Xanthohumol glycoside, its production method, and method for improving water solubility of xanthohumol

Abstract

A purpose of the present invention is to provide a xanthohumol glycoside in which a sugar chain constructed from two or more hexoses is bonded to xanthohumol and a method for producing the same, a xanthohumol glycoside in which one hexose is bonded to xanthohumol and a method for producing the same, a method for improving the water solubility of xanthohumol, a method for reducing the bitter taste of xanthohumol, and a method for improving the stability of xanthohumol, etc. The present invention relates to a xanthohumol glycoside in which a sugar chain constructed from 2-10 hexoses is bonded to the 4'-position or 4-position of xanthohumol.

Description

[Technical Field]

[0001] The present invention relates to a xanthohumol glycoside in which a sugar chain composed of two or more hexoses is bound to xanthohumol, and a method for producing the same.The present invention also relates to a xanthohumol glycoside in which one hexose is bound to xanthohumol, and a method for producing the same.Furthermore, the present invention relates to a method for improving the water solubility of xanthohumol.The present invention further relates to a method for reducing the bitterness of xanthohumol, a method for improving the stability, etc. [Background technology]

[0002] Flavonoids, secondary plant metabolites, are found in a variety of plant-derived foods and beverages, many of which exist as glycosylated, highly water-soluble glycosides. On the other hand, xanthohumol (hereinafter sometimes abbreviated as XN) found in the female flowers of hops (Humulus lupulus) is biosynthesized in lupulin, a specialized cell group formed by the protruding epidermal cells of the female flowers, and accumulates as an aglycone. Xanthohumol is a type of prenylflavonoid containing a prenyl group.

[0003] Xanthohumol is known to have a variety of useful biological activities. However, because xanthohumol has extremely low polarity and is almost insoluble in water, the amount that can be added to beverages, for example, is sometimes limited.

[0004] There are known examples in which xanthohumol has been converted into a glucose glycoside (glucoside) in order to improve its solubility. Patent Document 1 discloses a method for producing xanthohumol glycoside by culturing a fungus of the genus Aspergillus in the presence of xanthohumol, and the produced xanthohumol glycoside is identified as xanthohumol 4'-O-β-glucopyranoside (XN4'G), a compound in which one glucose is bound to the 4' position of xanthohumol. Non-Patent Document 1 discloses that XN4'G and xanthohumol 4,4'-O-β-diglucopyranoside (XN4',4diG) were produced by culturing Penicillium chrysogenum 6933 in the presence of XN. On the other hand, it has been disclosed that when Rhizopus oryzae KCTC 6946 was cultured in the presence of xanthohumol, isoxanthohumol (IX) was produced. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-96750 [Non-patent literature]

[0006] [Non-Patent Document 1] J. Nat. Prod. 2006, 69, 1522-1524 Summary of the Invention [Problem to be solved by the invention]

[0007] Methods of using emulsifiers, dispersants, etc. are known as methods for increasing the solubility of poorly soluble components. However, depending on the beverage, emulsifiers or dispersants cannot be used, or the use of emulsifiers, dispersants, etc. in beverages may impart unintended flavors derived from the emulsifiers or dispersants. A technology that can improve the water solubility of xanthohumol without using emulsifiers, dispersants, etc. is useful, for example, in beverages.

[0008] An object of the present invention is to provide a xanthohumol glycoside in which a sugar chain composed of two or more hexoses is bound to xanthohumol, and a method for producing the same. Another object of the present invention is to provide a xanthohumol glycoside in which one hexose is bound to xanthohumol, and a method for producing the same. Another object of the present invention is to provide a method for improving the water solubility of xanthohumol, a method for reducing the bitterness of xanthohumol, a method for improving the stability of xanthohumol, etc. [Means for solving the problem]

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that a hexose (hexose residue) can be added to the 4' position of xanthohumol by a filamentous fungus belonging to the genus Rhizopus. Furthermore, the present inventors have discovered a plant-derived UDP (uridine diphosphate)-glycosyltransferase (UGT) that adds a hexose (hexose residue) to the 4' and / or 4 position of xanthohumol. They have also found that a xanthohumol glycoside having a sugar chain composed of two or more hexoses bound to the 4' or 4 position of xanthohumol can be produced by adding an additional hexose to the hexose residue of a xanthohumol glycoside produced by a fungus belonging to the genus Rhizopus or a UGT.

[0010] Furthermore, the present inventors have found that the water solubility and other properties of xanthohumol can be improved by adding a sugar chain consisting of two or more hexoses to the 4' or 4 position of xanthohumol. A xanthohumol glycoside in which a sugar chain consisting of two or more hexoses is bound to the 4' or 4 position of xanthohumol has not been reported to date.

[0011] That is, the present invention relates to the following xanthohumol glycoside, a method for producing a xanthohumol glycoside, a method for improving the water solubility of xanthohumol, and the like, although not limited thereto. [1] Xanthohumol glycoside in which a sugar chain consisting of 2 to 10 hexoses is bound to the 4' or 4' position of xanthohumol. [2] The xanthohumol glycoside according to [1] above, wherein the hexose is glucose. [3] Xanthohumol glycoside in which one hexose is bound to the 4th position of xanthohumol. [4] A composition containing the xanthohumol glycoside described in any one of [1] to [3] above. [5] The composition according to [4] above, which is a food or drink, a pharmaceutical product, a quasi-drug, a cosmetic, or a feed. [6] The composition according to [4] or [5] above, which is a packaged beverage. [7] A method for producing a xanthohumol glycoside in which a sugar chain consisting of 2 to 10 hexoses is bound to the 4' position of xanthohumol, comprising: a step (A1) of culturing a bacterium belonging to the genus Rhizopus in the presence of xanthohumol to produce a xanthohumol glycoside in which one hexose is bound to the 4' position of xanthohumol; and a step (B1) of adding 1 to 9 hexoses to the hexose residue of the xanthohumol glycoside produced in step (A1). [8] A method for producing a xanthohumol glycoside in which one hexose is bound to the 4' position of xanthohumol, comprising the step (A1) of culturing a fungus belonging to the genus Rhizopus in the presence of xanthohumol to produce a xanthohumol glycoside in which one hexose is bound to the 4' position of xanthohumol. [9] A method for producing xanthohumol glycosides according to [7] or [8] above, wherein in step (A1), a fungus belonging to the genus Rhizopus is cultured in the presence of xanthohumol and hexose.

[10] A method for producing a xanthohumol glycoside having a sugar chain composed of 2 to 10 hexoses bound to the 4' and / or 4' position of xanthohumol, comprising: a step (A2) of producing a xanthohumol glycoside having one hexose bound to the 4' and / or 4' position of xanthohumol from xanthohumol and UDP-hexose using one or more proteins selected from the group consisting of (p1), (p2), and (p3) below; and a step (B2) of adding 1 to 9 hexoses to the hexose residues of the xanthohumol glycoside produced in step (A2). (p1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (p2) A protein consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having the activity of adding hexose to the 4' and / or 4' positions of xanthohumol. (p3) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1 and having the activity of adding hexose to the 4' and / or 4' positions of xanthohumol.

[11] A method for producing a xanthohumol glycoside in which one hexose is bound to the 4'-position and / or the 4'-position of xanthohumol, comprising step (A2) of generating a xanthohumol glycoside in which one hexose is bound to the 4'-position and / or the 4'-position of xanthohumol from xanthohumol and UDP-hexose using one or more proteins selected from the group consisting of (p1), (p2), and (p3) below. (p1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (p2) A protein consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having the activity of adding hexose to the 4' and / or 4' positions of xanthohumol. (p3) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1 and having the activity of adding hexose to the 4' and / or 4' positions of xanthohumol.

[12] A method for improving the water solubility of xanthohumol, which comprises adding a sugar chain consisting of 2 to 10 hexoses to the 4' or 4 position of xanthohumol.

[13] A method for reducing the bitterness of xanthohumol, which comprises adding a sugar chain consisting of 2 to 10 hexoses to the 4' or 4 position of xanthohumol.

[14] A method for improving the stability of xanthohumol, which comprises adding a sugar chain consisting of 2 to 10 hexoses to the 4' or 4 position of xanthohumol.

[15] The method according to any one of [7] to

[14] above, wherein the hexose is glucose. [Effects of the Invention]

[0012] According to the present invention, there are provided a xanthohumol glycoside in which a sugar chain composed of two or more hexoses is bound to xanthohumol, and a method for producing the same. The xanthohumol glycoside of the present invention has improved solubility in water compared to the aglycone xanthohumol and a xanthohumol glycoside in which one hexose is added to xanthohumol (xanthohumol monoglycoside).

[0013] The present invention also provides a xanthohumol glycoside in which one hexose is bound to the 4-position of xanthohumol. The present invention also provides a method for producing a xanthohumol glycoside in which one hexose is bound to the 4'-position of xanthohumol. For example, a xanthohumol glycoside in which one hexose is bound to the 4'-position of xanthohumol can be produced by adding a hexose to xanthohumol using a filamentous fungus belonging to the genus Rhizopus. The present invention also provides a method for producing a xanthohumol glycoside in which one hexose is bound to the 4'-position and / or the 4'-position of xanthohumol by adding a hexose to xanthohumol using a UGT derived from a plant or the like.

