JP2003252895A - Rakanka (fruit of momordica grosvenori) glycoside with improved quality of taste and method of production for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sweetening with a high degree of sweetness, having lower energy than saccharose, having a good quality of taste close to that so saccharose and high safety, and having physiological and physical properties equal to that of the conventional sweetening. <P>SOLUTION: The sweetening is a compound with high glycosylation, which has more than one α bonding glucose residues to RAKANKA (fruit of Morordica grosvenori) glycoside, and the method of production for the compound with high glycosylation which contacts the RAKANKA glycoside with an alpha- glucan and a glycosyltransferase. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to Rakan fruit glycosides.
TECHNICAL FIELD The present invention relates to a highly glycosylated compound having one or more glucose residues bound thereto and a method for producing the same.

[0002]

2. Description of the Related Art In recent years, consumers' preference for low sweetness has increased, and they have taken too much energy (especially, too much sucrose).
Due to factors such as awareness of health concerns
Many foods that are labeled as "moderate in sugar" or "without added sugar" have been put on the market.

In fact, the food situation in Japan is "the age of satiety."
Reflecting that, excessive intake of energy is becoming commonplace. High energy intake and increased lipid energy ratio
It has been clarified that it causes the occurrence of lifestyle-related diseases.

Therefore, people who are restricted in energy intake (eg, obese patients and diabetic patients) and those who are on a diet improve their own diseases in order to prevent their own diseases. Therefore, it is said that it is important to suppress excessive intake of sucrose and lipids, improve the lifestyle and regain a healthy life in order to manage the health.

From these things, a sweetener replacing sucrose,
Above all, there has been a demand for the development of a high-potency sweetener capable of substantially suppressing energy as compared with sucrose.

Hereinafter, in this specification, a sweetener composition having substantially lower energy than sucrose is referred to as an energy-suppressing sweetener. Here, "energy" means
It refers to the amount of heat that is absorbed in the body and released into the body by metabolism when a person eats or drinks a certain amount (for example, 100 grams) of a certain substance.

However, energy control sweeteners have various problems. The biggest problem is the quality of sweetness. This is because human beings are very familiar with the sweetness of sucrose, and it is easy for a sweetener having a sweetness slightly different from that of sucrose to feel uncomfortable. The various conventional high intensity sweeteners will be specifically described below.

The high intensity sweetener has a sweetness intensity several hundred times that of sucrose. High-intensity sweeteners can be generally classified into artificial sweeteners (also called synthetic sweeteners) and natural sweeteners. Examples of artificial high-intensity sweeteners include saccharin, aspartame, sucralose, and acesulfame potassium.

Saccharin is an artificial sweetener that has been used for a long time. However, due to the suspicion of carcinogenicity, currently, the items to be used are limited in Japan and the standard amount for use is also limited.

Aspartame was established in 1981 by the US FDA.
Although it is an artificial sweetener licensed by, until it is licensed, it has been the subject of intense debate over its impaired neurotransmitter system. Furthermore, since aspartame is decomposed by heat, it has been pointed out that there is a defect in its stability.

[0011] Although artificial high-potency sweeteners such as sucralose and acesulfame potassium have not been the subject of debate regarding safety at the present time, their sweetness qualities are not sufficient. For example, sucralose has a significantly long sweetness onset time, and it is known that the sweetness trait is always followed. In contrast, acesulfame potassium has a very short sweetening time in the oral cavity and cannot be used alone as a sweetener. Acesulfame potassium also has the major drawback of having bitterness.

Thus, the artificial high-intensity sweetener includes
Not only is sweetness inadequate compared to sucrose, there is a constant debate over the safety assessment.

On the other hand, natural high-intensity sweeteners include licorice extract, stevia extract, Rakan fruit extract and the like.
These natural sweeteners are of plant origin and are highly safe for the human body.

Licorice is a perennial plant belonging to the legume family, and its sweetening component, glycyrrhizin, is contained in the rhizome of licorice. However, unlike the sweetness of sugars typified by sucrose, its sweetness remains forever, and when used in a large amount, bitterness may be felt or astringent taste may be felt on both cheek walls.

Stevia is a perennial plant of the Asteraceae family, and its sweetening ingredients include stevioside and rebaudioside. Among the sweet components of stevia, stevioside has a strong bitterness and astringency, and its sweetness has a marked aftertaste.

Among natural high-intensity sweeteners, especially Rakan fruit extract is known as a medicinal sweetener obtained from dried Rakan fruit and having a strong sweetness. Rakanka is
It is a perennial medicinal plant of the family Cucurbitaceae, which is one of the specialty products around Guilin, China. Originally, Lo Han Guo extract has been widely used as a sweetener and a folk medicine in China since ancient times. As the medicinal effects of Lo Han Guo extract, it is known to improve throat roughness, relieve pain, cough and expectorant. Rakan fruit extract can be expected to have a beneficial effect on humans at the same time as sweetness, and therefore it has been proposed to use it as a sweetening component of confectioneries, beverages, syrups, etc. (Japanese Patent Laid-Open No. 53-9352).
JP-A-53-9359). Specifically, for example, for drinks, the Rakan fruit paste extract obtained by concentrating the Rakan fruit extract in a paste form is diluted and used. This is because when the Rakan fruit extract is used without being concentrated, it takes a high cost to store and transport the Rakan fruit extract, and microorganisms are easily generated in the Rakan fruit extract, so that the quality of the Rakan fruit extract is easily deteriorated.

However, Rakan fruit extract has the following drawbacks. In other words, it has a charring taste similar to that of brown sugar such as brown sugar, a unique smell, a bitterness, and a residual sweetness, which is very uncomfortable when eating or drinking. In addition, the addition of Rakan fruit extract causes foods and drinks to turn yellowish brown in color, which is often not suitable for use in foods.

As described above, natural high-intensity sweeteners are highly safe, but on the other hand, the sweetness is insufficient as a sweetener used alone as a substitute for sucrose.

On the other hand, in order to improve the taste quality of the Rakan fruit extract, only the sweet component contained in the Rakan fruit extract is fractionated,
A syrup containing purified and powder-dried Rakan fruit glycosides and erythritol has a weak residue of sweetness, odor, bitterness and sweetness, which conventional Rakan fruit extract has, and exhibits good sweetness. , It is described as being suitable as a low-calorie syrup (Japanese Patent Laid-Open No. 11-467).
01 gazette; patent 3110005).

Further, the total content of mogroside V, mogroside IV, 11-oxo-mogroside V and siamenoside I as sweet components of the Rakan fruit glycoside is 33.
It has also been reported that the sweetness of the composition in an amount of not less than wt% is close to that of sucrose (Japanese Patent Laid-Open No. 2001-212854).
Issue).

However, even if these sweeteners are high-purity Rakan fruit glycosides, when compared with the sweetness of sucrose,
It is not equivalent to sucrose in any of the items of "bitterness, backsizing, persistentness, habit, astringency and refreshing feeling",
Insufficient as a sweetener used as a substitute for sucrose. Therefore, in order to further expand the consumption scale and application of these sweeteners, improvement and improvement of the sweetness quality of these sweeteners have been desired.

[0022]

[PROBLEMS TO BE SOLVED BY THE INVENTION] A high-potency sweetener capable of substantially suppressing energy as compared with sucrose, having a sweetness quality extremely close to that of sucrose, having high safety and having a conventional sweetness. It is an object of the present invention to provide a high-intensity sweetener having physiological and physical properties comparable to those of the sweetener.

[0023]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the conventional Rakan fruit glycoside can be obtained by adding a sugar residue to the Rakan fruit glycoside. The results are improved in taste quality items such as “bitterness, aftertaste, stubbornness, habit, astringency and refreshing feeling”, and have sweetness extremely similar to sucrose for all evaluations The present invention was completed based on the finding that a novel high-intensity sweetener having a physiological and physical property comparable to that of a sweetener can be obtained.

Furthermore, the present inventors have also referred to cyclodextrin synthase (EC 2.4.1.19; also called cyclodextrin glucanotransferase or cyclomaltodextrin glucanotransferase; hereinafter abbreviated as "CGTase"). Is used in the reaction, a highly glycosylated compound (herein also referred to as “glycosylated Rakan fruit glycoside”) in which one or more glucose residues are α-linked to Rakan fruit glycoside, It has been found to be very efficient and cheap to manufacture.

In the highly glycosylated compound of the present invention, one or more glucose residues are α-linked to the Rakan fruit glycoside.

[0026] In one embodiment, the number of glucose residues may be 1 to 45.

[0027] In one embodiment, the number of glucose residues may be 1 to 15.

[0028] In one embodiment, the hyperglycosylated compound may be selected from the group consisting of:

[0029]

[Chemical 10]

[0030]

[Chemical 11]

[0031]

[Chemical 12]

[0032]

[Chemical 13]

[0033]

[Chemical 14]

[0034]

[Chemical 15]

[0035]

[Chemical 16]

[0036]

[Chemical 17]

[0037]

[Chemical 18] In one embodiment, the hyperglycosylated compound has one or more glucose residues α in mogroside V.
May be combined.

The food composition of the present invention contains the above highly glycosylated compound.

The sweetener of the present invention contains the above-mentioned highly glycosylated compound.

The pharmaceutical composition of the present invention contains the above-mentioned highly glycosylated compound.

The quasi-drug composition of the present invention contains the above-mentioned highly glycosylated compound.

The cosmetic composition of the present invention contains the above highly glycosylated compound.

The method for producing a highly glycosylated compound of the present invention includes a step of contacting a Rakan fruit glycoside with an α-glucan and a glycosyltransferase to obtain a highly glycosylated compound.

In one embodiment, the glycosyltransferase is
It can be a cyclodextrin synthase.

[0045] In one embodiment, the Rakan fruit glycoside may be mogroside V.

The hyperglycosylated compound of the present invention is obtained by the above method.

According to the present invention, the method for producing a highly glycosylated compound in which 1 to 4 glucose residues are α-linked to the Rakan fruit glycoside, the Rakan fruit glycoside is contacted with α-glucan and a glycosyltransferase. And a step of obtaining a highly glycosylated compound in which five or more glucose residues are α-bonded to the Rakan fruit glycoside, and a high degree in which five or more glucose residues are α-bonded to the Rakan fruit glycoside The step of contacting a glycosylase with a glycosylated compound to obtain a hyperglycosylated compound in which 1 to 4 glucose residues are α-linked to the Rakan fruit glycoside is included.

In one embodiment, the glycolytic enzyme may be selected from the group consisting of glucoamylase, β-amylase and α-amylase.

In one embodiment, the Rakan fruit glycoside may be mogroside V.

In the present invention, 1 to 4 glucose residues are α
The bound hyperglycosylated compound is obtained by the method described above.

The method of improving the taste of a sweetener containing Rakan fruit glycoside according to the present invention includes the step of bringing the sweetener into contact with α-glucan and a glycosyltransferase.

