactic acid beverages, lactic acid bacteria beverages, coffee beverages, cocoa beverages, tea beverages, liqueurs, wines, sherbets, cereal foods, plant fiber-containing foods, sauces, soy sauces, misos, vinegars, dressings, mayonnaises, ketchups, curries, soups, rice confectioneries, arares, breads, biscuits, crackers, pancake mixes, canned fruits, canned vegetables, meat products, fish paste products, salty foods, pickles, compound seasonings, savory foods, cosmetics, etc., and calorie reduction, sugar reduction, melting point reduction, sweet taste quality improvement, and masking effects can be obtained, and other natural and artificial sweeteners, diluents, etc. can also be further added.

Example

[0023] Example 1 Production of RA extract (1) Extraction 100 g each of dried leaves obtained from Variety A, Variety B, and Variety C, each having RA as the main component, were extracted several times with 20 times the amount of water until no sweetness was felt. The extract was passed through a column filled with 300 ml of an adsorption resin (Diaion HP-20) to adsorb the sweet components, and after thorough washing with water, it was eluted with 900 ml of methanol. HP-20)300ml filled column to adsorb the sweet components, and after sufficient washing with water, methanol 900 ml of eluent was used. A column filled with 200 ml of an ion exchange resin (Diaion WA-30) was used. The eluent was passed through the column, and 10 g of activated carbon was added to the passing solution and stirred. The mixture was filtered, concentrated, and dried to obtain 13.0 g of RA-A extract (S T 35.4%, RA 41.7%, RC 9.8%), 11.5 g of RA-B extract (ST 19.5%, RA 58.1%, RC 8.8%), and 12 g of RA-C extract (ST 5.4%, RA 72.3%, RC 8.1%) respectively. (2) RA Recrystallization 5 g of the above RA-B extract and RA-C extract were heated and dissolved in 10 times the amount of 90% methanol, then cooled at 4°C for 6 days, the obtained crystals were separated, washed with cold methanol, and dried under reduced pressure to obtain 3.9 g of white RA-B crystals (ST 0.2%, RA 95.0%, RC 0.2%) and 4.5 g of RA-C crystals (ST 0.2%, RA 95.6%, RC 0.1%) respectively. obtained.

[0024] Example 2 Preparation of ST Extract For comparison, the same treatment was also carried out on a variety with ST as the main component to obtain 11.3 g of ST extract (ST 51.9%, RA 23.7%, RC 7.4%).

[0025] Example 3 RA-A Mother Liquid, ST Mother Liquid 10 g each of the above RA-A extract and ST extract were heated and dissolved in 10 times the amount of 90% methanol aqueous solution, then cooled at 4°C for 6 days, the obtained crystals were separated, washed with cold 98% methanol, and dried under reduced pressure to obtain 2.1 g of RA-ST crystals, which are white crystals of stevioside, and 3.8 g of ST-ST crystals respectively. 8.8 g of RA-A mother liquid with RA as the main component (ST 15.7%, RA 43.8%, RC 6.9%) and 6.1 g of ST mother liquid (ST 20.0%, RA 37.1%, RC 11.2%) were concentrated and dried to obtain mother liquid powders with light yellow RA as the main component respectively.

[0026] Example 4 RA-A Crystal, ST-RA Crystal The mother liquor powders of Example 3 were each dissolved by heating in 10 times the amount of a 90% methanol aqueous solution, left to cool at 4°C for 6 days, and the resulting crystals were separated. , washed with cold 98% methanol, and dried under reduced pressure to obtain 2.2 g of white RA-A crystals (ST 1.6%, RA 90.4%, RC 1.4%) and 1.2 g of ST-RA crystals (ST 96.9%, RC 1.4%).

[0027] Example 5 Structure Determination of Steviol Glycosides Analysis was performed using high performance liquid chromatography (HPLC) as follows. Separation of steviol glycosides in each extract was carried out using a Shimadzu LC-10ADvp liquid chromatograph, and a TSKgel Amide-80 column (4.6 x 250 mm, Tosoh) was used. The solvent used was a mixture of acetonitrile and water, and gradient elution was performed over 60 minutes such that the concentration of acetonitrile and water changed from 82:18 to 66:34. The flow rate was 0.65 ml / min, the column temperature was 40°C, and detection was performed using an ultraviolet absorption spectrum at 210 nm. Molecular weight measurement was performed using a Waters Alliance HPLC system 2695 and a Waters Quattro micro (triple quadrupole) electrospray ionization (ESI)-mass spectrometer. High performance liquid chromatography was carried out using a TSKgel Amide-80 column (2.0 x 250 mm, Tosoh) , and the solvent used was a mixture of acetonitrile and water, and gradient elution was performed over 60 minutes such that the concentration of acetonitrile and water changed from 82:18 to 66:34. The flow rate was 0.2 ml / min, and the column temperature It was carried out at 40 °C. Nitrogen gas was used as the desolvation gas and argon gas was used as the collision gas. The capillary voltage was 15.0 kV in the negative mode for the analysis of steviol glycosides and 13.5 kV in the positive mode for the analysis of ABEE-oligosaccharides. The cone voltage and the collision voltage during MS / MS analysis were set to voltages ranging from 10 to 80 V. The source temperature was 100 °C, the desolvation temperature was 400 °C, the cone gas flow rate was 50 l / hr, and the desolvation gas flow rate was 900 l / hr.