[0014] According to the present invention, there are provided a method for improving the water solubility of xanthohumol, a method for reducing the bitterness of xanthohumol, and a method for improving the stability of xanthohumol, and therefore, the beverage suitability of xanthohumol can be significantly improved. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the results of HPLC analysis of the reaction solution obtained by reacting UDP-glucose and xanthohumol (XN) with soybean-derived UGT (Gm_UGT1) expressed in Escherichia coli (detection: 350 nm). [Figure 2] FIG. 2 shows the results of NMR measurement of the reaction products of UDP-glucose and XN by soybean-derived UGT (Gm_UGT1) expressed in Escherichia coli. [Figure 3] FIG. 3 shows the results of NMR measurement of the reaction products of UDP-glucose and XN by soybean-derived UGT (Gm_UGT1) expressed in Escherichia coli. [Figure 4]Figure 4A shows the results of an analysis of PD Broth containing only the substrate (XN) without adding any Rhizopus bacteria (detection: 280 nm). Figure 4B shows the results of an analysis of the culture supernatant of Rhizopus oryzae (R. oryzae) IAM6049 (detection: 280 nm). [Figure 5] Figure 5A shows the results of analysis of the culture supernatant of R. oryzae IAM6256 cultured in PD Broth containing XN (detection: 320 nm). Figure 5B shows the results of analysis of the culture supernatant of R. oryzae IAM6256 cultured in PD Broth containing XN (detection: 280 nm). [Figure 6] Figure 6A shows the results of analysis of the cell extract of R. oryzae IAM6256 cultured in PD Broth containing XN (detection: 320 nm). Figure 6B shows the results of analysis of the cell extract of R. oryzae IAM6256 cultured in PD Broth containing XN (detection: 280 nm). [Figure 7] Figure 7A shows the results of analysis of the culture supernatant of R. oryzae IAM6256 cultured in a medium containing 0.2% yeast extract (YE) and 0.5% glucose (Glc) containing XN (detection: 320 nm). Figure 7B shows the results of analysis of the culture supernatant of R. oryzae IAM6256 cultured in a medium containing 0.2% YE and 0.5% Glc containing XN (detection: 280 nm). [Figure 8] Figure 8A shows the results of analysis of the cell extract of R. oryzae IAM6256 cultured in a medium containing 0.2% YE and 0.5% Glc containing XN (detection at 320 nm). Figure 8B shows the results of analysis of the cell extract of R. oryzae IAM6256 cultured in a medium containing 0.2% YE and 0.5% Glc containing XN (detection at 280 nm). [Figure 9] Figure 9A shows the analytical results of the eluate obtained after the first elution with 55% ethanol in the purification of XN4'G by solid-phase extraction (detection: 320 nm). Figure 9B shows the analytical results of the eluate obtained after the first elution with 55% ethanol in the purification of XN4'G by solid-phase extraction (detection: 280 nm). [Figure 10]Figure 10A shows the analytical results of the eluate obtained after the second elution with 55% ethanol in the purification of XN4'G by solid-phase extraction (detection: 320 nm). Figure 10B shows the analytical results of the eluate obtained after the second elution with 55% ethanol in the purification of XN4'G by solid-phase extraction (detection: 280 nm). [Figure 11] Figure 11A shows the analytical results of the eluate obtained after the third elution with 55% ethanol in the purification of XN4'G by solid-phase extraction (detection: 320 nm). Figure 11B shows the analytical results of the eluate obtained after the third elution with 55% ethanol in the purification of XN4'G by solid-phase extraction (detection: 280 nm). [Figure 12] Figure 12A shows the results of analysis of the culture supernatant of R. oryzae IAM6256 cultured in medium containing XN for one day (detection: 320 nm). Figure 12B shows the results of analysis of the culture supernatant of R. oryzae IAM6256 cultured in medium containing XN for two days (detection: 320 nm). Figure 12C shows the results of analysis of the reaction solution in which CGTase was added to the culture supernatant of R. oryzae IAM6256 and a reaction was carried out (detection: 320 nm). [Figure 13] Figure 13A shows the analytical results of the eluate obtained after the second elution with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 320 nm). Figure 13B shows the analytical results of the eluate obtained after the second elution with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 280 nm). [Figure 14] Figure 14A shows the analysis results of the eluate obtained after the third elution with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 320 nm). Figure 14B shows the analysis results of the eluate obtained after the third elution with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 280 nm). [Figure 15]Figure 15A shows the analysis results of the eluate obtained after the fourth elution with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 320 nm). Figure 15B shows the analysis results of the eluate obtained after the fourth elution with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 280 nm). [Figure 16] Figure 16A shows the analysis results of the eluate obtained after the fifth elution with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 320 nm). Figure 16B shows the analysis results of the eluate obtained after the fifth elution with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 280 nm). [Figure 17] Figure 17A shows the analytical results of the eluate obtained after the first elution with 55% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 320 nm). Figure 17B shows the analytical results of the eluate obtained after the first elution with 55% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 280 nm). [Figure 18] Figure 18A shows the analytical results of the eluate obtained after the second elution with 55% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 320 nm). Figure 18B shows the analytical results of the eluate obtained after the second elution with 55% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 280 nm). [Figure 19] Figure 19A shows the results of analysis of the eluate obtained from the third to fifth elutions with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 320 nm). Figure 19B shows the results of analysis of the eluate obtained from the third to fifth elutions with 45% ethanol in the purification of XN4'Gs by solid-phase extraction (detection: 280 nm). DETAILED DESCRIPTION OF THE INVENTION

[0016] The xanthohumol glycoside of the first aspect of the present invention is a xanthohumol glycoside in which a sugar chain composed of 2 to 10 hexoses is bound to the 4'-position or the 4-position of xanthohumol. In the xanthohumol glycoside of the first aspect of the present invention, the sugar chain is bonded to the 4'-position or the 4-position. The 2 to 10 hexoses (hexose residues) constituting the sugar chain may all be the same or different. In the xanthohumol glycoside of the first aspect of the present invention, the sugar chain may be composed of one type of hexose (hexose residue), or may be composed of two or more types of hexose. The xanthohumol glycoside according to the first aspect of the present invention has a sugar chain composed of 2 to 10 hexoses, and can also be called a xanthohumol polyglycoside.

[0017] In one embodiment, the xanthohumol glycoside of the first embodiment of the present invention is preferably a xanthohumol glycoside in which a sugar chain composed of 2 to 10 hexoses is bound to the 4'-position of xanthohumol. Examples of xanthohumol glycosides in which a sugar chain composed of 2 to 10 hexoses is bound to the 4'-position of xanthohumol include compounds represented by the following general formula (1A):

[0018] [ka]

[0019] In general formula (1A), X represents a hexose residue, (X)n represents a sugar chain composed of 2 to 10 hexose residues, and n represents an integer of 2 to 10. The xanthohumol glycoside of the first aspect of the present invention also includes a xanthohumol glycoside in which a sugar chain composed of 2 to 10 hexoses is bound to position 4 of xanthohumol. Examples of xanthohumol glycosides in which a sugar chain composed of 2 to 10 hexoses is bound to position 4 of xanthohumol include compounds represented by the following general formula (1B):

[0020] [ka]

[0021] In general formula (1B), X represents a hexose residue, (X)n represents a sugar chain composed of 2 to 10 hexose residues, and n represents an integer of 2 to 10. In the general formulae (1A) and (1B), the 2 to 10 hexose residues (X) may all be the same or different.

[0022] Examples of hexoses include glucose, galactose, rhamnose, and fructose. Among these, glucose, galactose, and glucuronic acid are preferred, with glucose being more preferred. In this specification, hexoses are preferably D-isomers. Preferred hexoses are D-glucose and D-galactose, with D-glucose being more preferred. The above-mentioned hexoses are preferred as the hexose residue.

[0023] In one embodiment, in the sugar chain, the first hexose bonded to xanthohumol is preferably glucose or galactose, more preferably glucose, and the second to tenth hexoses bonded to xanthohumol are preferably glucose.

[0024] In the xanthohumol glycoside of the first aspect of the present invention, the sugar chain composed of 2 to 10 hexoses is preferably a sugar chain composed of 2 to 7 hexoses. In general formulas (1A) and (1B), n is preferably 2 to 7. As the xanthohumol glycoside of the first aspect of the present invention, a xanthohumol glycoside in which a sugar chain composed of 2 to 7 hexoses is bound to the 4'-position of xanthohumol is more preferred.

[0025] The xanthohumol glycoside of the first aspect of the present invention is a xanthohumol glycoside in which a sugar chain composed of 2 to 10 (preferably 2 to 7) hexoses (hexose residues) is glycosidicly bonded (more specifically, O-glycosidically bonded) to the 4'- or 4'-position of xanthohumol. In the xanthohumol glycoside of the first aspect of the present invention, xanthohumol is O-glycosidically bonded to the 1-position hydroxyl group of the hexose at the 4'- or 4'-position (hydroxyl group). The glycosidic bond may be an α- or β-bond, preferably a β-bond. In the sugar chain, the bond between hexoses is not particularly limited, and may be an α-glycosidic bond or a β-glycosidic bond. In one aspect, the hexoses constituting the sugar chain are preferably bonded together via an α-1,4-glycosidic bond.

[0026] In the present invention, a xanthohumol glycoside having a sugar chain composed of 2 to 10 hexoses bound to the 4'-position of xanthohumol may have a sugar chain composed of 2 to 10 hexoses or one hexose bound to the 4'-position. In one aspect, the 4th position of the xanthohumol glycoside is preferably a hydroxyl group (neither the sugar chain nor one hexose is bound to the 4th position). A xanthohumol glycoside in which a sugar chain composed of 2 to 10 hexoses is bound to the 4-position of xanthohumol may or may not have a sugar chain composed of 2 to 10 hexoses or one hexose bound to the 4'-position. In one aspect, the 4'-position of the xanthohumol glycoside is preferably a hydroxyl group (neither the sugar chain nor one hexose is bound to the 4'-position). In one aspect, the xanthohumol glycoside of the first aspect of the present invention is preferably a xanthohumol glycoside represented by the above general formula (1A) or general formula (1B), and more preferably a xanthohumol glycoside represented by the above general formula (1A).

[0027] In one aspect, the xanthohumol glycoside of the first aspect of the present invention is preferably a xanthohumol glycoside (xanthohumol polyglucoside) in which a sugar chain composed of 2 to 10 (preferably 2 to 7) glucose residues (glucose residues) is bound to the 4'-position or 4'-position of xanthohumol, and more preferably a xanthohumol glycoside in which the sugar chain is bound to the 4'-position of xanthohumol. In one aspect, in the above general formulas (1A) and (1B), X preferably represents a glucose residue.

[0028] The xanthohumol glycoside of the first aspect of the present invention has significantly improved water solubility compared to the aglycone xanthohumol and the xanthohumol glycoside in which one hexose is bound to it (xanthohumol (XN) monoglycoside). The xanthohumol glycoside of the first aspect of the present invention has excellent solubility in water, and therefore can be contained at high concentrations in liquid foods and beverages such as beverages. Furthermore, an aqueous solution of the xanthohumol glycoside of the first aspect of the present invention has high clarity. While emulsifiers, dispersants, and the like have been used to increase the water solubility of xanthohumol, the xanthohumol glycoside of the first aspect of the present invention can be dissolved at high concentrations in water without the use of emulsifiers, etc. Furthermore, the xanthohumol glycoside of the first aspect of the present invention effectively reduces the bitterness of the aglycone xanthohumol. Furthermore, the xanthohumol glycoside of the first aspect of the present invention exhibits greater stability than the aglycone xanthohumol and its monoglycoside. Therefore, the xanthohumol glycoside of the first aspect of the present invention is considered to be less susceptible to decomposition during storage, etc.

[0029] The present invention also encompasses xanthohumol glycosides in which one hexose is bound to the 4-position of xanthohumol. A xanthohumol glycoside in which one hexose is bound to the 4-position of xanthohumol is also referred to as a xanthohumol glycoside of the second aspect of the present invention. The xanthohumol glycoside of the second aspect of the present invention is a compound represented by the following general formula (2B):

[0030] [ka]

[0031] In general formula (2B), X represents a hexose residue. The hexose of the hexose residue may be any of those mentioned above, preferably glucose or galactose, more preferably glucose. The xanthohumol glycoside of the second aspect of the present invention has improved water solubility compared to the aglycone xanthohumol. The xanthohumol glycoside of the second aspect of the present invention is a xanthohumol glycoside in which one hexose is bound to xanthohumol, and can also be called a xanthohumol (XN) monoglycoside.