The taste-improved sweetener of the present invention is obtained by the above-mentioned method.

[0053]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

<Highly Glycosylated Compound> In general, the highly glycosylated compound of the present invention has one or more glucose residues α-linked to the Rakan fruit glycoside.

“Highly glycosylated compound” as used herein
Means a compound having a structure of the following chemical structure 1: (Chemical structure 1)

[0056]

[Chemical 19] Here, R ₁ is -Glc-Glc- (Glc) n, and n is an integer of 1 or more, or R ₂ is-
Glc-Glc- (Glc) m, m is an integer of 1 or more, or R ₂ is

[0057]

[Chemical 20] And h + k is an integer of 1 or more, and R _3a is —OH.
And R _3b is —H, or R _3a and R _3b together are ═O, and Glc represents a glucose residue.

Here, the bond between the O atom and R ₁ , the bond between the O atom and R _2, and the bond between the first glucose residue and the second glucose residue of R ₁ . The bond between, and the bond between the first glucose residue in R ₂ and the branched glucose residue when R ₂ has a branch is a β bond. The bond between the other glucose residues is an α bond.
The relationship of these bonds is maintained even when not specifically mentioned in the present specification.

In the present specification, the "lohan fruit glycoside" refers to any glycoside contained in lokan fruit, and has high sweetness such as mogroside V, mogroside IV, 11-oxo-mogroside V, and siamenoside I. Included glycosides. Mogroside V is preferable. In general,
A mixture containing mogroside V as a main component and a small amount of mogroside IV, siamenoside I and 11-oxo-mogroside V is easily available and can be used in the present invention.

The Rakan fruit glycoside may be mixed with ingredients other than the Rakan fruit glycoside compound (for example, aglycone and water) other than the Rakan fruit glycoside compound in the production of the compound of the present invention. The term “glycoside” as used herein also includes a mixture of glycosides. Lo Han Guo Glycosides are the main cause of the sweetness of Lo Han Guo fruit. There are several types of Rakan fruit glycosides contained in Rakan fruit, and among them, the major content is 4 kinds of glycosides having the structure shown in the following chemical structure 2 (Takemoto, Arihara, Nakajima). , Okudaira, Pharmaceutical Journal 103:
1151-1154 (1983); Takemoto, Arihara, Nakajima,
Okudaira, Pharmaceutical Journal 103: 1155-1166 (198
3); Matsumoto, R .; Kasai, K .;
Ohtani and O.I. Tanaka, Chem. Ph
arm. Bull. , 38: 2030-2032 (19
90); Kasai, R .; -L. Nie,
K. Nashi, G .; -D. Tao and O.D. Tana
ka, Agric. Biol. Chem. , 53:33
47-3349 (1987)).

For example, in mogroside V, two skeletons in R ₁ and 3 in R ₂ are added to the skeleton represented by the following chemical structure 2.
Having a structure in which a total of 5 glucose residues are bound, or further having one or more glucose residues bound thereto, that is, a compound having a total of 6 or more glucose bound to R ₁ and R ₂ . Is called a highly glycosylated compound.

(Chemical structure 2)

[0063]

[Chemical 21] Among the mixture of Glucoside glycosides, which is usually obtained from Grape,
The highest content is in the glycoside called Mogroside V, and its sweetness intensity is about 300 times that of sucrose. Rakan fruit glycosides other than Mogroside V also have high sweetness intensity.

The sugar residue bound to the Rakan fruit glycoside is any sugar residue that can be bonded to the Rakan fruit glycoside. Examples of such sugar residues include glucosyl group, fructosyl group, galactosyl group, mannosyl group, xylosyl group, arabinosyl group, N-acetylglucosaminyl group, N-acetylgalactosaminyl group, glucosaminyl group, galactosaminyl group. , Glucuronyl group, galacturonyl group, rhamnosyl group and the like. The sugar residue is preferably a glucosyl group.

The bond between the Rakan fruit glycoside and the sugar residue is α
It may be a bond or a β bond, but preferably α
It is a bond, more preferably an α-1,4 bond.

The sugar residue is β-of R ₁ of the above chemical structure 1.
It is bound to the Rakan fruit glycoside at the D-glucopyranosyl terminal or the β-D-glucopyranosyl terminal of R ₂ . When a plurality of sugar residues are bound, these sugar residues may be bound to either R ₁ or R ₂ or R ₁
And R ₂ may be separately bonded. When R ₂ has two β-D-glucopyranosyl ends, it may be bound to one of them or to both.

The number of sugar residues bound to the Rakan fruit glycoside may be any number as the total of sugar residues bound to R ₁ and R ₂ , but is typically 1 to 45. , Preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 12, and even more preferably 1 to 4. Typically, the number of sugar residues attached at each position of R ₁ and R ₂ is 1 to
The number is 15, preferably 1 to 5, and more preferably 1 to 4. When R ₂ has two β-D-glucopyranosyl terminals, the number bonded to the first β-D-glucopyranosyl terminal is preferably 1 to 15, more preferably 1 to 5, and further preferably 1 to It is 4 and the number bonded to the second β-D-glucopyranosyl terminal is preferably 1 to 15, more preferably 1 to 5.
The number is more preferably 1 to 4. If the number of sugar residues bound to the lokhan fruit glycoside is too large, the sweetness intensity of the obtained highly glycosylated compound tends to be slightly lowered. When the number of sugar residues bound to the Rakan fruit glycoside is in the range of 1 to 4, substantially the same sweetness intensity as that of the Rakan fruit glycoside is obtained, and the sweetness is better than that of the Rakan fruit glycoside. Is improved, which is very preferable.

The highly glycosylated compound of the present invention in which one or more glucose residues are α-linked to the Rakan fruit glycoside is generally represented by the following "Chemical structure 3".

(Chemical structure 3)

[0070]

[Chemical formula 22] Among these highly glycosylated compounds, the highly glycosylated compound in which 1 to 4 glucose residues are α-linked to the Rakan fruit glycoside (hereinafter, referred to as “partially degradable glycosylated Rakan fruit glycoside” in the present specification). (Also referred to as) is generally represented by the following “Chemical Structure 4”.

(Chemical structure 4)

[0072]

[Chemical formula 23] The hyperglycosylated compounds of the present invention preferably have a structure selected from the group consisting of compound numbers 1-34 above.

The Rakan fruit glycoside portion in the highly glycosylated compound of the present invention may be derived from any Rakan fruit glycoside, but is preferably derived from mogroside V.

The highly glycosylated compound of the present invention may be pure consisting of only one compound,
It may be a mixture of multiple hyperglycosylated compounds.

<Raw Material for Highly Glycosylated Compound> Raw materials for the highly glycosylated compound of the present invention include Rakan fruit glycosides, sugar donor substrates, glycosyltransferases and glycolytic enzymes.

The Rakan fruit glycoside may be provided as a pure Rahan fruit glycoside compound or a mixture of Rahan fruit glycoside compounds, or a less pure Rakan fruit glycoside-containing composition containing a substance other than the Rakan fruit glycoside compound. It may be provided as a product. The Rakan fruit glycoside may consist of only one kind of Rakan fruit glycoside compound (for example, mogroside V) or a mixture of a plurality of Rakan fruit glycoside compounds (for example, mogroside V, mogroside IV, siamenoside I, 11-oxo-mogroside V mixture).

The Rakan fruit glycoside-containing composition may be used in any purity as long as it contains the Rakan fruit glycoside, but typically, it is the total of a plurality of kinds of Rakan fruit glycoside,
Based on the weight of the composition containing the Rakan fruit glycoside, the content of the Rakan fruit glycoside is about 5% by weight or more, preferably about 10% by weight or more,
More preferably about 15% by weight or more, more preferably about 2%.
0% by weight or more, more preferably about 30% by weight or more, more preferably about 40% by weight or more, more preferably about 50% by weight or more. If the content of Lokhan fruit glycoside is too low, the amount of the highly glycosylated compound of the present invention obtained may be too low. The Rakan fruit glycoside may be a pure product, but if a pure product is used, the cost may be too high.

Examples of the Rakan fruit glycoside-containing composition include crude extract of Rakan fruit, partially purified product, purified product of each glycoside component, and the like. Lokhan fruit glycosides are generally in the form of yellow to tan powder. Since fructose contained in the crude extract cannot serve as an acceptor substrate for the glycosyltransferase used in the present invention, there is no problem even if it is contained at a high concentration in the Rakan fruit glycoside-containing composition. The composition containing Rakan fruit glycosides is
A commercially available product may be used or the product may be manufactured. The Luo Han Glycoside-containing composition can be produced using extraction methods and separation methods known to those skilled in the art.

The Rakan fruit glycoside can be obtained by, for example, washing, crushing, and extracting water with a fruit of Rakan fruit, and subjecting the extract obtained to steps such as filtration, column absorption, column separation, recovery, concentration, and drying. It is manufactured by carrying out. The Rakan fruit glycoside is available as a commercial product in Japan. The Rakan fruit glycoside-containing composition can be produced, for example, by the following method. Methanol extraction is performed on the fruits of Lo Han fruit to obtain a methanol extract. The methanol extract is mixed with water and degreased with n-hexane. The defatted methanol extract is subjected to column chromatography and eluted with 100% water, 80% methanol, 100% methanol, and acetone successively to obtain a crude glycoside fraction, 80% methanol fraction.
The obtained crude glycoside fraction is dissolved in methanol, mixed with silica gel and dried, and then this silica gel is eluted with a mixed solvent of chloroform-methanol to obtain a glycoside fraction. The obtained glycoside fraction may be used as a Rakan fruit glycoside-containing composition or may be further purified. In the case of further purification, for example, the glycoside fraction of higher purity can be obtained by subjecting the obtained glycoside fraction to liquid chromatography.

The Rakan fruit glycoside-containing composition is preferably substantially free of substances that give an unpleasant taste, smell or the like to the resulting mixture containing the highly glycosylated compound. The term "substantially free" means that the resulting mixture containing a highly glycosylated compound is in an amount such that no unpleasant taste, smell or the like is perceived when subjected to a sensory test.

In the reaction system in which the Rakan fruit glycoside is contacted with glucan and glycosyltransferase, for example, when a purified Rakan fruit product containing about 30% mogroside V is used, the concentration range is typically 1 to 70%. (W / v) (in this case, the mogroside V is 0.3 to 21% w / v), preferably 5 to 40% (w / v) (in this case, the mogroside V is 1.5 to 12). % (W / v)). The total weight of the Rakan fruit glycoside compound in the reaction system is typically 0.01 to 50% (w / w), preferably 0.05 to 40% (w / w), and more preferably 0.2 to
20% (w / w). These concentration ranges should be experimentally determined according to the content of the Rakan fruit glycoside in the Rakan fruit glycoside-containing composition to be used, and are not limited thereto. When using a composition containing Rakan fruit glycoside containing a high concentration of Rakan fruit glycoside (ie, sugar acceptor substrate), the concentration of the sugar donor substrate, the enzyme concentration, the reaction time and the reaction temperature are appropriately increased. It is preferable.