[0028] Figures 1 to 10 show the HPLC analysis results for each extract and crystal. Tables 1 to 9 below show the analysis results for each peak of the chromatography shown in Figures 2 to 10, respectively. [Table 1]

[0029] [Table 2]

[0030] [Table 3]

[0031] [Table 4]

[0032] [Table 5]

[0033] [Table 6]

[0034] [Table 7]

[0035]

Table 8

[0036]

Table 9

[0037] The abbreviations used in each of the above tables are as follows. PKNO: Peak number T: Time (min) A: Peak area H: Peak height CONC: Concentration (%) N: Glycoside name TOT: Total Stev mono: Steviol monoside Stev bio: Steviol bioside Rebuso: Rubusoside Rebau: Rebaudioside Stev: Stevioside Dulco: Dulcoside The concentration is the concentration calculated from the total area of the ultraviolet absorption spectrum at 210 nm, and it is necessary to correct for the molecular weight when measuring the content. In addition, I to X in the chromatogram indicate novel steviol glycosides I to X.

[0038] Example 6 Evaluation of taste quality A 0.05% aqueous solution of each extract and a 0.03% aqueous solution of the crystal were evaluated by 10 persons familiar with the sensory inspection of stevia, and the average of the evaluation results is shown in Table 10 below. The evaluation was conducted in the following five grades. Evaluation 5; Very good, 4: Good, 3; Normal, 2; Slightly bad, 1; Bad

Table 10

[0039] Example 7 Determination of Variety When each extract or each crystal was analyzed by HPLC, the extract obtained from the variety with RA as the main component (hereinafter referred to as the RA variety) contained more rebaudioside D than the extract obtained from the variety with ST as the main component (hereinafter referred to as the ST variety), and furthermore, it contained steviol glycoside X (rebaudioside O). It was also found that this steviol glycoside X was contained in trace amounts in the RA crystals purified from the extract of the RA variety. On the other hand, the extract obtained from the ST variety does not contain steviol glycoside X. Naturally, since the ST-RA crystal obtained from the ST variety also does not contain steviol glycoside X, if the presence of steviol glycoside X is confirmed, it can be determined that it is an extract obtained from the RA variety or a crystal obtained therefrom.

[0040] Example 8 Analysis Method of Steviol Glycosides From the measurement conditions of high performance liquid chromatography (HPLC) described in Example 5 above, each steviol glycoside I to X can be confirmed. In principle, the presence of steviol glycosides can be confirmed from the retention time (hereinafter referred to as R.T.) of the HPLC analysis chart, but if necessary, each steviol glycoside I to X can be confirmed by separating these components and measuring the molecular weight.

[0041] Example 9 Table Sugar 1) 1 g of RA-A crystals and 99 g of powdered sugar were mixed to prepare table sugar. 2) 1 g of RA-B crystals and 99 g of erythritol were mixed to prepare table sugar. prepared. 3) 1 g of RA-C crystals and 99 g of grape fructose liquid sugar were mixed to prepare table sugar.

[0042] Example 10: Candy 0.3 g of RA-C extract, 100 g of paraffin, and an appropriate amount of flavor were used to prepare candy.

[0043] Example 11: Milk jelly 15 g of sugar, 0.08 g of RA-B extract, 250 g of milk, 5 g of gelatin, and an appropriate amount of milk flavor were used to prepare milk jelly.

[0044] Example 12: Sports drink A sports drink was prepared with 0.075% of RA-B crystals, 0.11% of calcium lactate, 0.045% of citric acid, 0.03% of trisodium citrate, 0.015% of magnesium chloride, 0.0055% of glutamic acid, and 99.72% of water.

[0045] Example 13: Carbonated drink 0.012% of RA-B crystals, 8.4% of fructose, 0.6% of citric acid, 0.12% of arginine, 0.1% of inositol, 0.0025% of caffeine, 0.0034% of calcium pantothenate, 0.003% of niacinamide, 0.002% of vitamin B6, 0.00009% of vitamin B2 0.000002% of vitamin B12, an appropriate amount of flavor, and water were made up to 100%, and carbon dioxide gas was injected to prepare a carbonated drink.

Industrial applicability

[0046] By measuring the novel steviol glycoside components contained in the sweetener provided by the present invention using HPLC, it is possible to produce sweeteners and other foods having a certain degree of sweetness, sweet quality, and mellow taste in the production of sweeteners, and furthermore, it becomes possible to estimate the raw material variety, making it easier to determine the appropriateness of the origin indication for stevia varieties and the infringement of rights.

Claims

[Claim 1] Provided is a novel steviol glycoside as described herein.

Images