[0032] The xanthohumol glycoside of the first aspect and the xanthohumol glycoside of the second aspect of the present invention can be used as ingredients in foods and beverages, pharmaceuticals, quasi-drugs, cosmetics, feed, etc. The xanthohumol glycoside of the present invention can be used alone or in the form of a composition containing ingredients other than the glycoside, and can be used as foods and beverages, pharmaceuticals, quasi-drugs, cosmetics, feed, etc.

[0033] A composition containing the xanthohumol glycoside of the present invention also constitutes one aspect of the present invention. The composition of the present invention comprises at least one selected from the group consisting of the xanthohumol glycoside of the first aspect of the present invention and the xanthohumol of the second aspect of the present invention. In one aspect, the composition of the present invention preferably comprises the xanthohumol glycoside of the first aspect of the present invention (a xanthohumol glycoside in which a sugar chain composed of 2 to 10 hexoses is bound to the 4'- or 4-position of xanthohumol). One xanthohumol glycoside may be used, or two or more xanthohumol glycosides may be used in combination. The composition of the present invention may be a food or beverage, a pharmaceutical product, a quasi-drug, a cosmetic, a feed, or the like. The composition of the present invention may contain optional additives and optional ingredients in addition to the xanthohumol glycoside. These additives and ingredients can be selected depending on the form of the composition, and those generally usable for food or beverage, a pharmaceutical product, a quasi-drug, a cosmetic, a feed, and the like can be used. The composition of the present invention may also contain water.

[0034] When the composition of the present invention is made into a food or beverage, various foods and beverages can be prepared by blending the xanthohumol glycoside with ingredients that can be used in foods and beverages (e.g., food ingredients, food additives used as needed, etc.). The foods and beverages are not particularly limited, and examples thereof include general foods and beverages, health foods, health drinks, functional foods, foods for specified health uses, health supplements, and foods and beverages for patients. Food and beverages also include food additives. The health foods, functional foods, foods for specified health uses, health supplements, and the like may be in various dosage forms, such as fine granules, tablets, granules, powders, capsules, chewable tablets, dry syrups, syrups, liquids, beverages, and liquid diets.

[0035] As described above, the xanthohumol glycoside of the first embodiment of the present invention and the xanthohumol glycoside of the second embodiment have improved water solubility compared to xanthohumol. Therefore, the xanthohumol glycoside of the present invention has excellent suitability for beverages. In particular, the xanthohumol glycoside of the first embodiment of the present invention exhibits excellent water solubility. In one embodiment, the composition of the present invention is preferably a liquid composition, more preferably a beverage. The form of the beverage is not particularly limited, and it can be a packaged beverage. The container for the packaged beverage is not particularly limited, and any container of any shape or material can be used. For example, any commonly used container can be used, such as metal containers such as aluminum cans and steel cans; resin containers such as PET bottles; paper containers such as paper cartons; glass containers such as glass bottles; and wooden containers such as barrels. The packaged beverage can be obtained by filling and sealing such a container with the beverage.

[0036] When the composition of the present invention is used as a pharmaceutical or quasi-drug, various dosage forms of the pharmaceutical or quasi-drug can be prepared by blending the xanthohumol glycoside with a pharmacologically acceptable carrier and, if necessary, additives. Such carriers, additives, etc. may be any pharmacologically acceptable carriers that can be used in pharmaceuticals or quasi-drugs. The pharmaceutical or quasi-drug may be administered orally or parenterally, preferably orally. Examples of dosage forms for oral administration include liquids, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewable tablets, etc. Examples of dosage forms for parenteral administration include injections, infusions, ointments, lotions, patches, suppositories, nasal preparations, and pulmonary preparations (inhalants). The pharmaceutical may also be a drug for non-human animals.

[0037] When the composition of the present invention is used as a cosmetic, the xanthohumol glycoside may be blended with a carrier, additives, etc. that are acceptable for cosmetics. The product form of the cosmetic is not particularly limited. When the composition of the present invention is used as a feed, the xanthohumol glycoside may be added to the feed. The feed also includes feed additives.

[0038] The content of the xanthohumol glycoside in the composition of the present invention is not particularly limited, and can be, for example, 0.0001 to 48% by weight in terms of xanthohumol, the aglycone, in the composition. In one embodiment, in the case of a beverage, the content of the xanthohumol glycoside may be, for example, 0.0001 to 1% by weight in terms of xanthohumol (aglycone). The content of the xanthohumol glycoside is the total content of xanthohumol glycosides. When a composition containing the xanthohumol glycoside of the present invention is used in the form of a food or drink, a drug, a quasi-drug, a cosmetic, a feed, or the like, the method for producing the composition is not particularly limited, and the composition can be produced by a general method. For example, in the production of a food or drink, a drug, a quasi-drug, a cosmetic, or a feed, a food or drink, a drug, a quasi-drug, a cosmetic, or a feed containing the xanthohumol glycoside can be produced by a production method including a step of blending the xanthohumol glycoside of the present invention. As the xanthohumol glycoside, either or both of the xanthohumol glycoside of the first aspect of the present invention described above and the xanthohumol glycoside of the second aspect of the present invention can be used. In one aspect, it is preferable to use the xanthohumol glycoside of the first aspect of the present invention.

[0039] <Method for producing xanthohumol glycoside> The present invention also includes a method for producing the xanthohumol glycoside according to the first aspect of the present invention. A xanthohumol glycoside having a sugar chain composed of 2 to 10 hexoses attached to the 4'-position of xanthohumol can be produced by a method for producing a xanthohumol glycoside, comprising the steps of: (A1) culturing a fungus belonging to the genus Rhizopus in the presence of xanthohumol to produce a xanthohumol glycoside having one hexose attached to the 4'-position of xanthohumol; and (B1) adding 1 to 9 hexoses to the hexose residue of the xanthohumol glycoside produced in step (A1). This method allows the production of a xanthohumol glycoside having a sugar chain composed of 2 to 10 hexoses (hexose residues) attached to the 4'-position of xanthohumol, which is an example of the xanthohumol glycoside of the first aspect of the present invention. Xanthohumol glycosides and preferred embodiments thereof are as described above.

[0040] The present invention also includes a method for producing a xanthohumol glycoside in which a sugar chain composed of 2 to 10 hexoses is bound to the 4'-position of xanthohumol, the method comprising the above steps (A1) and (B1). The method for producing a xanthohumol glycoside comprising the above steps (A1) and (B1) is also referred to as the production method of the first aspect of the present invention. The production method of the first aspect of the present invention may include steps other than steps (A1) and (B1) as desired.

[0041] By culturing a fungus belonging to the genus Rhizopus in the presence of xanthohumol, the fungus can produce a xanthohumol glycoside in which one hexose is bound to the 4'-position of xanthohumol. The xanthohumol glycoside produced in the above step (A1) is a xanthohumol glycoside in which one hexose is bound to xanthohumol, and can also be called a xanthohumol (XN) monoglycoside. In step (A1), a fungus belonging to the genus Rhizopus may be cultured in a medium containing xanthohumol. The medium may be any medium that can culture a fungus belonging to the genus Rhizopus and contains xanthohumol. The medium is preferably a liquid medium. In step (A1), for example, a fungus belonging to the genus Rhizopus may be cultured in the presence of xanthohumol and a hexose source. When culturing in the presence of xanthohumol and a hexose source, the fungus belonging to the genus Rhizopus may be cultured in a medium containing xanthohumol and a hexose source. The medium may be any medium capable of culturing a fungus belonging to the genus Rhizopus and containing xanthohumol and a hexose source. Furthermore, in step (A1), the fungus belonging to the genus Rhizopus cultured in the presence of a hexose source may be cultured in the presence of xanthohumol. Preferred embodiments of step (A1) include, for example, culturing a fungus belonging to the genus Rhizopus in the presence of xanthohumol and hexose, or culturing a fungus belonging to the genus Rhizopus cultured in the presence of a hexose source in the presence of xanthohumol. When cultured in the presence of a hexose source, the hexose source is taken up into the cells of fungi, usually belonging to the genus Rhizopus, and used to produce xanthohumol glycosides. Examples of hexoses include the above-mentioned hexoses. The hexoses may be one type or a combination of two or more types. Preferred hexoses are glucose and galactose, and more preferred is glucose.

[0042] The fungus belonging to the genus Rhizopus is not particularly limited as long as it is capable of adding a hexose (preferably glucose) to the 4'-position of xanthohumol (forming a glycosidic bond) when cultured in the presence of xanthohumol or in the presence of xanthohumol and a hexose source. The fungus belonging to the genus Rhizopus may be a wild-type strain, a mutant strain obtained by conventional mutation treatment such as UV irradiation or NTG treatment, or a recombinant strain induced by genetic techniques such as gene recombination. One or more species of fungus belonging to the genus Rhizopus may be used. Fungi belonging to the genus Rhizopus have long been used in food processing in Asia, for example, in the production of rice cake koji in China and tempeh in Indonesia. As such, since fungi belonging to the genus Rhizopus have long been used in the production of food and beverages, it can be said that xanthohumol glycosides obtained using these microorganisms are suitable for use in food and beverage production from a safety perspective.

[0043] Examples of fungi belonging to the genus Rhizopus include Rhizopus microsporus (e.g., IFO31988), Rhizopus oligosporus (e.g., IFO8631, IFO31987, IFO32002, IFO32003), Rhizopus chinensis (e.g., IFO4768, IFO30499, IFO4737), Rhizopus delemar (e.g., IAM6038, IAM6252, ATCC34612), Rhizopus oryzae (e.g., Rhizopus oryzae (e.g., IAM6006, IAM6010, IAM6022, IAM6036, IAM6049, IAM6061, IAM6067, IAM6256, NRRL395), Rhizopus niveus (e.g., SAM543), etc. are preferred. Among these, Rhizopus oryzae, Rhizopus oligosporus, etc. are preferred.

[0044] The culture conditions in step (A1) are not particularly limited except that a culture medium containing xanthohumol is used, and the culture can be carried out under the culture conditions used when culturing bacteria belonging to the genus Rhizopus. In step (A1), the concentration of xanthohumol in the medium is not particularly limited and may be, for example, 1 mg / L to 10 g / L. In one embodiment, the concentration of xanthohumol may be the above concentration at the start of culture. Xanthohumol may be added to the medium all at once, or may be added to the medium in multiple portions during culture. Xanthohumol can be dissolved in a solvent such as ethanol, N,N-dimethylformamide (DMF), or dimethyl sulfoxide (DMSO) and then added to the medium.