As used herein, the term "sugar donor substrate" refers to a substance capable of providing a sugar residue to another molecule. Examples of sugar donor substrates include glycans (ie, polysaccharides or oligosaccharides) and glycosides. As used herein, the term "glycan" refers to a compound formed by dehydration condensation of two or more monosaccharides. Usually, a compound formed by dehydration condensation of 2 to 9 monosaccharides is called an oligosaccharide, and a compound formed by dehydration condensation of 10 or more monosaccharides is called a polysaccharide. The glycan may be a simple polysaccharide or a complex polysaccharide. The simple polysaccharide refers to a polysaccharide in which a monosaccharide serving as a constitutional unit is one type. The complex polysaccharide refers to a polysaccharide having two or more types of monosaccharides as constituent units. Examples of glycans are glucan,
Examples include galactan, mannan, xylan, arabinan, chitin and chitosan.

As used herein, the term "glucan" refers to a polysaccharide composed of D-glucose. Glucans are classified into α-glucan and β-glucan according to the arrangement of anomeric carbon atoms of glucose residues. The glucan is preferably α-glucan. Examples of α-glucans include starch, amylose, amylopectin, dextrin, cyclodextrin, glycogen, dextran, pullulan, nigeran, isoligenan and their inclusions. Examples of starches include soluble starch, potato starch, corn starch, tapioca starch and the like. As the sugar donor substrate, it is also possible to use malto-oligosaccharides such as maltose and maltotriose or a mixture thereof, low molecular weight dextrin and the like, in which case the amount of reducing sugar in the product is starch or the like. It is higher than that when using high molecular weight glucan. When using high molecular weight glucan,
Since it does not reduce the yield of glycosylated Rakan Glycosides,
It is preferable not to include a reducing sugar that can be a good acceptor substrate for CGTase such as glucose or maltose. When preparing a solution of a substrate containing a polysaccharide such as starch that is difficult to dissolve in water, it is preferable to sufficiently gelatinize the starch by boiling it before adding the glycosyltransferase.

An example of β-glucan is cellulose.

In the present specification, the "glycoside" refers to a compound formed by dehydration condensation of sugar and a substance other than sugar. Examples of glycosides include phenyl α-glucoside, paranitrophenyl, β-galactoside and the like.

The sugar donor substrate is 0.1 to 50% (w / v), preferably 1 to 3 in this reaction system when the Rakan fruit glycoside is contacted with the sugar donor substrate and transferase.
It is preferably used in a concentration range of 0% (w / v), but is not limited thereto. In the reaction system used in the production method of the present invention, if the sugar donor substrate concentration is set to about 1/2 to 1/4 of the acceptor substrate concentration, a sufficiently high synthesis rate of glycosylated Rahan fruit glycosides ( When the acceptor substrate used is 100%, about 80-90%) can be achieved. When a high-concentration sugar donor substrate is used, a long-chain sugar residue is transferred to the sugar-acceptor substrate in the early stage of the reaction, and a disproportionation reaction causes a short-chain sugar residue to be transferred over time. Since it is transferred to the sugar acceptor substrate, if the reaction is stopped at an early stage, a product having a large degree of polymerization of glucose residues and a large amount of glycosylated product can be obtained. Furthermore, as the reaction time elapses, the proportion of glycosylated products having a low degree of polymerization of glucose residues gradually increases. However, in these cases as well, it goes without saying that it depends on other conditions such as an appropriate enzyme concentration and reaction temperature, and it is desirable to make a determination by conducting a preliminary experiment.

As used herein, the term "glycosyltransferase" refers to an enzyme capable of transferring a sugar residue from a sugar donor substrate to a sugar acceptor substrate. Any conventionally known glycosyltransferase can be used in the present invention. Examples of glycosyltransferases include cyclodextrin synthase (CGTase), α-galactosidase, β-galactosidase, β-fructosidase, α-glucosidase, α-mannosidase, β
-Mannosidase, α-amylase, α-pullulanase, dextrin dextranase, D enzyme, amylomaltase, dextranase, phosphorylase, sucrose phosphorylase, maltose phosphorylase, trehalose phosphorylase, α-glucosyltransferase, amylosucrase, dextranslase,
Levansucrase, inulinase, levanfructotransferase, galactanase, α-galactosidase, β-galactosidase, mannanase, xylanase, arabinase, cellulase, α-xylosidase,
β-xylosidase, α-arabinosidase, cyclodextran synthase, lysozyme, agarase, laminarinase, lichenase, β-glucuronidase, α-glucuronidase, hyaluronidase, chitosanase, chitinase, N-acetylhexosaminidase, rhamnosidase and α-fucosidase. Is mentioned.

As the glycosyltransferase, CGTase is more preferable. This is because CGTase can catalyze the glycosylation of Rakan fruit glycoside with extremely high efficiency. Depending on the reaction conditions, CGTase can glycosylate 90% or more of the Rakan fruit glycosides used in the reaction. This high efficiency is due to the fact that CGTase hardly catalyzes the hydrolysis reaction that competes with the glycosyl transfer reaction. With conventional hydrolases, both hydrolysis and transfer occur at a constant rate. CGTase
Then, hydrolysis occurs only within 5% of the Rakan fruit glycosides used in the reaction. Luohan Glycoside is CGTas
It may be a very good receptor substrate in the method of the present invention using e.

Any commercially available CG may be used as CGTase.
Tase may be used, or CGTase may be purified from microbial cells, microbial culture (for example, culture supernatant) and used. Examples of commercially available CGTases include Bacillus stearothermophilus (Bacill)
CSTase (produced by Hayashibara Biochemical Laboratories) derived from us stearothermophilus, Thermomo aerobacter or Thermoanaerobium (The)
rmoanaerobacterium sp. , Therm
oanaerobium sp. ) -Derived CGTase
(“CGTa” manufactured by Novo Nordisk Industries Ltd.
se ACN0002 ", Japanese Patent Application No. 2-500247,
B. E. Norman and S.M. T. Jφrense
n, Denpun Kagaku, 1992; 39:10.
1-108, in the present specification, hereinafter referred to as "CGTase derived from" Termoanaeobacter "", Bacillus
Circulance (Bacillus circulan)
s) -derived CGTase (Hayashibara Biochemical Laboratories, Inc.), Bacillus maceras (Bacillus macera)
ns) -derived CGTase (Amano Enzyme's "Contizyme"), Brevibacterium genus (Breviva)
cterium sp. ) -Derived CGTase (manufactured by Amano Enzyme Inc.) and the like. Since CGTase is an enzyme that is industrially manufactured and sold by a plurality of manufacturers, it can be obtained in large quantities at a relatively low cost, and it can be appropriately selected from enzymes of different origins.

As the α-galactosidase, any commercially available α-galactosidase may be used, or the α-galactosidase may be purified from microbial cells, microbial culture (eg, culture supernatant), and used. . Α to be marketed
-Examples of galactosidase include α from Mortierella vinacea
-Galactosidase (manufactured by Sigma), α-galactosidase derived from almond (manufactured by Sigma), Aspergillus niger (Aspergillus niger)
r) -derived α-galactosidase (manufactured by Amano Enzyme) and the like. α-Galactosidase uses melibiose, raffinose and the like as sugar donor substrates.

As β-galactosidase, any commercially available β-galactosidase may be used, or β-galactosidase may be purified from microbial cells, microbial culture (eg, culture supernatant), and used. . Beta marketed
-Examples of galactosidases include E. coli
β-galactosidase (manufactured by Sigma) derived from Riccia coli, Aspergillus oryzae (Asper)
β-galactosidase derived from gillus oryzae (manufactured by Yakult), Penicillium multicolor (Penicillium multico)
or β) -galactosidase (Kai Kasei Co., Ltd.), Bacillus circulans (Bacillus)
and β-galactosidase (manufactured by Daiwa Kasei). β-galactosidase is
Lactose, galactooligosaccharide, and galactoside glycoside are used as sugar donor substrates.

As the β-fructosidase, any commercially available β-fructosidase may be used, or the β-fructosidase may be purified from microbial cells, a microbial culture (eg, culture supernatant), or the like. You may use it. Beta marketed
-Examples of fructosidase include β-fructosidase derived from Arthrobacter genus (manufactured by BICO (manufactured by Saltwater Port Sugar)), Saccharomyces cerevisiae (Saccharomyces cer).
β-fructosidase (manufactured by Sigma) derived from Evisiae, baker's yeast, brewer's yeast, β-fructosidase derived from Aspergillus niger (Aspergillus niger, Aspergillus niger) (Hidaka, Hirayama; “Chemistry and organisms”, 23) , 600 (1985)). β-
Fructosidase uses sucrose, fructooligosaccharide, etc. as a sugar donor substrate.

As the α-glucosidase, any commercially available α-glucosidase may be used, or the α-glucosidase may be purified from microbial cells, microbial culture (eg, culture supernatant), and used. . Examples of commercially available α-glucosidases include α-glucosidase derived from Aspergillus niger (manufactured by Amano Enzyme Inc.) and Bacillus stearothermophilus (Bacillus stearoro).
α-glucosidase (thermophilus) -derived (manufactured by Sigma), Saccharomyces cerevisiae (Sac
Examples include α-glucosidase (manufactured by Sigma) derived from charomyces cerevisiae. α-
Glucosidase uses maltose, dextrin and the like as sugar donor substrates.

As the α-mannosidase, any commercially available α-mannosidase may be used, or natural α-mannosidase may be purified from microbial cells, microbial culture (for example, culture supernatant) and used. Good. Α to be marketed
-Examples of mannosidases include alpha from jack bean
-Mannosidase (manufactured by Sigma). The α-mannosidase uses a mannoside glycoside, a manno-oligosaccharide, etc. as a sugar donor substrate.

The glycosyltransferase may be of any purity so long as it does not contain other enzyme activity that adversely affects the glycosyltransferase reaction. The glycosyltransferase can be used in any form such as a mere inclusion or an immobilized enzyme as long as it can exert its glycosyltransferase activity.

The amount of the glycosyltransferase may be any amount as long as it can catalyze the glycosyl transfer reaction to the Rakan fruit glycoside.
Appropriate amounts can be appropriately determined by those of ordinary skill in the art. It is preferably 0.01 to 10000 units, and more preferably 0.1 to 5000 units per 1 g of the substrate. For example, CGTase used in the transglycosylation reaction is Bacillus
In the case of CGTase derived from Stearothermophilus or Thermoanaerobacterium, 0.1 to 2000 units are preferable per 1 g of starch.

The larger the amount of glycosyltransferase, the shorter the reaction time required for the glycosyl transfer reaction. When CGTase derived from Bacillus macerans is used, even if the reaction is carried out at a lower temperature (50 ° C) than CGTase derived from Bacillus stearothermophilus or Thermoanaerobacterium, the yield is almost the same as that of other origin. Glycosylated Rahan fruit glycosides can be obtained at a rate.

In one embodiment, a hyperglycosylated compound in which five or more glucose residues are bound to the Rakan fruit glycoside is once produced, and then a glycolytic enzyme is allowed to act to give 1 to 4 to the Rakan fruit glycoside. A hyperglycosylated compound with attached glucose residues is produced.

<Highly Glycosylated Compound Containing 1-4 Glucose Residues in Rakan Glycoside> Partially Degraded Glycosylation Any glycolytic enzyme can be used for the production of Rakan glycoside. As such a glycolytic enzyme, α
-Amylase, β-amylase, glucoamylase, α
-Glucosidase, maltotriose-producing amylase,
Examples include maltotetraose-forming amylase, maltopentaose-forming amylase, maltohexaose-forming amylase, isoamylase, and pullulanase, as long as they have the action of shortening the glycosyl sugar chain bound to the Rakan fruit glycoside. Not limited.

The α-amylase is an amylase that randomly hydrolyzes the inside of the sugar chain of a glycosyl group. As the α-amylase, any commercially available α-amylase may be used, or the α-amylase may be purified from microbial cells, microbial culture (for example, culture supernatant), and used. Examples of commercially available α-amylases include α-amylase derived from Bacillus subtilis, Aspergillus oryzae (As).
and α-amylase (manufactured by Daiwa Kasei Co., Ltd. and Novo Nordisk Industry Co., Ltd.) derived from pergillus oryzae).

In particular, α-amylase having a strong saccharifying power is more preferable than amylase having a strong liquefying power of starch for the production of partially-degraded glycosyl sugar conjugate.

Β-amylase is an amylase that hydrolyzes maltose units from the non-reducing end side of a glycosyl group. As the β-amylase, any commercially available β-amylase may be used, or the β-amylase may be purified from microbial cells, microbial culture (for example, culture supernatant) and used. Examples of commercially available β-amylase include barley-derived β-amylase and wheat-derived β-amylase.
Examples include amylase, soybean-derived β-amylase (manufactured by Amano Enzyme Inc. and Nagase Chemtech), and sweet potato-derived β-amylase (manufactured by Sigma).

Glucoamylase is an amylase that hydrolyzes in glucose units from the non-reducing end side of the glycosyl group. As the glucoamylase, any commercially available glucoamylase may be used, or the glucoamylase may be purified from microbial cells, microbial culture (for example, culture supernatant), and used. Examples of commercially available glucoamylases include Rhizopus
glucoamylase derived from niveus (manufactured by Seikagaku Corporation), Aspergillus niger
s niger) -derived glucoamylase (manufactured by Hankyu Bio Industry).

<Other Components> As the solvent, any solvent can be used. For example, water can be used.

The pH of the reaction solution during the glycosyl transfer reaction and the partial decomposition reaction can be arbitrarily set within a pH range in which an enzyme that catalyzes each reaction can act, but typically, p
H3 to 11, and preferably 5 to 7. The pH of the reaction solution can be appropriately adjusted in consideration of the optimum pH of the enzyme used in the reaction. The method for adjusting the pH of the reaction solution is well known to those skilled in the art.

A buffer solution is not always necessary, but any buffer solution may be used if necessary. For example, 10 to 500 mM acetate buffer and phosphate buffer in the above pH range can be used.

Even if the reaction system contains about 5 to 10% (v / v) of a water-soluble organic solvent such as methanol, ethanol or isopropanol, the yield of the highly glycosylated compound is hardly affected, but it is very high. Since it becomes an extremely weak receptor substrate, it is desirable that it does not exist. It is not particularly necessary to add a metal salt such as calcium or magnesium 2.

<Method for Producing Highly Glycosylated Compound> In the method for producing the compound of the present invention, unless otherwise specified, the glycoside extraction and fractionation methods and sweetener known in the art are well known. A preparation method or the like can be adopted. These techniques can be performed using a commercially available column or the like.

The hyperglycosylated compound of the present invention is produced by a method including a step of contacting a Rakan fruit glycoside with a sugar donor substrate such as glucan and a glycosyltransferase to obtain a hyperglycosylated compound. Typically, the highly glycosylated compound of the present invention will be used to convert Rakan fruit glycosides to α-
It is produced by a method including a step of contacting a sugar donor substrate such as glucan and a glycosyltransferase to obtain a hyperglycosylated compound.

In the method of the present invention, first, Rakan fruit glycosides,
A sugar donor substrate such as glucan and a glycosyltransferase are mixed. The order of mixing these may be in any order. That is, first, the Rakan fruit glycoside and the sugar donor substrate may be mixed, and then a glycosyltransferase may be added to and mixed therewith, or after the Rakan fruit glycoside and the glycosyltransferase are mixed, The sugar-donor substrate may be added to and mixed with this, or the glycosyltransferase and the sugar-donor substrate may be mixed first, and then the Rakan fruit glycoside may be added and mixed therein. The glycoside, sugar donor substrate and glycosyltransferase may be mixed together. When an enzyme other than CGTase is used as the glycosyltransferase, and the Rakan fruit glycoside is added after mixing the glycosyltransferase and the sugar donor substrate, from the mixture of the glycosyltransferase and the sugar donor substrate,
It is preferable that the time until the addition of Rakan fruit glycoside is as short as possible. Mixing may be performed using any method as long as the Rakan fruit glycoside, the sugar donor substrate and the glycosyltransferase are substantially uniformly confused, and the mixing time is appropriately selected. Can be done.

By mixing in this way, the Rakan fruit glycoside can come into contact with the sugar donor substrate and the glycosyltransferase. During the contact, the mixture is preferably kept at a temperature suitable for the transglycosylation reaction. Reaction temperature is typically 1
It is 0 to 100 ° C, preferably 40 to 90 ° C. The reaction temperature suitable for the glycosyltransferase used is known to those skilled in the art and can be appropriately selected by those skilled in the art. For example, since CGTase derived from Bacillus stearothermophilus or Thermoanaerobacter is highly heat-resistant, CGTase derived from Bacillus stearothermophilus is a CGTase derived from Thermoanaerobacter for several days in a reaction at 80 ° C.
Se can be used for several days in a 90 ° C. reaction. On the other hand, CGTase derived from Bacillus macerans
Is not so high in heat resistance, so it is preferable to use it at a reaction temperature of 50 ° C. or lower.

The time for which the Rakan fruit glycoside is contacted with the sugar donor substrate and the glycosyltransferase can be appropriately selected by those skilled in the art in consideration of the glycosyltransferase to be used. The contact time is typically 5 minutes to 10 days, preferably 30 minutes to 5 days, more preferably 1 hour to 3 days.

By contacting the Rakan fruit glycoside with the sugar donor substrate and the glycosyltransferase, a glycosyl residue is transferred to the Rakan fruit glycoside, and one or more glucose residues are transferred to the Rakan fruit glycoside. A bound hyperglycosylated compound is formed. The reaction system containing the highly glycosylated compound may be used as it is for the intended purpose, or may be subjected to a treatment for inactivating the glycosyltransferase to prevent further glycosyltransferase reaction. The highly glycosylated compound may be partially or completely purified from the system.

For example, when contacting mogroside V, which has the highest content among Rakan fruit glycosides, with starch and CGTase, a plurality of R ₁ and R ₂ of the above-mentioned chemical structure 1 is produced by the action of CGTase. The number of glucose residues newly bonded to the glucose residue (i.e., the degree of polymerization) is usually 1 to 15, respectively.
There are 5 items relatively. However, these ratios are not limited because they change depending on the reaction conditions. CGTase
By using, the anomeric form of the bond between the glucose residue of mogroside V and the newly bound glucose residue is limited to the α form. The degree of polymerization of newly bound glucose residue obtained by the action of CGTase is 2
In the above cases, the binding mode between glucose residues is usually α
Although there are only -1,4 bonds, α-1,6 bonds (that is, a branched structure) may be formed.

The highly glycosylated compound in which 5 or more glucose residues are α-linked to the Rahan fruit glycoside is a highly glycosylated compound in which 1 to 4 glucose residues are α-linked to the Rahan fruit glycoside. It can be contacted with a glycolytic enzyme in order to obtain (ie a partially degraded glycosylated Luohan glycoside).

Glycosylated Rahan Glycosides can be contacted with a glycolytic enzyme by being mixed with the glycolytic enzyme in, for example, an aqueous solution. The reaction solution containing the glucosylated Rakan fruit glycoside may be directly mixed with the glycolytic enzyme, but it is preferably mixed with the glycolytic enzyme after the treatment for removing or deactivating the glycosyltransferase. . Examples of the treatment for inactivating the glycosyltransferase include heating, pH change,
Examples include addition of an organic solvent such as ethanol.
Examples of heating include boiling for 15 minutes. Ordinary glycosyltransferase is almost inactivated by boiling for 15 minutes. When the glycosylated Rakan fruit glycoside is treated with a glycolytic enzyme after deactivating the glycosyltransferase by adding an organic solvent, it is preferable to distill off the organic solvent before adding the glycolytic enzyme. . When glycosylated Rahan Glycosides are contacted with a glycolytic enzyme without treatment for deactivation, glucose, oligosaccharides, etc. produced by the decomposition reaction of the glycolytic enzyme are sugar acceptors of the glycosyl transfer reaction. Since the glycosylated Rakan Gyocoside once produced becomes a body substrate and can function as a sugar donor substrate to return to a Rakanka glycoside, the yield of partially degraded glycosylated Rakan Gyocoside may decrease.

Glycosylation During the contacting of the Luohan glycoside with the glycolytic enzyme, the mixture may be kept at a temperature suitable for the hydrolysis of glycosyl residues by the glycolytic enzyme.
A suitable temperature range is typically 30-80 ° C.

Glycosylation The pH of the mixture during the contact between the sugarcane glycoside and the glycolytic enzyme is in the range of pH 5 to 7 in which the glycosyl transfer reaction is carried out, and there is no need to readjust the pH. The partial decomposition reaction can be continued as it is.

The time for contacting the glycosylated Ra Han Glycoside with the glycolytic enzyme is determined in consideration of the nature of the glycolytic enzyme used in the hydrolysis reaction and the desired composition of the partially degradable glycosylated Ra Han Glycoside. Alternatively, it can be appropriately determined by those skilled in the art by measuring the progress of the reaction using a method known in the art. The degree of production of partially decomposed glycosylated Rakan fruit glycosides can be determined, for example, by high performance liquid chromatography (H
PLC) or thin layer chromatography (TLC) can be used to measure (quantify, analyze, etc.).