[0045] In one embodiment, the culture in step (A1) can be carried out in the presence of a hexose source that provides the hexose residues of xanthohumol glycosides. Alternatively, for example, prior to step (A1), a fungus belonging to the genus Rhizopus may be cultured in the presence of a hexose source. Hexose or a carbon source containing hexose units that provide the hexose source can be used as the hexose source. One type of hexose source may be used, or two or more types may be used in combination. In the present invention, hexose and / or a carbon source containing hexose units can be used as the hexose source. Preferred hexose sources include a glucose source and a galactose source, with a glucose source being more preferred. In culturing in the presence of a hexose source, hexose may be added to the medium, or a carbon source containing hexose units may be added as a hexose source. Examples of carbon sources that can be used as such hexose sources include sucrose, lactose, dextrin, cyclodextrins (α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin), amylose, amylopectin, and starch. The hexose source can be selected depending on the hexose residue of the xanthohumol glycoside. In one embodiment, when producing a xanthohumol monoglycoside having glucose attached at the 4'-position (xanthohumol monoglucoside), a glucose source can be used. Examples of glucose sources include glucose and carbon sources containing glucose units (e.g., sucrose, lactose, dextrin, cyclodextrin, amylose, amylopectin, and starch). One or more glucose sources can be used. In one embodiment, the hexose source preferably contains hexose, and more preferably contains glucose. The concentration of the hexose source in the medium is preferably 0.1 to 20% by weight, more preferably 0.2 to 10% by weight. In one embodiment, the concentration of the hexose source may be the above concentration at the start of culture. The hexose source may be added to the medium all at once, or may be added in portions during culture.

[0046] If necessary, the medium may contain nitrogen sources (e.g., various peptones (potato peptone, etc.), various extracts, ammonium sulfate, urea, etc.), inorganic substances (dipotassium hydrogen phosphate, potassium phosphate, magnesium sulfate, zinc sulfate, iron, manganese, molybdenum, sodium chloride, potassium chloride, magnesium chloride, etc.), etc. In the production method of the first aspect of the present invention, for example, potato dextrose medium (PD medium) or the like can be used as the medium.

[0047] A fungus belonging to the genus Rhizopus can be grown by directly inoculating a small amount of fungal cells into the medium, but it is preferable to inoculate a spore suspension of the fungus. The spore suspension may be prepared by a commonly used method. In one embodiment, the spore suspension can also be prepared by the method described in the Examples below. The production method of the first embodiment of the present invention may include a step of preparing a spore suspension of a fungus belonging to the genus Rhizopus. In one embodiment, the fungus belonging to the genus Rhizopus used in step (A1) is preferably a fungus (fungal cells) cultured in the presence of a hexose source. The culture time for the fungus belonging to the genus Rhizopus in step (A1) can be, for example, 0.5 to 120 hours. When the fungus belonging to the genus Rhizopus is cultured in the presence of a hexose source before step (A1), the culture time can be, for example, 0.5 to 120 hours.

[0048] The culture is preferably aerobically cultured in a liquid medium. The method for aerobic culture is not particularly limited, and a liquid medium inoculated with a bacterium belonging to the genus Rhizopus may be cultured, for example, by shaking or stirring. If desired, bubbling with sterilized air or oxygen may be performed. The culture format may be batch culture, fed-batch culture, or continuous culture, with batch culture being preferred. In the production method of the present invention, stationary culture may also be performed.

[0049] In the production method of the first aspect of the present invention, immobilized cells of bacteria belonging to the genus Rhizopus immobilized on a carrier can also be used. The method for preparing immobilized cells is not particularly limited, and known methods can be used. For example, spores of bacteria belonging to the genus Rhizopus are added to an aqueous sodium alginate solution, stirred to form a suspension, and then dropped into an aqueous calcium chloride solution to obtain a bead-like gel in which the spores are immobilized. This bead-like gel can be cultured in a medium and under culture conditions that are used to culture bacteria belonging to the genus Rhizopus, thereby obtaining immobilized cells. In one aspect, in step (A1), immobilized cells of bacteria belonging to the genus Rhizopus may be cultured in a medium containing xanthohumol. Alternatively, in step (A1), immobilized cells of bacteria belonging to the genus Rhizopus may be cultured in a medium containing xanthohumol and a hexose source.

[0050] In step (A1), by carrying out the above-described culture, a xanthohumol glycoside in which one hexose is bound to the 4'-position of xanthohumol is produced. The produced xanthohumol glycoside in which one hexose is bound to the 4'-position usually accumulates in the medium and in the fungal cells. The xanthohumol glycoside in which one hexose is bound to the 4'-position of xanthohumol produced in step (A1) is a compound represented by the following general formula (2A): In the following general formula (2A), X represents a hexose residue. Examples of the hexose in the hexose residue include those mentioned above, preferably glucose or galactose, and more preferably glucose.

[0051] [ka]

[0052] In the production method of the first aspect of the present invention, 1 to 9 hexoses are added to the hexose residue of the xanthohumol glycoside having one hexose (hexose residue) bound to the 4'-position of xanthohumol (step (B1)). By adding 1 to 9 hexoses to the hexose residue at the 4'-position of the xanthohumol glycoside produced in step (A1), it is possible to produce a xanthohumol glycoside having a sugar chain composed of 2 to 10 hexoses bound to the 4'-position of xanthohumol.

[0053] The xanthohumol glycoside (XN monoglycoside) produced in step (A1) may be separated from the cultured cells, medium, etc., purified, and then used in step (B1). The separation and purification methods are not particularly limited, and methods such as centrifugation, filtration, and ion exchange resins can be used. Step (B1) can also be performed using a culture supernatant containing the xanthohumol glycoside, a cell extract of a fungus belonging to the genus Rhizopus, or the like. In one embodiment, the production method of the first embodiment of the present invention may further include, after step (A1), step (C1) of separating the fungal cells from the culture supernatant containing the xanthohumol glycoside (XN monoglycoside). The method for separating the fungal cells from the culture supernatant is not particularly limited, and methods such as centrifugation and filtration can be employed. In one embodiment, step (B1) can be performed using the culture supernatant. When a fungal cell extract containing the xanthohumol glycoside (XN monoglycoside) is used, step (C2) of preparing a fungal cell extract of a fungus belonging to the genus Rhizopus may be performed. The method for preparing the fungal cell extract is not particularly limited, and for example, the fungal cells after culture can be collected and extracted with an organic solvent (e.g., ethyl acetate) to obtain the fungal cell extract containing the xanthohumol glycoside (XN monoglycoside). Either one of steps (C1) and (C2) or both may be performed. After carrying out step (A1), step (B1) can also be carried out using the medium containing the produced xanthohumol glycoside and the fungus belonging to the genus Rhizopus as it is.

[0054] The xanthohumol glycoside of the first aspect of the present invention can also be produced by a method including the following steps (A2) and (B2). The present invention also encompasses a method for producing a xanthohumol glycoside, including the following steps (A2) and (B2). A method for producing a xanthohumol glycoside having a sugar chain composed of 2 to 10 hexoses attached to the 4' and / or 4' position of xanthohumol, comprising: step (A2) of producing a xanthohumol glycoside having one hexose attached to the 4' and / or 4' position of xanthohumol from xanthohumol and UDP-hexose using one or more proteins selected from the group consisting of (p1), (p2) and (p3) below; and step (B) of adding 1 to 9 hexoses to the hexose residues of the xanthohumol glycoside produced in step (A2). (p1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (p2) A protein consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having the activity of adding hexose to the 4' and / or 4' positions of xanthohumol. (p3) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1 and having the activity of adding hexose to the 4' and / or 4' positions of xanthohumol. The method for producing a xanthohumol glycoside, which includes the steps (A2) and (B2), is also referred to as the second aspect of the present invention. The second aspect of the present invention allows for the production of a xanthohumol glycoside in which a sugar chain composed of 2 to 10 hexoses (hexose residues) is bound to the 4'-position and / or the 4-position of xanthohumol. The xanthohumol glycoside and preferred aspects thereof are as described above.

[0055] Examples of the hexose in UDP-hexose include glucose, galactose, rhamnose, fructose, and glucuronic acid, preferably glucose and galactose, and more preferably glucose. UDP-hexose is preferably UDP-glucose or UDP-galactose, and more preferably UDP-glucose.

[0056] In the second aspect of the production method of the present invention, a xanthohumol glycoside in which one hexose is bound to the 4'-position and / or the 4-position of xanthohumol is produced from xanthohumol and UDP-hexose using one or more proteins selected from the group consisting of (p1), (p2) and (p3) above as a catalyst (step (A2)). The above proteins (p1), (p2), and (p3) are UDP-glycosyltransferases (UGTs) that catalyze the transfer of a hexose residue from UDP-hexose (a glycosyl donor) to xanthohumol (a glycosyl acceptor substrate) to produce xanthohumol glycosides. These proteins typically add (transfer) a hexose (hexose residue) to the 4'- and / or 4'-position (hydroxyl group) of xanthohumol. More specifically, they bond the 4'- and / or 4'-hydroxyl group of xanthohumol to the 1'-hydroxyl group of the hexose. Hereinafter, proteins (p1), (p2), and (p3) are also referred to as UGT proteins. The UGT proteins described above convert xanthohumol and UDP-hexose to produce at least one xanthohumol glycoside, either a xanthohumol glycoside (XN monoglycoside) in which one hexose (hexose residue) is bound to the 4' or 4 position of xanthohumol, or a xanthohumol glycoside (xanthohumol (XN) diglycoside) in which one hexose is bound to each of the 4' and 4 positions of xanthohumol. In one aspect, the UGT protein preferably has the activity of adding a hexose (preferably glucose) to the 4' or 4' position of xanthohumol. In one aspect, it is more preferable that the UGT protein has the activity of adding a hexose (preferably glucose) to the 4' position of xanthohumol. In another aspect, it is more preferable that the UGT protein has the activity of adding a hexose (preferably glucose) to the 4' position of xanthohumol.

[0057] The number of deleted, substituted, inserted and / or added amino acids in the protein (p2) above is preferably 1 to 8, 1 to 7 or 1 to 6, more preferably 1 to 5, even more preferably 1 to 4, particularly preferably 1 to 3, particularly preferably 1 or 2, and most preferably 1.

[0058] As used herein, the phrase "one or more amino acids have been deleted, substituted, inserted, and / or added" in the amino acid sequence of a protein means that one or more amino acids have been deleted, substituted, inserted, and / or added at any one or more positions in the same amino acid sequence, and two or more of the deletions, substitutions, insertions, and additions may occur simultaneously.

[0059] In the protein (p3) above, the identity (sequence identity) of the amino acid sequence to the amino acid sequence shown in SEQ ID NO: 1 is preferably 91% or more, 92% or more, 93% or more, or 94% or more, more preferably 95% or more, 96% or more, or 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. Amino acid sequence identity can be calculated using, for example, analysis software such as BLAST with default parameters.

[0060] In one embodiment, it is preferable to use (p1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in step (A2).

[0061] The above proteins (p1) to (p3) are not limited by their origin or production method. For example, they can be produced by introducing a polynucleotide encoding the protein into a non-human host such as a microorganism using known genetic engineering techniques, and then culturing the resulting transformant to express the protein. More specifically, the desired protein can be obtained by purifying the protein from a culture obtained by culturing the transformant. Protein purification can be carried out according to conventional methods. For example, an example of a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2. The degree of purification of the proteins (p1) to (p3) is not particularly limited, as long as they have the activity of adding a hexose (preferably glucose) to the 4'-position and / or 4-position of xanthohumol. The above cultures and crudely purified proteins can also be used as long as they achieve the effects of the present invention. The culture refers to any of the culture solution, cultured bacterial cells or cultured cells, and disrupted cultured bacterial cells or cultured cells.

[0062] Specifically, when a UGT protein accumulates within cultured bacterial cells or bacteria, after cultivation, the bacterial cells or cells can be disrupted by a conventional method (e.g., ultrasonication, lysozyme, freeze-thawing, etc.), and then a crude protein extract can be obtained by a conventional method (e.g., centrifugation, filtration, etc.). When the protein accumulates in the culture medium, after cultivation, the bacterial cells or cells can be separated from the culture supernatant by a conventional method (e.g., centrifugation, filtration, etc.), and the culture supernatant containing the desired protein can be obtained. The proteins contained in the extract or culture supernatant thus obtained can be purified by a conventional separation or purification method, such as ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reversed-phase high performance liquid chromatography, dialysis, or ultrafiltration, which can be used alone or in combination.

[0063] UGT proteins can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

[0064] In step (A2), xanthohumol and UDP-hexose are reacted using the above-mentioned UGT protein as a catalyst to produce a xanthohumol glycoside in which one hexose is bound to the 4'-position and / or 4-position of xanthohumol. The above reaction is preferably carried out in a liquid (reaction liquid), for example, in an aqueous medium such as water or a buffer solution. The pH of the reaction liquid can be, for example, 7 to 8, preferably 7.2 to 7.7. The reaction temperature can be 25 to 40°C, preferably 30 to 37°C. The reaction time can be set appropriately. The pH is the pH at 25°C. The pH can be measured using a commercially available pH meter.

[0065] In step (A2), the initial concentration of xanthohumol in the reaction solution may be, for example, 0.1 to 50 mM. Xanthohumol can be dissolved in a solvent such as ethanol, DMF, or dimethyl sulfoxide (DMSO) and then added to the reaction solution.

[0066] The concentration of UDP-hexose in the reaction solution at the start of the reaction (initial concentration) is preferably 5 to 20 times, and more preferably 5 to 15 times, the molar ratio of xanthohumol.

[0067] The xanthohumol glycoside produced in step (A2) is one or more xanthohumol glycosides selected from the group consisting of xanthohumol glycosides (XN monoglycosides) in which one hexose (hexose residue) is bound to the 4'-position of xanthohumol, xanthohumol glycosides (XN monoglycosides) in which one hexose is bound to the 4'-position of xanthohumol, and xanthohumol glycosides (XN diglycosides) in which one hexose is bound to the 4'-position and the 4'-position of xanthohumol. In the XN diglycoside, one hexose is bound to each of the 4'-position and the 4'-position of xanthohumol. The glycoside is preferably one or more xanthohumol glycosides selected from the group consisting of xanthohumol glycosides (XN monoglucosides) in which one glucose is bound to the 4' position of xanthohumol, xanthohumol glycosides (XN monoglucosides) in which one glucose is bound to the 4' position of xanthohumol, and xanthohumol glycosides (XN diglucosides) in which one glucose is bound to each of the 4' and 4 positions of xanthohumol. In one aspect, the xanthohumol glycoside produced in step (A2) may be at least one glycoside selected from the group consisting of xanthohumol glycosides having one hexose bonded to the 4'-position of xanthohumol and xanthohumol glycosides having one hexose bonded to the 4'-position of xanthohumol. Furthermore, in one aspect, the xanthohumol glycoside produced in step (A2) preferably includes a xanthohumol glycoside having one hexose bonded to the 4'-position of xanthohumol. In another aspect, the xanthohumol glycoside produced in step (A2) preferably includes a xanthohumol glycoside having one hexose bonded to the 4'-position of xanthohumol. The hexose is preferably glucose.

[0068] A xanthohumol glycoside having one hexose bonded to the 4'-position of xanthohumol is a compound represented by the general formula (2A) above. A xanthohumol glycoside having one hexose (hexose residue) bonded to the 4-position of xanthohumol is a compound represented by the general formula (2B) above. The xanthohumol monoglycoside is usually a xanthohumol glycoside in which one hexose is O-glycosidically linked to the 4'-position or 4'-position of xanthohumol. The XN diglycoside produced in step (A2) is usually a xanthohumol glycoside in which one hexose is O-glycosidically linked to each of the 4'-position and the 4'-position of xanthohumol.

[0069] In the production method of the second aspect of the present invention, 1 to 9 hexoses are added to the hexose residue of the xanthohumol glycoside produced in step (A2), which has one hexose (hexose residue) bound to the 4'-position and / or 4-position of xanthohumol (step (B2)). When the xanthohumol glycoside produced in step (A2) is, for example, a xanthohumol glycoside (XN monoglycoside) in which one hexose (hexose residue) is bound to the 4'- or 4'-position of xanthohumol, 1 to 9 hexoses are added to the hexose residue at the 4'- or 4'-position of the xanthohumol glycoside, thereby producing a xanthohumol glycoside in which a sugar chain composed of 2 to 10 hexoses is bound to the 4'- or 4'-position of xanthohumol. When the xanthohumol glycoside produced in step (A2) is a xanthohumol glycoside (XN diglycoside) having one hexose (hexose residue) bound to each of the 4' and 4 positions, 1 to 9 hexoses are added to the hexose residues at the 4' and / or 4 positions of the xanthohumol glycoside. This allows the production of a xanthohumol glycoside having a sugar chain consisting of 2 to 10 hexoses bound to the 4' and / or 4 position of xanthohumol.

[0070] In step (B1) of the production method of the first embodiment and step (B2) of the production method of the second embodiment of the present invention, the method for adding 1 to 9 hexoses to the hexose residue of the xanthohumol glycoside produced in step (A1) or step (A2) is not particularly limited. For example, hexoses can be added to the xanthohumol glycoside using an enzyme having transglycosylation activity as a catalyst. In one aspect, in step (B1), it is preferable to add 1 to 9 hexoses to the hexose residue (the hexose residue bound to the 4'-position) of xanthohumol monoglycoside using an enzyme having transglycosylation activity. In one aspect, in step (B2), it is preferable to add 1 to 9 hexoses to the hexose residues (hexose residues bound to the 4'- and / or 4-positions) of the xanthohumol glycoside (XN monoglycoside and / or XN diglycoside) using an enzyme having transglycosylation activity. Examples of enzymes having transglycosylation activity that can be used include cyclodextrin glucosyltransferase (CGTase), dextrinase, amylase, α-glucosidase, and glucoamylase. Enzymes having transglycosylation activity may be used singly or in combination of two or more. In one embodiment, it is preferable to use CGTase. Examples of hexoses to be added include the same as those mentioned above, with glucose, galactose, and the like being preferred, and glucose being more preferred.

[0071] The reactions in steps (B1) and (B2) can be carried out in a liquid (reaction liquid), preferably in an aqueous medium such as water or a buffer solution. The reaction conditions can be set appropriately depending on the enzyme used. For example, the pH of the reaction liquid is preferably 5 to 7, more preferably 5.5 to 6.75. The reaction temperature in steps (B1) and (B2) can be 40 to 60°C, preferably 45 to 55°C. The reaction time can be set appropriately, but can be, for example, 1 to 10 hours, preferably 1.5 to 7 hours, more preferably 2 to 7 hours, and even more preferably 3 to 5 hours. The reaction solution typically contains a hexose source that provides the hexose residues of xanthohumol glycosides. Examples of the hexose source include hexose and sugar sources that provide hexose. The hexose source may be one type or two or more types. A hexose such as glucose may be added to the reaction solution, but a sugar source that serves as a supply source of hexose may also be used. The hexose source can be selected depending on the types of hexose and enzyme. Examples of glucose sources include glucose, cyclodextrin, dextrin (e.g., cluster dextrin), and starch. The amount of hexose source added to the reaction solution is preferably, for example, 10 to 1,000 times the molar ratio of xanthohumol monoglycoside in the reaction solution. In one aspect, the amount of hexose source in the reaction solution at the start of the reaction is preferably 10 to 1,000 times the molar ratio of xanthohumol monoglycoside.

[0072] In one aspect, in step (B1), 1 to 9 hexose residues can be added to the hexose residue of the xanthohumol glycoside produced in step (A1) using an enzyme having transglycosylation activity as a catalyst in a reaction solution containing a hexose source. In one aspect, in step (B2), 1 to 9 hexose residues can be added to the hexose residue of the xanthohumol glycoside produced in step (A2) using an enzyme having transglycosylation activity as a catalyst in a reaction solution containing a hexose source.

[0073] The xanthohumol glycoside (XN monoglycoside) produced in step (A1) may be purified from the above-mentioned medium by a known method and then subjected to step (B1). Alternatively, steps (A1) and (B1) may be performed consecutively without purification. For example, step (B1) may be performed by adding an enzyme having transglycosylation activity and, if necessary, a hexose source to a culture broth containing the xanthohumol glycoside obtained in step (A1) and a bacterium belonging to the genus Rhizopus. Alternatively, step (B1) may be performed by concentrating the culture broth containing the xanthohumol glycoside produced in step (A1), and then adding the enzyme having transglycosylation activity and, if necessary, a hexose source to the resulting concentrate. Prior to step (B1), it is preferable to adjust the pH of the culture broth or concentrate containing the xanthohumol glycoside to a pH suitable for the enzyme used in step (B1).

[0074] The xanthohumol glycoside (XN monoglycoside and / or XN diglycoside) produced in step (A2) may be purified from the reaction solution by a known method and then subjected to step (B2). Alternatively, steps (A2) and (B2) may be performed consecutively without purification. Step (B2) may also be performed by adding the enzyme having transglycosylation activity used in step (B2) and, if necessary, a hexose source to a reaction solution containing the xanthohumol glycoside obtained in step (A2) and the UGT protein. Step (B2) may also be performed by concentrating the reaction solution containing the xanthohumol glycoside produced in step (A2), and then adding the enzyme having transglycosylation activity and, if necessary, a hexose source to the resulting concentrate. Prior to step (B2), it is preferable to adjust the pH of the reaction solution containing the xanthohumol glycoside or a concentrate of the reaction solution to a pH suitable for the enzyme used in step (B2).

[0075] The hexose source used in step (B1) and step (B2) can be selected depending on the enzyme. In one embodiment, a glucose source is preferred. In one embodiment, it is preferred to use dextrin such as cluster dextrin as the glucose source. When glucose is added using CGTase, preferred glucose sources include dextrin such as cluster dextrin, cyclodextrin such as α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and soluble starch. One or more glucose sources may be used.