[0120] In this manner, the partially-degraded glycosylated Rahan-glycoside is obtained. The mixture containing the partially-degraded glycosylated Rahan-glycoside may be used as it is for the intended purpose, or may be a glycolytic enzyme May be inactivated, or the partially-degraded glycosylated Rakan fruit glycoside may be partially or completely purified. An example of the treatment for deactivating the glycolytic enzyme is boiling.

<Method for Isolating, Analyzing and Identifying Hyperglycosylated Compound> The hyperglycosylated compound of the present invention can be analyzed by methods known in the art. Examples of such analysis methods include high performance liquid chromatography (HPLC), TLC, silica gel chromatography and the like.

Columns that can be used for HPLC include
An amide column can be used. Examples of the amide column include Asahi Pack NH2P-50 (manufactured by Shoredex), Amide 80 (manufactured by Tosoh Corporation) and the like. When using these columns, it is preferable to add the same volume of acetonitrile, ethanol, or the like to the sample solution to be analyzed, and remove unreacted high-molecular-weight glucan or the like as a precipitate in advance.

Eluents that can be used in HPLC include aqueous acetonitrile, typically 55-85.
A% (v / v) aqueous acetonitrile solution is suitable. With regard to the highly glycosylated compound, the higher the degree of polymerization of the glucose residue bound to the Rakan fruit glycoside, the longer the retention time on the column.

Other columns that can be used for HPLC include reverse phase columns. Since the reverse phase column does not require a precipitation treatment, the analytical operation is simpler than when an amide column is used. Examples of the reverse phase column include YMC-Pack ODS-AQ (manufactured by YMC),
Shim-pack CLC-ODS (manufactured by Shimadzu Corporation) can be mentioned. When using a reversed-phase column, the retention time on the column will be shorter as the highly glycosylated compound with more glucose residues α-linked to the Rakan fruit glycoside,
Receptor substrates, Rakan fruit glycosides, tend to have longer retention times than highly glycosylated compounds.

When the reaction solution is analyzed by thin layer chromatography, it can be qualitatively analyzed as follows, for example. A part of the reaction solution is spotted on a thin layer plate (Kieselgel 60, manufactured by Merck & Co., Inc.) and developed by an ascending method using ethyl acetate: acetic acid: water (3: 1: 1, v / v) as a developing solvent. An appropriate time since the deployment started (typically,
5 minutes to 1 hour, more preferably 15 minutes to 30 minutes)
After the passage of, the thin plate is taken out from the developing solvent and air dried. In order to detect the highly glycosylated Rakan fruit glycoside, 50% sulfuric acid / methanol solution is sprayed on the air-dried thin plate and heated at 120 ° C. As a result, the portion of the thin plate containing the glycoside changes to brown. In glycosylated Rakan-glycoside, the greater the number of glucose residues bound to the Rakan-kaido glycoside, the smaller the expansion mobility.

The hyperglycosylated compounds of the present invention (glycosylated Rahan glycosides and partially degraded glycosylated Rakan glycosides) can be purified using purification methods well known to those skilled in the art. The highly glycosylated compound of the present invention is subjected to adsorption chromatography using a reversed-phase column (for example, ODS), gel filtration chromatography, etc. to remove glucose and oligosaccharides mixed in a small amount. The purity of the highly glycosylated compound of the present invention can be increased.

For example, in ODS chromatography,
Equilibrate the ODS column with water. Glucose and short chain oligosaccharides in the sample are not adsorbed on the ODS column,
It is collected in the non-adsorbed fraction and the water-washed fraction. Glycosylated loquat glycosides can be eluted by increasing the ethanol concentration or methanol concentration in the eluent stepwise or linearly. At that time, the glycosylated Rakan fruit glycosides having a large number of glucose residues bound to the Rakan fruit glycoside are eluted in order.

At the highest concentration, the ethanol concentration in the eluent preferably does not exceed 90% (v / v), and is suitably about 20 to 50% (v / v).

Examples of gel filtration carriers that can be used are Sephadex G-15 or Sephadex G-25 (Pharmacia) and Biogel P-2 (BioRad).
Etc.

As an eluent for gel filtration chromatography, distilled water, 5% ethanol or the like can be used.

<Industrial Level Production of Glycosylated Rahan Glycoside or Partially Degraded Glycosylated Rahan Glycoside> Examples of production steps of glycosylated Rahan glycoside and partially degraded glycosylated Rahan glycoside at an industrial level As shown in FIG.
Hereinafter, this example will be described.

As the acceptor substrate, a mixture of any purity such as a crude extract of Rakan fruit, a partially purified product and a purified product of each glycoside component can be used. Also, fructose contained in the crude extract is a glycosyltransferase (eg, CGTas).
Since it cannot serve as the receptor substrate of e), there is no problem even if it is contained in the receptor substrate at a high concentration.

In a temperature-controlled reaction kettle having a capacity corresponding to the production amount,
Add Lo Han Guo extract, starch and CGTase, and make up with water. The optimum reaction temperature (for example, 40 ° C. to 90 ° C.) and the optimum reaction time (for example, 6 to 48 hours) according to the CGTase to be used, and then the CGTas used.
The glycosyltransferase activity is inactivated by heating at 70 ° C to 100 ° C for 15 minutes depending on the heat resistance of e. Glucose-degrading enzyme is added as necessary and reacted at an optimum reaction temperature (for example, 30 ° C to 80 ° C) for the carbohydrate-degrading enzyme to be used, and 1 to 4 glucose residues are contained in the Rakan fruit glycoside. It is also possible to obtain linked partially degradative glycosylated Rakan fruit glycosides.

Next, in order to isolate and purify the highly glycosylated Rakan Gyocoside or the partially degraded glycosylated Rakan Gyocoside having 1 to 4 glucose residues bound to the Rakanka Glycoside, obtained above. The reaction product is poured into a column packed with ODS reverse phase resin at an optimum flow rate. After washing this column with water, the glycosylated Rakan-glycoside or the partially decomposed glycosylated Rakan-glycoside is eluted with an aqueous solution having an optimum alcohol concentration.

In the concentration step, a reduced-pressure concentration apparatus for alcohol recovery having a volume corresponding to the amount of the obtained eluate is used for the above-mentioned eluate, and the alcohol is recovered under the optimum vacuum degree and concentration temperature conditions necessary for alcohol recovery. Are collected and the eluate is concentrated. The recovered alcohol is recycled for use.

Regarding the powdering step, the concentrated eluate described above was applied to a spray dryer having a water evaporation amount corresponding to the amount of the concentrated eluate, and the glycosylated Rakan fruit glycoside or partially decomposed glycosylated Rakan fruit was obtained. A powdered dried product of glycoside is obtained.

The chemical structures of the isolated glycosylated Rahan-glycoside and partially degraded glycosylated Rakan-glycoside can be confirmed by mass spectrum. The chemical structure of mogroside V confirmed by the mass spectrum is shown in chemical structure 4 below. The coupling mode between glucose is C
Due to the specificity of GTase, there are only α-1,4 bonds, and it is not necessary to analyze the isolated product of each polymerization degree in detail by NMR or the like.

(Chemical structure 4)

[0139]

[Chemical formula 24] In the method of the present invention, since the glycosyltransferase hardly catalyzes the hydrolysis reaction, the proportion of products having different degrees of polymerization of sugars bound to Rakan fruit glycosides changes with the progress of the reaction,
The overall yield of glycosylated Rakan Glycosides hardly decreased over the reaction time. Therefore, strict control of reaction conditions and frequent monitoring of products during the reaction are unnecessary.

Preferably, starch is used as the sugar donor substrate. In this case, combined with high glycosylation efficiency, it is possible to reduce the production cost of glycosylated Rakan fruit glycoside.

<Use of Highly Glycosylated Compound> By bringing a sweetener containing a Rakan fruit glycoside into contact with α-glucan and a glycosyltransferase, the Rakan fruit glycoside contained in the sweetener is highly glycosylated. Converted to a compound.
Since the highly glycosylated compound has a better sweetness, which is closer to sucrose than the Rakan fruit glycoside, this improves the taste of this sweetener.

The highly glycosylated compound of the present invention can be used as a food composition, a sweetener, a pharmaceutical composition, a quasi drug composition, and a cosmetic composition.

The food grade composition of the present invention contains a highly glycosylated compound. As used herein, "food composition"
The term refers to anything that can be used for food. Examples of food compositions include cooked foods; drinks such as soft drinks, functional drinks and jelly drinks; confectioneries such as Western confectionery and Japanese confectionery; dairy products such as yogurt; seasonings; health foods; special-purpose foods (Foods for specified health use). The content of the highly glycosylated compound in the food composition of the present invention is
It depends on the form and use of the food composition and can be appropriately selected by those skilled in the art. For example, common soft drinks,
In the case of beverages such as functional beverages and jelly beverages, the content of the highly glycosylated compound in the entire beverage is typically about 0.001 to about 2.0% by weight, preferably about 0.
005 to about 1.0% by weight, more preferably about 0.01
Is about 0.5% by weight.

The food composition of the present invention can be manufactured using methods known to those skilled in the art. Those skilled in the art can select an appropriate manufacturing method depending on the form and type of the food composition. Here, the hyperglycosylated compound may be incorporated into the food composition in any manner.

The highly glycosylated compound contained in the food composition of the present invention is used as a sweetener in place of general sucrose as a cooked food, Western sweets, Japanese sweets, beverages, dairy products,
It can be widely used for food applications such as seasonings, health food applications, special-purpose foods (foods for specified health uses), and the like. The highly glycosylated compound of the present invention is particularly useful for the above applications because it is stable to heat, does not cause browning and coloration, and is stable in acidic foods.

The sweeteners of the present invention are energy-suppressing sweeteners containing highly glycosylated compounds. As used herein, the term “sweetener” refers to a composition used for sweetening foods. The sweetener of the present invention may be a low energy sweetener or a zero energy sweetener. Alternatively, the sweetener of the present invention has an energy per unit weight of the sweetener which is almost the same as that of sucrose, but has a significantly higher sweetness intensity than sucrose, so that the amount of the sweetener used is extremely small. It may be a sweetener that can reduce the absolute amount used. According to the Nutrition Improvement Act, as a standard for labeling that emphasizes that nutritional content is low,
Energy indications such as "Low", "Light", "Himeme", "Reduction", "Cut" and "Off" are 100% sweetener.
The energy per g is specified to be 40 kcal or less (however, the food to be consumed is 20 kcal or less).
Energy indications such as "none", "zero", "non",
Energy per 100 g of sweetener is set to 5 kcal or less. In a preferred embodiment, the sweetener of the present invention may be a sweetener which can be displayed by highlighting "zero" or "low" energy per 100 g.