[0076] In the production method of the first aspect of the present invention, step (B1) produces a xanthohumol glycoside in which a sugar chain consisting of two or more, typically 2 to 10 (preferably 2 to 7) hexoses (hexose residues) is bound to the 4' position of xanthohumol. In the production method of the second aspect of the present invention, step (B2) produces a xanthohumol glycoside in which a sugar chain composed of two or more, typically 2 to 10 (preferably 2 to 7) hexoses (hexose residues) is bound to the 4'-position and / or 4-position (preferably the 4'-position or 4-position) of xanthohumol. By carrying out step (A1) and step (B1), or step (A2) and step (B2), the xanthohumol glycoside of the first aspect of the present invention described above can be produced. The produced xanthohumol glycoside can be separated or purified from the solution by a known method. Specifically, for example, separation using a synthetic adsorbent, separation by reversed-phase chromatography, precipitation utilizing a change in solubility, etc. can be used alone or in combination as appropriate. The production method of the first aspect and the production method of the xanthohumol glycoside of the second aspect of the present invention may also include a step (D) of purifying the xanthohumol glycoside produced in step (B1) or step (B2).

[0077] Whether the desired glycoside has been obtained can be confirmed by known methods (for example, mass spectrometry, nuclear magnetic resonance spectroscopy (NMR), high performance liquid chromatography (HPLC), etc.). The xanthohumol glycoside obtained by the production method of the present invention may be one type or two or more types, and may be, for example, a mixture of two or more types of glycosides that differ in the type and / or number of hexoses constituting the sugar chain. The xanthohumol glycoside thus obtained is useful as a raw material for foods and beverages, pharmaceuticals, quasi-drugs, cosmetics, feeds, etc.

[0078] The present invention also includes the following method for producing xanthohumol glycosides. A method for producing a xanthohumol glycoside in which one hexose is bound to the 4' position of xanthohumol, comprising the step (A1) of culturing a fungus belonging to the genus Rhizopus in the presence of xanthohumol to produce a xanthohumol glycoside in which one hexose is bound to the 4' position of xanthohumol. The above-mentioned method for producing xanthohumol glycosides (xanthohumol monoglycosides) using a fungus belonging to the genus Rhizopus is also referred to as the production method of the third embodiment of the present invention. In the third production method of the present invention, step (A1) and its preferred embodiments are the same as step (A1) and its preferred embodiments in the production method of the first embodiment of the present invention described above. In step (A1), a fungus belonging to the genus Rhizopus may be cultured in the presence of xanthohumol and a hexose source. In one embodiment, in step (A1), a fungus belonging to the genus Rhizopus cultured in the presence of a hexose source may be cultured in the presence of xanthohumol. A third aspect of the production method of the present invention includes a method for producing a xanthohumol glycoside in which one hexose is bound to the 4' position of xanthohumol, which includes a step (A1) of culturing a fungus belonging to the genus Rhizopus in the presence of xanthohumol and a hexose source to produce a xanthohumol glycoside in which one hexose is bound to the 4' position of xanthohumol.

[0079] The present invention also includes the following method for producing xanthohumol glycosides. A method for producing a xanthohumol glycoside having one hexose bound to the 4' and / or 4' position of xanthohumol, comprising step (A2) of generating a xanthohumol glycoside having one hexose bound to the 4' and / or 4' position of xanthohumol from xanthohumol and UDP-hexose using one or more proteins selected from the group consisting of (p1), (p2) and (p3) above. The method for producing xanthohumol glycoside using the above protein and UDP-hexose is also referred to as the production method of the fourth aspect of the present invention. In the production method of the fourth aspect of the present invention, step (A2) and its preferred aspects are the same as step (A2) and its preferred aspects in the production method of the second aspect of the present invention.

[0080] In the production method of the third aspect of the present invention, the xanthohumol glycoside produced in step (A1) can be separated or purified from the culture medium by a known method. In the production method of the fourth aspect of the present invention, the xanthohumol glycoside produced in step (A2) can be separated or purified from the solution by a known method. Specifically, for example, separation using a synthetic adsorbent, separation by reversed-phase chromatography, precipitation utilizing changes in solubility, etc. can be used alone or in appropriate combination. The production method of the third aspect of the present invention may include a step of purifying the produced xanthohumol glycoside. The production method of the fourth aspect of the present invention may include a step of purifying the produced xanthohumol glycoside. The fact that the desired glycoside has been obtained can be confirmed by a known method (e.g., mass spectrometry, nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), etc.) based on the structural information of the known xanthohumol monoglycoside.

[0081] The xanthohumol glycoside obtained by the production method of the third aspect of the present invention is a xanthohumol monoglycoside in which one hexose is bound to the 4'-position of xanthohumol. The xanthohumol glycoside in which one hexose is bound to the 4'-position and / or the 4'-position obtained by the production method of the fourth aspect of the present invention is the same as the xanthohumol glycoside produced in step (A2) of the production method of the second aspect of the present invention, and is preferably either or both of a xanthohumol glycoside in which one hexose is bound to the 4'-position of xanthohumol and a xanthohumol glycoside in which one hexose is bound to the 4'-position of xanthohumol. The hexose is preferably glucose. In one embodiment, the production method of the fourth aspect of the present invention can be used as a method for producing the xanthohumol glycoside of the second aspect of the present invention. The xanthohumol glycosides obtained by the production methods of the third and fourth aspects of the present invention can be used, for example, as raw materials for foods and drinks, pharmaceuticals, quasi-drugs, cosmetics, feeds, etc. They can also be used as raw materials for the xanthohumol glycosides (xanthohumol polyglycosides) of the first aspect of the present invention.

[0082] The xanthohumol used in the production methods of the first, second, third, and fourth aspects of the present invention is not limited in any way by its production method, etc. Xanthohumol is a type of prenylflavonoid contained in hops and the like. Xanthohumol can be prepared, for example, by purifying a hop extract using a known method. Commercially available xanthohumol products can also be used. In the present invention, purified xanthohumol may be used, or a plant-derived material rich in xanthohumol may be used, as long as the effects of the present invention are achieved.

[0083] <Methods for improving water solubility, reducing bitterness, and improving stability of xanthohumol> By adding a sugar chain consisting of 2 to 10 hexoses (hexose residues) to the 4' or 4 position of xanthohumol, the polarity of the xanthohumol can be changed. This can improve the water solubility of xanthohumol. Furthermore, by adding the sugar chain, the bitterness of xanthohumol can be reduced. Furthermore, the stability of xanthohumol can be improved. The present invention also encompasses a method for improving the solubility of xanthohumol by adding a sugar chain composed of 2 to 10 hexoses to the 4'- or 4'-position of xanthohumol; a method for reducing the bitterness of xanthohumol by adding a sugar chain composed of 2 to 10 hexoses to the 4'- or 4'-position of xanthohumol; and a method for improving the stability of xanthohumol by adding a sugar chain composed of 2 to 10 hexoses to the 4'- or 4'-position of xanthohumol. In these methods, a sugar chain composed of 2 to 10 hexoses is typically O-glycosidically linked to the 4'- or 4'-position of xanthohumol. The number of hexoses constituting the sugar chain is preferably 2 to 7. The hexose is the same as that described above, with glucose being preferred.

[0084] The method for adding a sugar chain consisting of 2 to 10 hexoses to the 4'-position or 4-position of xanthohumol can be the same as the method for the production method of the first aspect or the production method of the second aspect of the present invention. By producing a xanthohumol glycoside in which one hexose is O-glycosidically linked to the 4'-position and / or 4-position of xanthohumol by the above-mentioned step (A1) or (A2), and then adding 1 to 9 hexoses to the hexose residue of the xanthohumol glycoside, a sugar chain consisting of 2 to 10 hexoses can be added to the 4'-position and / or 4-position of xanthohumol. All scientific and patent literature cited herein is hereby incorporated by reference. [Example]

[0085] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples. Unless otherwise specified, the molecular biological techniques used in the examples were in accordance with the methods described in Molecular Cloning (Sambrook et al., Cold Spring Harbour Laboratory Press, 2001).

[0086] Example 1 Production of xanthohumol glycosides (XNG) by β-glucosylation of xanthohumol (XN) with plant enzymes

[0087] (UGT Selection) Based on previously reported information on the regiospecificity of flavonoid UGTs (UDP-glycosyltransferases), we selected soybean-derived Gm_UGT1 / IF7G1cT / UGT83E3 as a candidate UGT for glycosylation at the 4' position of xanthohumol (XN) (Reference 1: Noguchi et al. (2007) J. Biol. Chem., 282(32):23581-23590 and Reference 2: Funaki et al. (2015) Plant Cell Physiol., 56(8):1512-1520). The amino acid sequence of the soybean-derived UGT Gm_UGT1 / IF7GlcT / UGT88E3 (ACCESSION AB292164) (also referred to as Gm_UGT1) is shown in SEQ ID NO: 1, and the nucleotide sequence of the DNA encoding it is shown in SEQ ID NO: 2.

[0088] (Recombinant protein expression and purification) According to a previously reported method (Reference 2), the Gm_UGT1 cDNA was cloned into the E. coli expression vector pET15b (Novagen) with a HisTag attached to the N-terminus, and then transformed into E. coli One Shot TM BL21 Star TM The transformants were then transformed into E. coli (DE3) cells (Thermo Fisher Scientific) and recombinant E. coli was selected with ampicillin. The transformants were cultured in LB medium (2 L) and then incubated with Overnight Express TM Proteins were expressed using Autoinduction System 1 (Novagen). Recombinant proteins were extracted from the collected E. coli using BugBuster (registered trademark) Protein Extraction Reagent (Merck Millipore). TM The target UGT protein (Gm_UGT1) was purified using a High Performance (GE Healthcare) column. Expression of the purified protein was confirmed by SDS-PAGE and CBB staining. The resulting Gm_UGT1 purified enzyme was used to react xanthohumol (XN) with UDP-glucose under the following conditions.

[0089] (XN's reaction) Substrate: XN solution: Xanthoflav lot. 9310 (Hopsteiner, XN approximately 90%) 20 mg / 40 mL DMF (N,N-dimethylformamide) Enzyme: Gm-UGT1 purified enzyme, 2 mL Cosubstrate: Uridine-5'-diphosphoglucose Disodium Salt (UDP-Glc, Sigma Aldrich) Temperature: 35℃ Buffer: 0.1M potassium phosphate buffer (pH 7.5)

[0090] The reaction volume was adjusted to 450 mL. 69 mg of UDP-Glc was dissolved in the buffer, and 13 mL of XN solution was added. 2 mL of purified enzyme was then added to initiate the reaction. 13 mL of XN solution was added 0.5 hours after the start of the reaction, and 14 mL was added 1 hour later. Another 69 mg of UDP-Glc was added 1.5 hours later, and the reaction was continued for 3 hours. XN has low solubility, and some of it was observed to remain insoluble in the reaction solution. The reaction solution was diluted 20-fold with 40% acetonitrile and analyzed by HPLC under analytical conditions (A). The results are shown in Figure 1.

[0091] Typical HPLC conditions are as follows: Analysis conditions (A) Column: Shim-pack FC-ODS 150 mm x 4.6 mm φ3 μm (Shimadzu Corporation) Mobile phase: Solution A: 0.1% TFA (trifluoroacetic acid) / water, Solution B: 0.1% TFA / 100% CH3CN Flow rate: 0.6mL / min Gradient program (B solution concentration (%) is vol%): B solution 20% → 70% (10 min), B solution 70% (6 min), B solution 70% → 20% (0.5 min), B solution 20% (13.5 min) Column oven: 40℃ PDA detection: Measures from 230 to 500 nm Sample injection volume: 10 μL

[0092] Figure 1 shows the results of HPLC analysis of the reaction mixture obtained by reacting UDP-Glc and XN with soybean-derived UGT (Gm_UGT1) expressed in Escherichia coli (detection: 350 nm). In Figure 1, XN represents xanthohumol, and A and B represent the products. IXG represents isoxanthohumol glucoside, which is isoxanthohumol to which glucose has been added.

[0093] (Purification of XN glucoside) The product was purified by stepwise elution on a Sep-PAK column. The XN enzyme reaction mixture was centrifuged at 8,000 rpm for 10 minutes, and the supernatant was loaded onto a 20 cc Sep-pak C18 (Waters). It was washed with water for 3 CV (column volume), and eluted with 3 CV of 5% acetonitrile, 3 CV of 30% acetonitrile, 3 CV of 40% acetonitrile, and 3 CV of 80% acetonitrile. No peaks were observed in the water wash or 5% acetonitrile fractions. 1.4 mg of isoxanthohumol-7-glucoside (IXG), an isomer of XN, was eluted in the 30% acetonitrile elution fraction. 5 mg of XN glucoside was eluted in the 40% acetonitrile elution fraction. Isoxanthohumol-7-glucoside is a compound in which one glucose is attached to the 7-position of isoxanthohumol. The monoglucosides of XN consisted of two components (peaks A and B in Figure 1), which were purified by preparative HPLC and analyzed by NMR. NMR measurements were performed in CD3OD.

[0094] Tables 1, 2, Figures 2 and 3 show the results of NMR measurements of the reaction products of UDP-glucose and xanthohumol using soybean-derived UGT (Gm_UGT1) expressed in E. coli. 1 H and 132 and 3 show the results of HMBC (Heteronuclear Multiple Bond Correlation) and NOE (Nuclear Overhauser Effect), which are key to determining the sugar binding position to XN. In Tables 1 and 2, the units of chemical shifts (δ) are ppm, and the units of coupling constants (J) are Hz.

[0095] [Table 1]

[0096] Table 1 and Figure 2 show the results of NMR measurement of the product indicated by A in Figure 1. This confirmed that the main product, fraction A (RT: 11.21 min), was xanthohumol-4'-glucoside (XN4'G), in which one glucose is bound to the 4' position of XN.

[0097] [Table 2]

[0098] Table 2 and Figure 3 show the results of NMR measurement of the product shown as B in Figure 1. This confirmed that the minor component, fraction B (RT: 11.84 min), was a compound with one glucose bonded to the 4-position of XN (xanthohumol 4-O-β-glucopyranoside (XN4G)).

[0099] <Example 2> (Study on XN glycoside-forming activity of Rhizopus fungi) The 14 strains of Rhizopus filamentous fungi shown in Table 3 were collected and used.

[0100] [Table 3]

[0101] Rhizopus fungi were inoculated onto Potato Dextrose Agar (PDA, Difco, Becton Dickinson) plates and cultured at 25°C for 6 days. To obtain a spore suspension, 0.1% Tween 80 and 0.8% NaCl were added to the plate. After filtering through Miracloth, conidia were collected by centrifugation, washed with 0.1% Tween 80 and 0.8% NaCl, and suspended in sterile water until the spore count reached 10 per mL. 7 The resulting mixture was used as a spore suspension.

[0102] To 2 mL of Potato Dextrose (PD) Broth (Difco, Becton Dickinson), 4 μL of a 50 mg / mL xanthoflav ethanol solution was added, and 2 μL of a Rhizopus spore suspension listed in Table 3 was inoculated. The mixture was then shaken at 25°C for 3 days. The culture supernatant and bacterial cells were collected by centrifugation. The culture supernatant and acetonitrile were mixed in a 1:1 ratio and filtered through a GL Chromatodisc 4P 0.45 μm column to prepare the culture supernatant for HPLC analysis. The culture supernatant was analyzed by HPLC under analytical conditions (B).

[0103] Analysis conditions (B) Column: COSMOSIL 5C 18 -AR-II 4.6mm I.D. x 250mm (Nacalai Tesque, Inc.) Solution A: 0.1%TFA 2:8(v / v)CH3CN:H2O B solution: 0.1%TFA 8:2(v / v)CH3CN:H2O Flow Rate: 0.6 mL / min Gradient program: Solution B 0% → 85% (15 min), Solution B 85% (10 min), Solution B 85% → 0% (0.5 min), Solution B 0% (15.5 min) Detection: 320nm or 280nm Sample injection: 10 μL

[0104] The analysis results for the culture supernatant of Rhizopus oryzae (R. oryzae) IAM6049 are shown in Figure 4B. Figure 4A shows the analysis results for PD Broth containing only the substrate (XN) without any Rhizopus bacteria. The detection wavelength for both Figures 4A and 4B was 280 nm. The analytical results of the culture supernatants of the strains shown in Table 3 were all similar. When the Rhizopus fungi shown in Table 3 were cultured in the presence of XN, two main products were produced. One of them had an elution time identical to that of xanthohumol-4'-glucoside (XN4'G) in Example 1. The other had an elution time identical to that of isoxanthohumol-7-glucoside (IXG). Although isoxanthohumol (IX) and XN4'G had similar elution times under analytical condition (B), detailed analysis revealed that IX was almost absent. This suggests that Rhizopus fungi may have not only xanthohumol glycosyltransferase activity but also flavanone lyase activity.

[0105] Example 3 (Preparation of XN4´G by Rhizopus fungi) 100 mL of the medium shown in Table 4 was placed in a 500 mL Sakaguchi flask, and 200 μL of XN solution (50 mg / mL xanthoflav / ethanol solution) was added as a substrate. At this time, XN precipitated and the mixture became turbid. After stirring well, 1 × 10 spores of R. oryzae IAM6256 were added. 4 The cells were inoculated at 0.1% ethanol at 25°C for 2 days with shaking. After incubation, the mixture was filtered through Kiriyama filter paper (5A) to separate the culture supernatant from the bacterial cells. The culture supernatant and acetonitrile were mixed at a 1:1 ratio and filtered through a GL Chromatodisc 4P 0.45 μm column to prepare the HPLC analysis sample for the culture supernatant, which was analyzed under analytical conditions (B). The bacterial cells were peeled from the filter, collected, transferred to a glass test tube, and extracted twice with 10 mL of ethyl acetate. The obtained bacterial extract was dried using a centrifugal concentrator and dissolved in 2 mL of 50% acetonitrile. The extract was diluted 10-fold with 50% acetonitrile and analyzed under analytical conditions (B).

[0106] Examples of HPLC data for analyzing culture supernatants or bacterial cell extracts are shown in Figures 5A, 5B, 6A, 6B, 7A, 7B, 8A, and 8B. Figures 5A and 5B show the results of analyzing culture supernatants from cultures in PD Broth. Figures 6A and 6B show the results of analyzing bacterial cell extracts from cultures in PD Broth. Figures 7A and 7B show the results of analyzing culture supernatants from cultures in a medium containing 0.2% yeast extract (YE) and 0.5% glucose (Glc) (No. 12 in Table 4). Figures 8A and 8B show the results of analyzing bacterial cell extracts from cultures in a medium containing 0.2% YE and 0.5% Glc. Table 4 summarizes the amounts of XN4'G and XN in the culture supernatants and bacterial cells when R. oryzae IAM6256 was cultured in various media.

[0107] [Table 4]

[0108] The amount of XN4'G shown in Table 4 is the amount converted into xanthohumol (XN equivalent amount). The medium ingredients shown in Tables 4 and 5 are described below. PP: Potato Peptone CP (Kyokuto Pharmaceutical Industries Co., Ltd.) YE: Yeast Extract (Difco, Becton Dickinson) Glc: glucose Media other than PD Broth were prepared by mixing the ingredients shown in Table 4 with water. PD Broth was prepared using Potato Dextrose Broth (Difco, Becton Dickinson). PD Broth (1) to (3) were from different lots.

[0109] In cultures No. 4 and No. 9, which had low C / N ratios, the bacterial mass and XN4´G production were low, but in other media, no clear correlation was observed between bacterial mass and XN4´G production. The XN4´G conversion rate for the combined medium and bacterial mass was as high as 90%. The low XN recovery rate was thought to be due to undissolved precipitated XN adsorbed onto the filter paper when the culture medium and bacterial mass were separated. The medium containing Yeast Extract (Difco) had the highest recovery of XN4´G in the medium. Furthermore, the strain was cultured in the medium shown in Table 5 with the addition of XN, and the culture supernatant was analyzed in the same manner as above. XN in the culture supernatant was almost entirely converted to XN4'G.

[0110] [Table 5]

[0111] YP-21CM and Himax GL listed in Table 5 are yeast extracts manufactured by Fuji Food Industry Co., Ltd. Media were prepared by mixing the media ingredients shown in Table 5 with water. The results shown in Table 5 are the analysis results of the culture supernatant.

[0112] XN4'G was purified from the culture supernatants Nos. 11-14 listed in Table 4 by solid-phase extraction using a Sep-Pak C18 20cc Vac cartridge, 5g (Waters). Approximately 100 mL of each sample was loaded onto each cartridge and washed with 20 mL of distilled water, followed by 60 mL of 40% ethanol. The sample was then eluted once with 10 mL of 40% ethanol and four times with 10 mL of 55% ethanol. The eluate was mixed 1:1 with acetonitrile and analyzed under analytical conditions (B). Examples of elution results are shown in Figures 9A, 9B, 10A, 10B, 11A, and 11B. The samples Nos. 11-14 from the second 55% ethanol elution were combined, concentrated, and lyophilized to obtain the XN4'G sample. Unless otherwise specified, the ethanol concentration (%) refers to the ethanol concentration (vol%) in the aqueous ethanol solution. Figures 9A and 9B show the analytical results of the eluate obtained from the first elution with 55% ethanol. Figures 10A and 10B show the analytical results of the eluate obtained from the second elution with 55% ethanol. Figures 11A and 11B show the analytical results of the eluate obtained from the third elution with 55% ethanol. Figures 9A, 10A, and 11A were taken at a detection wavelength of 320 nm. Figures 9B, 10B, and 11B were taken at a detection wavelength of 280 nm. By solid-phase extraction, XN4´G was mainly recovered in the second elution with 55% ethanol.