The sweeteners of the present invention may be in liquid (ie, syrup-like), semi-solid or solid (eg powdered, granular, crystalline, hexagonal, etc.) form. Those skilled in the art can appropriately select the form of the sweetener depending on the application of the sweetener.

The content of the highly glycosylated compound in the sweetener of the present invention varies depending on the form and use of the product and can be appropriately selected by those skilled in the art. For example, when used as a solid high-potency sweetener for tabletop use, the sweeteners of the present invention may consist of the hyperglycosylated compound alone. That is, the content of highly glycosylated compound is 100% by weight.
Can be. When used as a general tabletop powder or granular low-energy sweetener and low-energy syrup, etc., the content of the highly glycosylated compound in the whole sweetener is typically about 0.001 to about 5 wt. %, Preferably about 0.005 to about 2% by weight, more preferably about 0.01 to about 0.5% by weight.

The sweeteners of the present invention are prepared using methods known to those of ordinary skill in the art appropriate to the form. For example, in the case of a sweetener in a solid form, it is typically prepared by mixing a highly glycosylated compound, and optionally other components. The solid sweetener may be shaped if desired. Liquid sweeteners are typically prepared by mixing and dissolving the highly glycosylated compound, and optionally other ingredients with the required amount of water.
Those skilled in the art can select an appropriate production method depending on the intended form and use of the sweetener composition.

The pharmaceutical composition of the present invention contains a hyperglycosylated compound. As used herein, the term "pharmaceutical composition" refers to any composition that can be used for medicine. Examples of the pharmaceutical composition include a preparation to be orally administered; a preparation to be applied sublingually (eg, sublingual tablet); an external preparation for dental use and an oral preparation (eg, gargle). The content of the highly glycosylated compound in the pharmaceutical composition of the present invention varies depending on the form and use of the pharmaceutical composition,
It can be appropriately selected by those skilled in the art. For example, in the case of a typical orally administered formulation, the content of highly glycosylated compound in the entire formulation is typically about 0.001 to
It is about 5% by weight, preferably about 0.05 to about 2% by weight, more preferably about 0.01 to about 0.5% by weight.

The pharmaceutical composition of the present invention can be manufactured using methods known to those skilled in the art. Those skilled in the art can select an appropriate production method depending on the form and type of the pharmaceutical composition. Here, the hyperglycosylated compound may be incorporated into the pharmaceutical composition in any manner.

The quasi drug composition of the present invention contains a highly glycosylated compound. In the present specification, the “quasi-drug composition” refers to any composition that can be provided for quasi-drugs. Examples of quasi-drug compositions include oral refreshing agents (eg, throat refreshing agents, stomach refreshing agents); medicated cosmetics; medicated toothpastes. Examples of the quasi drug composition include vitamin C agents, vitamin E agents, and vitamin E agents.
It may be a C agent, a vitamin-containing health agent, or a calcium agent. In the present specification, the quasi drug composition includes both a composition used as a quasi drug and a composition used as a new designated quasi drug. The content of the highly glycosylated compound in the pharmaceutical composition of the present invention varies depending on the form and use of the pharmaceutical composition and can be appropriately selected by those skilled in the art. For example, in the case of a general throat cooling agent, the content of the highly glycosylated compound in the whole throat cooling agent is typically about 0.001 to about 5% by weight, preferably about 0.05 to about 2% by weight. %, And more preferably about 0.01 to about 0.5% by weight.

The pharmaceutical composition of the present invention can be manufactured using methods known to those skilled in the art. Those skilled in the art can select an appropriate production method depending on the form and type of the pharmaceutical composition. Here, the hyperglycosylated compound may be incorporated into the pharmaceutical composition in any manner.

The cosmetic composition of the present invention contains a highly glycosylated compound. As used herein, the term "cosmetic composition" refers to any composition that can be used for cosmetics. Examples of cosmetic compositions include lipsticks and toothpastes. The content of the highly glycosylated compound in the cosmetic composition of the present invention varies depending on the form and application of the cosmetic composition and can be appropriately selected by those skilled in the art.
For example, in the case of general toothpaste, the content of the highly glycosylated compound in the whole toothpaste is typically about 0.005 to about 5% by weight, preferably about 0.005 to about 2% by weight, Preferably, the proportion is about 0.01 to about 0.5% by weight.

The cosmetic composition of the present invention may be manufactured using methods known to those skilled in the art. Those skilled in the art can select an appropriate production method depending on the form and type of the cosmetic composition. Here, the hyperglycosylated compound may be incorporated into the cosmetic composition in any manner.

The glycosyltransferase and the glycolytic enzyme used in the present invention include materials and additives such as cyclodextrin, coupling sugar (glycosyl sucrose) and the like for foods (eg sweeteners), pharmaceuticals, cosmetics and the like. Since it has been used in the industrial production of, there is no problem in terms of safety.

[0157]

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to the following examples.

<Example 1: Glucose transfer rate to mogroside V when various CGTases were used> Various CGTases
The following experiment was conducted in order to investigate the glycosyl transfer rate to mogroside V. First, 50 mM acetate buffer (pH
Composition containing 20% (w / v) of Rakan fruit glycosides (about 6.0% of mogroside V as Rakan fruit glycosides).
/ V) Included: Obtained from Guilin Sino-Tech Co., Ltd. of China) and 10% (w / v) soluble starch (Kanto Chemical Co., Inc.)
CGTase derived from Bacillus stearothermophilus against the solution of Rakan fruit glycoside (0.95 ml) containing
(Example 1-1), C derived from the genus Thermoanaerobacterium
GTase (Example 1-2), CGTase derived from Bacillus circulans (Example 1-3) or CGTase derived from Bacillus macerans (Example 1-4) (each 20 units) were added and mixed at 60 ° C. 24 hours and 4
The reaction was carried out for 8 hours. As a blank, the same operation was performed without adding CGTase.

After 24 hours or 48 hours of reaction, the reaction solution was boiled at 100 ° C. for 15 minutes to inactivate CGTase. The reaction solution after boiling was used as HPL equipped with an ODS column.
Analyzed in C. The amount of mogroside V in the blank reaction solution (MogV (bla)) was set to 100%, and the amount of mogroside V in the reaction solution after the transglycosylation reaction (MogV (rea
The reduction rate of ct)) was considered as the glycosyl transfer rate. That is,

[0160]

[Equation 1] The results are shown in Table 1.

[0161]

[Table 1] No matter which CGTase was used, mogroside V
Glycosylation was observed. Glycosyl transfer rates are shown to be extremely high when CGTase derived from Bacillus stearothermophilus, CGTase derived from Thermoanaerobacter, and CGTase derived from Bacillus circulans are used, and these three types of glycosyl transfer rates are shown. With the enzyme, the transglycosylation rate was as high as 80% or more even in the reaction for 24 hours (Table 1-
, Table 1- and Table 1-). Therefore, these enzymes can be preferably used in the production method of the present invention.

On the other hand, CGT derived from Bacillus macerans
In the case of using ase, when 20 units were added and reacted at 60 ° C., the glycosyl transfer rate was 50% or less, which was not so high. Therefore, the enzyme amount was increased to 100 units and the reaction temperature was 50 ° C. An experiment was conducted in the same manner as described above (Example 1)
-5) However, the synthesis rate after 24 hours increased to 80% (Table 1-).

From these, any microbial cell,
CGTa derived from microbial culture (eg, culture supernatant)
It was found that even in the case of using se, it can be preferably used for the production of glycosylated Rakan fruit glycoside by appropriately setting the amount of enzyme and the reaction temperature.

<Example 2: Effect of the amount of enzyme added and the reaction temperature on the glycosyl transfer to mogroside V> The following experiment was conducted to examine the influence of the amount of enzyme added and the reaction temperature on the glycosyl transfer to mogroside V. went. First, 50 mM
20% (w / v) composition of Rakan fruit glycoside in acetic acid buffer (pH 6.0) (containing about 30% (w / v) of mogroside V as Rakan fruit glycoside; China Guilin Special Technology Rakan fruit glycoside solution (0.95 m) containing 10% (w / v) soluble starch (produced by Kanto Chemical Co., Inc.)
l), CGTase enzyme (from Thermoanaerobacter sp., manufactured by Novo Nordisk) was added to 0.
67 units / g starch (Sample A), 1.33 units / g starch (Sample B), 3.33 units / g starch (Sample C), 6.67 units / g starch (Sample D), 13.3
Add and mix at a concentration of either 3 units / g starch (Sample E), 33.33 units / g starch (Sample F) or 66.67 units / g starch (Sample G), reaction temperature 60 ° C and At 80 ° C., reaction time 24 hours and 48
Reacted for hours. As a blank, the same operation was performed without adding CGTase.

After the reaction for 24 hours or 48 hours, the reaction solution was boiled at 100 ° C. for 15 minutes to inactivate CGTase. The reaction solution after boiling was used as HPL equipped with an ODS column.
It analyzed by C and judged the influence of the enzyme amount and reaction temperature which affect glycosyl transfer. In the same manner as in Example 1, the amount of mogroside V (MogV (bla)) in the blank reaction solution was 100.
%, And the rate of decrease in the amount of mogroside V (MogV (react)) in the reaction solution after the transglycosylation reaction was regarded as the transglycosylation rate.

Table 2 below shows the transglycosylation rates at various amounts of CGTase added, reaction temperature and reaction time.

[0167]

[Table 2] As can be seen from Table 2, with the increase in the amount of added enzyme (Sample A → Sample G), the main component of Rakan fruit glycoside (mogroside V)
The transglycosylation rate of (Mogroside V) was increased.
The transglycosylation rate was also increased by increasing the reaction temperature from 60 ° C to 80 ° C. The transglycosylation rate was also increased by increasing the reaction time from 24 hours to 48 hours. Therefore, the glycosyl transfer rate to mogroside V is
It can be increased by appropriately setting the amount of added enzyme, the reaction temperature and the reaction time. Therefore, a person skilled in the art can appropriately set the conditions suitable for synthesizing the highly glycosylated compound.

Example 3: Effect of Concentration of Sugar Donor Substrate on Glycosyl Transfer Rate Cyclodextrin synthase (CGTa
se, Thermoanae spp. (Thermoana)
erobacter sp. The effect of the concentration of the sugar donor substrate (starch) on the rate of glycosyl transfer to mogroside V.

First, a composition containing 20% (w / v) of Rakan Glycoside in each test tube (obtained from Guilin Sci-Tech Co., Ltd., China)
And an equal amount of soluble starch solution of various concentrations,
Composition containing 10% (w / v) Rakan Glycoside, 0.5%
(W / v), 1.0% (w / v), 2.5% (w /
v), 5.0% (w / v), 7.5% (w / v) or 9.6% (w / v) soluble starch solution was prepared. After 10 units of CGTase was added to and mixed with the reaction solution, the reaction was carried out at 60 ° C. for 20 hours and 44 hours.

After the reaction for 24 hours or 44 hours, the reaction solution was boiled at 100 ° C. for 15 minutes to inactivate CGTase. The reaction solution after boiling was used as HPL equipped with an ODS column.
Analysis at C determined the effect of the amount of starch on the transglycosylation. In the same manner as in Example 1, the amount of mogroside V (MogV (bla)) in the blank reaction solution was set to 100%,
Amount of mogroside V in the reaction solution after the glycosyl transfer reaction (Mog
The rate of decrease in V (react) was regarded as the rate of transglycosylation. The transglycosylation rate was measured by the area value of the main component (Mogroside V) of the Rakan fruit glycoside.

The results are shown in Table 3.

[0172]

[Table 3] Table 3 shows the effect of the concentration of the sugar donor substrate (starch) on the sugar transfer rate to mogroside V by CGTase. In HPLC, a decrease in the main component (Mogroside V) of the Rakan fruit glycoside was observed at any starch concentration, and glycosyl transfer was confirmed. Especially when the starch concentration is 2.5% or more, the sugar transfer rate (%) after 20 hours is
It was as high as 75% or more. Furthermore, when the starch concentration is 2.5% or more, the sugar transfer rate (%) is 78 to 44 hours after 44 hours.
It was extremely high at 93%. From the above results, it was suggested that even if a relatively low starch concentration is used, a sugar transfer product is produced in a high yield within 20 hours of the reaction, which is an extremely excellent reaction.

Example 4 Synthesis and Analysis of Partially Degraded Glycosylate> Synthesis and analysis of a partially degradable glycosylated product by treatment of a highly glycosylated compound with β-amylase was performed as follows.

First, 50 mM acetate buffer (pH 6.0)
Rakan fruit glycoside containing 30% (w / v) composition of Rakan fruit glycoside (obtained from Guilin Sino-Tech Co., Ltd.) and 15% (w / v) soluble starch (manufactured by Kanto Chemical Co., Inc.) For an aqueous solution (50 ml), the CGTase enzyme (Bacillus stialothermophilus (Bacillus st
1,400 units of Hayashibara Biochemical Laboratories) derived from eathermophilus) were added and mixed, and reacted at a reaction temperature of 60 ° C. for 18 hours to synthesize a highly glycosylated compound. After reacting for 18 hours, the reaction solution was heated at 100 ° C for 1 hour.
CGTase was inactivated by boiling for 5 minutes, and 99.5% ethanol (manufactured by Nakarai Co., Ltd.) was added in an amount of 2 times to precipitate the glucan mixture. This precipitate was removed by centrifugation to obtain a supernatant. This supernatant was evaporated to remove ethanol.

Β-Amylase (derived from soybean, manufactured by Hankyu Bio Industry Co., Ltd.) was added to and mixed with this solution at 40 ° C.
The reaction was carried out for 16 hours at 37 ° C. to partially decompose the added sugar residue from the synthesized glycosylated Rakan Gyocoside. After reacting for 16 hours, the reaction solution was subjected to HPLC to obtain a chromatogram.

The obtained chromatogram is shown in FIG. In FIG. 2, the horizontal axis represents the peak height and the vertical axis represents the retention time. The number near the peak indicates the retention time at which the peak appears. The molecular weight of the component contained in each peak was measured by LC mass, and the number of glucose residue bonds was determined.

A peak of the main component (Mogroside V) of the Rakan fruit glycoside was confirmed near the retention time of 4.5 minutes. After that, at 6 minutes, the peak of the glycosylated product in which 1 residue of glucose was bound to mogroside V was observed, in the vicinity of 8 minutes, the peak of the glycosylated product in which 2 residues of glucose were bound to mogroside V was found at the vicinity of 11 minutes, The peak of glycosylated product in which 3 residues of glucose are bound to mogroside V, and at about 15 minutes, mogroside V
The peaks of the glycosylated product in which four residues of glucose were bound were confirmed.

From FIG. 2, it is apparent that the partially-degraded glycosylated Rakan-glycoside is produced and separated by HPLC according to the number of sugars added.

<Example 5: Measurement of sweetness intensity> (1) Synthesis of glycosylated Rahan fruit glycoside Rahan fruit glycoside (Rahan fruit glycoside containing about 30% (w / v) of mogroside V as Rahan fruit glycoside) Containing composition: 15g of starch (obtained from Guilin Sci-Tech Co., Ltd. of China) and 7.5g of starch (soluble, grade 1, Kanto Chemical Co., Ltd.) were prepared to 50mL with 50mM acetate buffer (pH 6.0), and CGTase was prepared.
Enzyme (trade name; THERMOPHILICCGTas
e, origin; Bacillus stearotherm
Ophooilus, 1,400 units / g) (1 mL) was added, and the mixture was reacted at 60 ° C. for 18 hours to synthesize glycosylated Rakan fruit glycosides. Then, this reaction solution is heat treated to C
After deactivating GTase, at 10,000 rpm,
Centrifuge for 20 minutes, and add the supernatant to the ODS column (Or
gano, φ30 mm × 450 mm), and after removing impurities by eluting 500 mL with deionized water, ethanol / water with a linear concentration gradient from water (0 v / v% ethanol) to 60 v / v% ethanol Using 3
An eluate of L was obtained and the eluate was freeze-dried to obtain a powdered glycosylated Rakan fruit glycoside.

(2) Synthesis of partially-degraded glycosylated Rakan fruit glycosides Rakan fruit glycosides (Rahan fruit glycoside-containing composition containing about 30% (w / v) of mogroside V as Rakan fruit glycosides; 15 g of starch (obtained from Technology Co., Ltd.) and 7.5 g of starch (soluble, first grade, manufactured by Kanto Chemical Co., Inc.) were adjusted to 50 mL with 50 mM acetate buffer (pH 6.0), and CGTase enzyme (trade name; THERMOPHILICCGGTase,
Origin; Bacillus stearothermop
hoilus, 1,400 units / g) 1 mL, add 6
The reaction was carried out at 0 ° C. for 18 hours to synthesize glycosylated Rakan fruit glycoside. Then, this reaction solution is heat-treated to perform CGTa.
After deactivating the se enzyme, ethanol (special grade reagent, Na
Karai (manufactured by Karai) was added to precipitate starch, followed by centrifugation at 10,000 rpm for 20 minutes. The supernatant liquid was recovered, ethanol was removed by applying an evaporator, and the whole was concentrated to 40 mL. 40 mL of this concentrated solution was added to a degrading enzyme (trade name: β-amylase # 1500S, manufactured by Nagase Chemtex Co., Ltd., 1
5,000 AUN / g) 20 mg and added at 40 ° C for 16
The reaction was allowed to proceed for a period of time to synthesize a partially-degraded glycosylated Rakan fruit glycoside. After heat-treating this reaction solution to inactivate the decomposing enzyme, this reaction solution was subjected to ODS column (Organo, φ3
After charging to 0 mm × 450 mm) and eluting 1.5 L with deionized water to remove impurities, water (0 v /
(v% ethanol) to 60 v / v% ethanol in a linear concentration gradient of ethanol / water to obtain 3 L of eluate, and the eluate was freeze-dried to partially decompose glycosylated Rahan fruit glycosides in powder form. Got

(3) Glycosylated Rahan Glycoside and Partially Degraded Glycosylation Measurement of Sweetness Intensity of Rahan Glycoside and Glycosylated Rahan Glycoside and Partially Degraded Glycosylation Obtained in (1) and (2) Above, respectively The sweetness intensity of the Rakan fruit glycoside was measured by the following method. Using 12 healthy subjects (6 men, 6 women, average age 31.2 years),
Each sample when the sweetness intensity of sucrose is set to 1 by tasting a glycosylated Rakanka glycoside or a partially degraded glycosylated Rakanka glycoside for 10% sucrose aqueous solution Was calculated.

As a result, the concentration of the glycosylated Ra Han Glycoside aqueous solution necessary to obtain the sweetness intensity equivalent to that of the 10% sucrose aqueous solution was 0.141% by weight, which is equivalent to that of the partially decomposed glycosylated Ra Han Gly glycoside aqueous solution. The concentration was 0.056% by weight. Therefore, it was found that the sweetness intensity of glycosylated Rakan Gyocoside is about 70 times that of sucrose, and that of partially degraded glycosylated Rakan Gyocoside is about 180 times that of sucrose.

<Comparative Example 1: Measurement of sweetness intensity of Rakan fruit glycoside> Similar to Example 5, Rakan fruit glycoside (Rakan fruit glycoside containing about 30% (w / v) of mogroside V as Rakan fruit glycoside). The sweetness intensity of the body-containing composition (obtained from Guilin Sino-Tech Co., Ltd., China) was measured. As a result, the concentration of the Rakan fruit glycoside aqueous solution necessary to obtain the sweetness intensity equivalent to that of the 10% sucrose aqueous solution was 0.047% by weight. Therefore, Rakan fruit glycoside has a sweetness intensity about 210 times that of sucrose.

[0184]

[Table 4] <Example 6 and Comparative Example 2> Various sweetener aqueous solutions were prepared by the methods described below.

Example 6 Preparation of Glycosylated Luohan Glycoside and Partially Degraded Glycosylated Luohan Glycoside Aqueous Solution>
In a glass beaker having a volume of 50 mL, the glycosylated Rakan-caicoside glycoside and the partially-degraded glycosylated Rakan-cause glycoside obtained in (1) and (2) of Example 5 were each adjusted to 0.1% by weight. Preparation was performed to obtain an aqueous solution of glycosylated Rahan-glycoside and a partially decomposed aqueous solution of glycoside Rakan-glycoside.

<Comparative Example 2: Preparation of Rakan Glycoside Aqueous Solution>
In the same manner as in Example 6, 0.1% by weight of the Rakan fruit glycoside aqueous solution was added.
Was prepared so that

<Experimental Example 1: Evaluation of sweetener aqueous solution> 10 healthy subjects (5 males, 5 females, average age 31.4)
Sensory test was performed on the 6-element taste of the sweetener aqueous solutions obtained in Example 2 and Comparative Example 2. Using the sucrose aqueous solution as the standard solution, various sweetener aqueous solutions (glycosylated Rahanka glycoside aqueous solution or partially decomposed glycosylated Rahanka glycoside aqueous solution or Rakanka glycoside aqueous solution) were sampled, and "bitterness, backwashing, persistentness, Each of "dullness, astringency, and refreshing feeling" was evaluated on a scale of 7 (0 to 6). The evaluation score is 0 for "excellently better than sucrose solution", 2 for "excellently better than sucrose solution", 2 for "slightly better than sucrose solution", 3 points “equal to sucrose solution” and 4 points “slightly inferior to sucrose solution”
5 points, "It is considerably inferior to the sucrose solution"
A score of 6 was "very inferior to the sucrose solution". Therefore, the evaluation score of the sweetness quality of sucrose is 3.0 for all the factors.