[0113] Example 4 (Preparation of XN4´Gs) 100 mL of 0.2% YP-21CM and 0.5% Glc medium (No. 20 in Table 5) was placed in a 500 mL Sakaguchi flask, and 200 μL of XN solution (50 mg / mL xanthoflavin / ethanol solution) was added as a substrate. 1 × 10 spores of R. oryzae IAM6256 were then added. 4 The cells were inoculated at 0.5% CO₂ / mL and cultured at 25°C for 2 days with shaking. After the culture was completed, the culture supernatant was obtained by filtration through Kiriyama filter paper (5A). The culture supernatants from the first day and the second day of culture were analyzed by HPLC under analytical conditions (B). The results are shown in Figures 12A and 12B. Ten culture supernatants obtained after two days of culture were mixed to obtain approximately 1 L of culture supernatant, which was then adjusted to pH 5.5 by adding sodium hydroxide solution. Five grams of cluster dextrin (Ezaki Glico Co., Ltd.) was added, and the mixture was incubated in an incubator at 50°C for a period of time. Then, 900 μL of CGTase (Amano Enzyme Inc., product name "CGT-SL, 400 u / mL") was added, and the mixture was allowed to react at 50°C for 4 hours. The reaction mixture after this enzyme reaction was analyzed by HPLC using analytical method (B). The analytical results of the reaction mixture after the enzyme reaction are shown in Figure 12C. As a result, it was found that xanthohumol glycosides, in which two or more glucose units were added to xanthohumol, were produced (Figure 12C). In Figures 12A and 12B, XN4'G is xanthohumol 4' monoglucoside, and XN is xanthohumol. In Figure 12C, XN4'Gs is xanthohumol glycosides in which two or more glucose units were added to the 4' position of XN.

[0114] XN4'Gs was purified from the enzyme reaction mixture by solid-phase extraction using a Sep-Pak C18 20cc Vac cartridge (5g, Waters). The sample was loaded onto the cartridge and washed with 20 mL of distilled water, followed by 40 mL of 35% ethanol. The sample was then eluted five times with 10 mL of 45% ethanol and three times with 10 mL of 55% ethanol. The eluate was mixed 1:1 with acetonitrile and analyzed under analytical conditions (B). Examples of elution results are shown in Figures 13A, 13B, 14A, 14B, 15A, 15B, 16A, 16B, 17A, 17B, 18A, and 18B (detection: 320 nm or 280 nm).

[0115] Figures 13A and 13B show the analytical results of the eluate from the second elution with 45% ethanol. Figures 14A and 14B show the analytical results of the eluate from the third elution with 45% ethanol. Figures 15A and 15B show the analytical results of the eluate from the fourth elution with 45% ethanol. Figures 16A and 16B show the analytical results of the eluate from the fifth elution with 45% ethanol. Figures 17A and 17B show the analytical results of the eluate from the first elution with 55% ethanol. Figures 18A and 18B show the analytical results of the eluate from the second elution with 55% ethanol.

[0116] The eluates from the purified enzyme reaction mixture, the third to fifth eluates from 45% ethanol elutions, were combined and lyophilized to prepare a sample containing XN4'Gs (xanthohumol glycosides with two or more glucose groups attached to the 4' position of XN). A portion of this sample was dissolved in 50% acetonitrile and analyzed by HPLC under analytical condition (B). The HPLC analysis results are shown in Figures 19A and 19B (Figure 19A: detection at 320 nm, Figure 19B: detection at 280 nm). Based on the area of ​​each peak, the average number of glucose groups attached to XN in this sample was 2.5.

[0117] <Example 5> (Preparation of immobilized cells and production of XN4´G and XN4´Gs) 1 × 10 spores of R. oryzae IAM6256 5 The beads were added to 100 mL of 2% sodium alginate solution to a concentration of 0.1 cells / mL, and slowly stirred to suspend the mixture. The resulting suspension was then added dropwise to 400 mL of ice-cooled 2% calcium chloride solution to form a gel, which then remained at 4°C for 2 hours. After removing the solution, the beads were washed several times with sterile water. 500 mL of medium (0.2% yeast extract (YP-21CM, Fuji Food Industry Co., Ltd.), 2% glucose) was added to the beads, and the mixture was cultured at 24°C with slow stirring to obtain immobilized bacteria. After removing the medium, the immobilized bacteria were washed with sterile water and stored in 400 mL of sterile water at 4°C until use in the reaction.

[0118] (Immobilized Bacterial Cell Reaction 1) Approximately one-quarter of the immobilized cells prepared as described above was placed in a 300 mL Erlenmeyer flask and 200 mL of sterile water was added. Glucose was added to the flask at concentrations of 0.05%, 0.1%, or 0.25%. 100 μL of a 50 mg / mL xanthoflavone-ethanol solution was then added. The reaction mixture was stirred at 25°C for 24 hours, after which the reaction mixture was collected and a portion was subjected to HPLC analysis under analytical conditions (B) (Table 6). The remaining immobilized cells were immersed in sterile water and stored at 4°C until further reaction.

[0119] (Immobilized Bacterial Cell Reaction 2) The same reaction was performed using immobilized cells stored after Reaction 1. The reaction mixture was sampled 24 and 48 hours after the start of the reaction and analyzed by HPLC (Table 6). The XN4'G and XN concentrations (mg / L) in the reaction mixture after 24 and 48 hours of reaction are shown in Table 6. The XN4'G concentration in Table 6 is the concentration converted to XN. After 48 hours, the entire reaction mixture was collected, and the remaining immobilized cells were immersed in sterilized water and stored at 4°C until the next reaction. The glycosylation rate was high in the absence of added glucose (no added Glu). This is thought to be because adding sugar to the reaction accelerated deterioration of the gel in which the cells were immobilized, affecting the activity of the cells. The glucose used for glycosylation was thought to be sufficient as the glucose taken up by the cells during the culture for preparing the immobilized cells.

[0120] [Table 6]

[0121] (Enzyme reaction of immobilized bacterial cell reaction solution) Approximately 200 mL of the recovered immobilized cell reaction solution was added with sodium hydroxide solution to adjust the pH to 5.5. Cluster dextrin was added to 5 g / L, and the mixture was incubated in an incubator at 50°C for a while. Then, 180 μL of CGTase (Amano Enzyme Co., Ltd., product name "CGT-SL, 400 μL") was added, and the mixture was allowed to react at 50°C for 4 hours. Analysis of the reaction solution using analytical method (B) revealed that xanthohumol glycosides (XN4'Gs), in which two or more glucose groups were added to the 4' position of xanthohumol, were produced.

[0122] Example 6 (Water-soluble XN glycoside) The water solubility of xanthohumol and its glycosides was investigated as follows. Water was added to xanthoflav to a concentration of 0.02 mg / mL, XN4'G obtained in Example 3 to a concentration of 0.05 mg / mL, and XN4'Gs (average glucose addition number 2.5) obtained in Example 4 to a concentration of 1.0 mg / mL, and the mixture was dissolved by vigorously stirring at room temperature. Precipitation occurred in each case, so the supernatant was centrifuged to remove the precipitate, and the supernatant was analyzed and quantified by HPLC. The results are shown in Table 7. The dissolved amounts shown in Table 7 are the dissolved amounts converted to xanthohumol (XN).

[0123] [Table 7]

[0124] As shown in Table 7, while xanthohumol is almost insoluble in water, XN4'G was able to dissolve 95 times more xanthohumol, and XN4'Gs was able to dissolve more than 760 times more xanthohumol. Furthermore, XN4'Gs was able to dissolve 8.1 times more xanthohumol than XN4'G. In other words, XNGs has higher water solubility than XN4'G, and by adding glucose to XN4'G to prepare a glycoside, its water solubility could be significantly improved. The glycoside XN4G, which has one glucose attached to the 4-position of XN, was also predicted to have improved water solubility compared to xanthohumol. Furthermore, adding an additional glucose to the glucose residue of XN4G can improve water solubility compared to XN4G, similar to XN4´Gs.

[0125] By glycosidizing XN, (1) water solubility can be imparted without the addition of emulsifiers, (2) taste (bitterness and unpleasant flavors) can be improved, and (3) liquids can be clarified. Furthermore, (4) glycosidization is expected to have the effect of inhibiting the decomposition of XN. Therefore, glycosidization provides a technology that allows the addition of XN and similar functional substances to beverage products with high clarity, which cannot be treated with emulsifiers or dispersants.

Claims

1. (A2) producing a xanthohumol glycoside having one hexose bonded to the 4'-position and / or the 4-position of xanthohumol from xanthohumol and UDP-hexose by using one or more proteins selected from the group consisting of the following (p1), (p2), and (p3); and A method for producing a xanthohumol glycoside in which a sugar chain consisting of 2 to 10 hexoses is bound to the 4'-position and / or 4-position of xanthohumol, the method comprising step (B2) of adding 1 to 9 hexoses to the hexose residue of the xanthohumol glycoside produced in step (A2). (p1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (p2) A protein consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having the activity of adding hexose to the 4'-position and / or 4-position of xanthohumol. (p3) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1 and having the activity of adding hexose to the 4'-position and / or 4-position of xanthohumol.

2. A method for producing a xanthohumol glycoside in which one hexose is bound to the 4'-position and / or the 4'-position of xanthohumol, comprising step (A2) of generating a xanthohumol glycoside in which one hexose is bound to the 4'-position and / or the 4'-position of xanthohumol from xanthohumol and UDP-hexose using one or more proteins selected from the group consisting of (p1), (p2), and (p3) below. (p1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (p2) A protein consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having the activity of adding hexose to the 4'-position and / or 4-position of xanthohumol. (p3) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1 and having the activity of adding hexose to the 4'-position and / or 4-position of xanthohumol.

3. A method for improving the water solubility of xanthohumol, comprising adding a sugar chain consisting of 2 to 10 hexoses to the 4'-position or 4-position of xanthohumol, (A2) producing a xanthohumol glycoside having one hexose bonded to the 4'-position and / or the 4-position of xanthohumol from xanthohumol and UDP-hexose by using one or more proteins selected from the group consisting of the following (p1), (p2), and (p3); and A method comprising a step (B2) of adding 1 to 9 hexoses to the hexose residue of the xanthohumol glycoside produced in step (A2). (p1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (p2) A protein consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having the activity of adding hexose to the 4'-position and / or 4-position of xanthohumol. (p3) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1 and having the activity of adding hexose to the 4'-position and / or 4-position of xanthohumol.

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