Further, a radar chart was prepared based on the evaluation scores obtained for each element. That is, the evaluated 6-element taste was represented by 6 axes, and the average value of the evaluation scores of 10 subjects was plotted on each axis, and the plots were connected by a straight line to draw a hexagon. Further, it means that the sweetness quality is better as the plotted points of all six elements are closer to the inside, and conversely, the sweetness quality is poorer toward the outside.

The evaluation score by each subject of each sample,
Their average values are shown in the table and graph below.

[0190]

[Table 5] As is clear from the above table and FIG. 4, the evaluation score of the Rakan fruit glycoside aqueous solution of Comparative Example 2 was 4.0 or more for most of the elements, but the evaluation score of the glycosylated Rakan fruit glycoside aqueous solution was high. Was about 4.0 in all the elements, and was found to have an excellent sweetness quality as compared with Rakan fruit glycoside. In addition, the evaluation score of the partially decomposed glycosylated aqueous solution of Rakan glycoside is 3.1 to 3.
5 and similar evaluations to sucrose were obtained, and it was found that the partially-degraded glycosylated Rakan-kaito glycoside exhibits even better sweetness than the glycosylated Rakan-kaito glycoside.

<Example 7> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. After HPLC, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 1.

The partially decomposed glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 8> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. After HPLC, the structure was confirmed by NMR and MASS spectra to obtain a partially decomposed glycosylated Rakan fruit glycoside having the following structure of Compound No. 2.

The partially-degraded glycosylated Lo-han fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

Example 9 In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. After performing HPLC, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 3.

The partially-degraded glycosylated Luo Han Gyo glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 10> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 4.

The partially-degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 11> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 5.

[0200] The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 12> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rahan fruit glycoside having the structure of Compound No. 6.

The partially degraded glycosylated Rakan Gyocoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 13> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 7.

The partially degraded glycosylated Rakan glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 14> In the same manner as in Example 4, a large amount of partially degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially decomposed glycosylated Rakan fruit glycoside having the structure of Compound No. 8.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

Example 15 In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 9.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 16> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially decomposed glycosylated Rahan fruit glycoside having the structure of Compound No. 10.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 17> [0211] In the same manner as in Example 4, a large amount of partially-degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially degraded glycosylated Rahan fruit glycoside having the structure of Compound No. 11.

[0212] The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 18> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 12.

[0214] The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 19> A large amount of partially-degraded glycosylated Rakan fruit glycoside was obtained in the same manner as in Example 4. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rahan fruit glycoside having the structure of Compound No. 13.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 20> In the same manner as in Example 4, a large amount of partially degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 14.

[0218] The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 21> A large amount of partially-degraded glycosylated Rakan fruit glycoside was obtained in the same manner as in Example 4. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 15.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 22> In the same manner as in Example 4, a large amount of partially degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 16.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 23> In the same manner as in Example 4, a large amount of partially-degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 17.

The partially decomposed glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

Example 24 In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 18.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 25> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 19.

The partially degraded glycosylated Rakan Gyocoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 26> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 20.

The partially decomposed glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 27> In the same manner as in Example 4, a large amount of partially degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 21.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 28> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 22.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

Example 29 In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rahan fruit glycoside having the structure of Compound No. 23.

The partially degraded glycosylated Rakan Gyocoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the persistence, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 30> In the same manner as in Example 4, a large amount of partially degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 24.

The partially degraded glycosylated Rakan Gyocoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 31> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rahan fruit glycoside having the structure of Compound No. 25.

[0240] The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 32> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 26.

[0242] The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the persistence, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 33> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially degraded glycosylated Rahan fruit glycoside having the structure of Compound No. 27.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 34> A large amount of partially-degraded glycosylated Rakan fruit glycoside was obtained in the same manner as in Example 4. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rahan fruit glycoside having the structure of Compound No. 28.

The partially degraded glycosylated Rakan Gyocoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the persistence, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

Example 35 In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 29.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 36> In the same manner as in Example 4, a large amount of partially degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rahan-glycoside having the structure of Compound No. 30.

The partially degraded glycosylated Rakan Gyocoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 37> In the same manner as in Example 4, a large amount of partially degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 31.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the persistence, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 38> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 32.

[0254] The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 39> In the same manner as in Example 4, a large amount of partially-degraded glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially-degraded glycosylated Rakan fruit glycoside having the structure of Compound No. 33.

The partially degraded glycosylated Rakan Gyocoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

<Example 40> In the same manner as in Example 4, a large amount of partially decomposed glycosylated Rakan fruit glycoside was obtained. HPLC
After that, the structure was confirmed by NMR and MASS spectra to obtain a partially degraded glycosylated Rahan fruit glycoside having the structure of Compound No. 34.

The partially degraded glycosylated Rakan fruit glycoside was evaluated for sweetness in the same manner as in Example 6.
As a result, this partially decomposed glycosylated Rakan fruit glycoside,
It was found that it has an excellent sweetness property in which the bitterness, the aftertaste, the firmness, the habit, the astringency, and the refreshing feeling are significantly improved, as compared with the taste quality of the Rakan fruit glycoside.

[0259]

Industrial Applicability According to the present invention, a glycosyltransferase such as cyclodextrin synthase is allowed to act on a sugar donor substrate such as starch to produce a glycosylated Rakan fruit glycoside by a glycosyl transfer reaction very efficiently and inexpensively. And a method for producing a partially-degraded glycosylated Rakan fruit glycoside obtained by partially decomposing it by a glycolytic enzyme or the like.

According to the present invention, bitterness, after-taste, persistence,
Provided are a method for greatly improving the taste quality of a Rakan fruit glycoside, which is a soft and hypoallergenic taste quality, which is improved in taste items such as habit, astringency, and a refreshing feeling, and the novel glycosylated Rakan fruit glycoside. .

The glycoside obtained according to the present invention or the sugar containing the same can be used as a high-intensity sweetener for foods, pharmaceuticals, cosmetics and the like. The glycosylated Rahan-glycoside of the present invention has a particularly bitter taste compared to the taste quality of Rakan-glycoside,
After pulling, stubbornness, habit, astringency and refreshing feeling were significantly improved.

The highly glycosylated compound of the present invention can be used as an additive to foods such as tabletop sweeteners, beverages, confectionery, seasonings, pharmaceuticals, cosmetics and the like.

[Brief description of drawings]

FIG. 1 is a schematic view of an industrial production method of glycosylated Rakan-glycoside and partially degraded glycosylated Rakan-glycoside.

FIG. 2 is a high-performance liquid chromatogram of the Rakan fruit glycoside solution after treatment with CGTase and β-amylase enzyme.

FIG. 3 is a graph showing the sweetness intensity of sucrose as 1.
It is a graph which shows the sweetness intensity | strength of a glycosylated Luohan fruit glycoside and a partial decomposition glycosylated Luohan fruit glycoside.

FIG. 4 is a radar chart showing the results of Example 6 and Comparative Example 2.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C08B 37/00 C08B 37/00 G 4C090 C12P 33/00 C12P 33/00 4C091 (72) Inventor Shinichi Yoshikawa Osaka 474-2 Ami, Mihara-cho, Kawara-gun, Minami-ken (72) Aimi Nakamura Inventor Aimi Nakamura 2-4 Suehiro-cho, Izumiotsu-shi, Osaka (72) Inventor Sumio Kitabata 1-1-6 Mikumayo, Kumatori-cho, Sennan-gun, Osaka (72) ) Inventor Hirofumi Nakano 3-15-20 Hattori Nishimachi, Toyonaka City, Osaka Prefecture (72) Inventor Taro Kiso 5-250-7 Hikitacho, Nara City, Nara Prefecture (72) Motohiro Shizuma 6-tani Town, Chuo-ku, Osaka City, Osaka Prefecture 3-4 F-term (reference) 4B018 MD42 MD61 ME01 MF12 4B047 LB06 LB08 LG31 LG37 LP18 4B064 AH07 CA21 DA07 DA10 4C076 AA36 BB02 DD70 EE30 FF52 4C083 AA111 AD221 AD391 AD491 CC13 CC41 EE06 EE31 4C090 AA02 AA05 AA08 AA09 BA06 BB12 BB32.

Claims (20)

[Claims] 1. A highly glycosylated compound in which one or more glucose residues are α-linked to Rakan fruit glycoside. 2. The hyperglycosylated compound according to claim 1, wherein the number of glucose residues is 1 to 45. 3. The hyperglycosylated compound according to claim 1, wherein the number of glucose residues is 1 to 15. 4. A hyperglycosylated compound according to claim 1, selected from the group consisting of: [Chemical 2] [Chemical 3] [Chemical 4] [Chemical 5] [Chemical 6] [Chemical 7] [Chemical 8] [Chemical 9] 5. The hyperglycosylated compound according to claim 1, wherein one or more glucose residues are α-linked to mogroside V. 6. A food grade composition comprising the hyperglycosylated compound of claim 1. 7. A sweetener containing the hyperglycosylated compound according to claim 1. 8. A pharmaceutical composition containing the hyperglycosylated compound according to claim 1. 9. A quasi-drug composition comprising the highly glycosylated compound according to claim 1. 10. A cosmetic composition comprising the hyperglycosylated compound according to claim 1. 11. A method for producing a highly glycosylated compound, which comprises the step of contacting a Rakan fruit glycoside with an α-glucan and a glycosyltransferase to obtain a highly glycosylated compound. . 12. The method according to claim 11, wherein the glycosyltransferase is a cyclodextrin synthase. 13. The Rakan fruit glycoside is mogroside V.
The method of claim 11, wherein 14. A hyperglycosylated compound obtainable by the method of claim 11. 15. A method for producing a highly glycosylated compound in which 1 to 4 glucose residues are α-bonded to Rakan fruit glycoside, which comprises converting Rakan fruit glycoside into α-glucan and sugar. When contacted with a transferase, 5 or more glucose residues were
A step of obtaining a hyperglycosylated compound bound thereto, and bringing a glycolytic enzyme into contact with the hyperglycosylated compound wherein 5 or more glucose residues are α-bonded to the Rakan fruit glycoside; 1 to 4 glucose residues are α
A method comprising the step of obtaining a hyperglycosylated compound attached. 16. The method of claim 15, wherein the glycolytic enzyme is selected from the group consisting of glucoamylase, β-amylase and α-amylase. 17. The above-mentioned Rakan fruit glycoside is Mogroside V.
16. The method of claim 15, wherein 18. A hyperglycosylated compound having 1 to 4 glucose residues α-linked, obtainable by the method according to claim 15. 19. A method for improving the taste of a sweetener containing Rakan fruit glycoside, which comprises the step of contacting the sweetener with α-glucan and a glycosyltransferase. 20. A taste-improved sweetener obtained by the method of claim 19.