Composition containing hydroxytyrosol 4-o-glucoside

Abstract

To provide a composition capable of obtaining antioxidant activity similar to that of hydroxytyrosol and capable of being stably stored, and to provide a method for producing hydroxytyrosol 4-O-glucoside in improved productivity.SOLUTION: In one aspect, the present disclosure relates to a composition containing hydroxytyrosol 4-O-glucoside, wherein the content of hydroxytyrosol 4-O-glucoside in the composition is 85% by weight or more. In another aspect, the present invention relates to a method for producing hydroxytyrosol 4-O-glucoside, comprising mixing hydroxytyrosol, UDP-glucose, and UDP glucosyltransferase 89B1 or a homologue thereof to obtain hydroxytyrosol 4-O-glucoside.SELECTED DRAWING: Figure 12

Description

[Technical Field]

[0001] The present disclosure relates to compositions containing hydroxytyrosol 4-O-glucoside and methods for producing hydroxytyrosol 4-O-glucoside. [Background technology]

[0002] Hydroxytyrosol is a polyphenol with antioxidant activity 10 times greater than that of vitamin C. Hydroxytyrosol is one of the most powerful antioxidants with potential biological functions as an antitumor agent, antiatheroma agent, anti-inflammatory agent, and / or antiplatelet aggregation agent, and is used in cosmetics, health foods such as functional foods and nutritional supplements, animal feed, etc. In recent years, a process for producing highly pure hydroxytyrosol from glucose and glycerin by microbial fermentation has also been developed (e.g., Patent Document 1, etc.).

[0003] However, because hydroxytyrosol has extremely high antioxidant capacity, it is easily oxidized (e.g., Non-Patent Document 1, etc.), making its use and storage difficult. For example, a method has been proposed in which hydroxytyrosol is encapsulated in cyclodextrin to ensure stable use. However, this method has the problem that the antioxidant activity of hydroxytyrosol cannot be fully exerted. Another proposed storage method is to purge the bottle containing hydroxytyrosol with nitrogen gas or argon gas to remove oxygen, and then store it in a desiccator containing an oxygen scavenger. However, this method has the problem of being extremely cumbersome.

[0004] There is a need for a method that enables hydroxytyrosol to be stored stably while still allowing it to fully exert its antioxidant activity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-130128 [Non-patent literature]

[0006] [Non-Patent Document 1] "Evaluation of Polyphenols in Olive Pomace - Examination of Preservation Pretreatment of Olive Pomace by Microwave Irradiation" Kagawa Industrial Support Foundation, Regional Collaborative Research Department Annual Report, 2019 Summary of the Invention [Problem to be solved by the invention]

[0007] Hydroxytyrosol is a colorless, transparent substance, but it can turn yellow and become discolored. This discoloration is thought to be caused in part by oxidation of hydroxytyrosol. Hydroxytyrosol contains three hydroxyl groups, which are oxidized to form quinone compounds, which then form quinone bridges, resulting in discoloration. This oxidation can occur significantly when hydroxytyrosol is stored in solution.

[0008] In the course of extensive research into methods for stably storing hydroxytyrosol, the present inventors attempted to produce hydroxytyrosol glycosides using a glycosyltransferase-mediated UDP (uridine diphosphate glucose) glucosyltransferase reaction. As a result, they found that hydroxytyrosol 3-O-glucoside, in which the hydroxyl group at the 3-position of hydroxytyrosol is protected with glucose, was not sufficiently stable, whereas hydroxytyrosol 4-O-glucoside, in which the hydroxyl group at the 4-position is protected with glucose, was extremely stable and could be stored stably, even in solution, for example. Furthermore, the inventors discovered that when hydroxytyrosol 4-O-glucoside is applied to a living body, free hydroxytyrosol is produced by the body's glycosidases, and this produces an antioxidant effect similar to that achieved when hydroxytyrosol is applied to a living body. Therefore, it was found that by storing hydroxytyrosol as hydroxytyrosol 4-O-glucoside, it can be stored stably and the excellent antioxidant ability of hydroxytyrosol can be utilized.

[0009] Hydroxytyrosol 4-O-glucoside is known to be contained in solutions extracted from olives and other plants, but its content accounts for less than 40% of the phenolic compounds contained in the extract (J. Agric. Food Chem. 2002, 50, 3835-3839, and Molecules 2022, 27, 8380), and no compositions containing a high content of hydroxytyrosol 4-O-glucoside are known.

[0010] The present inventors attempted to produce hydroxytyrosol 4-O-glucoside using UDP-glucosyltransferase, but discovered that UDP-glucosyltransferase was unable to protect the hydroxyl group at the 4-position, resulting in insufficient productivity of hydroxytyrosol 4-O-glucoside. In the course of further research aimed at solving this problem, we discovered that by using a specific UDP-glucosyltransferase, glucose can be regioselectively introduced into the 4-hydroxyl group of hydroxytyrosol, thereby providing a composition containing hydroxytyrosol 4-O-glucoside that has antioxidant activity similar to that of hydroxytyrosol and is highly stable.

[0011] The present disclosure provides a composition that can provide antioxidant activity similar to that of hydroxytyrosol and that can be stably stored, as well as a method for producing hydroxytyrosol 4-O-glucoside with improved productivity. [Means for solving the problem]

[0012] In one aspect, the present disclosure relates to a composition containing hydroxytyrosol 4-O-glucoside, wherein the content of hydroxytyrosol 4-O-glucoside in the composition is 85% by weight or more.

[0013] In another aspect, the present disclosure relates to a method for producing hydroxytyrosol 4-O-glucoside, the method comprising mixing hydroxytyrosol, UDP-glucose, and UDP-glucosyltransferase 89B1 or a homolog thereof to obtain hydroxytyrosol 4-O-glucoside. [Effects of the Invention]

[0014] According to one aspect of the present disclosure, there is provided a composition that can provide antioxidant activity similar to that of hydroxytyrosol and that can be stably stored, as well as a method for producing hydroxytyrosol 4-O-glucoside with improved specific productivity. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows an example of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the UDP-glucosyltransferase 89B1 derived from Raphanus sativus (radish) obtained in the Examples. [Figure 2] FIG. 2 shows an example of SDS-PAGE of a sample (Rs89B1) obtained by desalting fractions 1 and 2 used in the SDS-PAGE of FIG. 1 on a PD-10 column. [Figure 3] FIG. 3 shows an example of SDS-PAGE of the NUS-AtUGT71C1 protein (Arabidopsis thaliana-derived UDP-glucosyltransferase 71C1 to which an NUS tag has been added at the N-terminus) obtained in the Examples. [Figure 4] FIG. 4 shows an example of SDS-PAGE of the NUS-AtUGT71C1 protein (NUS-UGT71C1) and the NUS-AtUGT71C1 protein treated with thrombin (NUS-UGT71C1+Thrombin). [Figure 5] FIG. 5 shows HPLC chromatograms confirming the transglycosylation activity of enzyme Rs89B1 on catechol compounds (dihydroxyphenyllactic acid (DHPLA), caffeic acid (HCA), and hydroxytyrosol (HTY)). [Figure 6] FIG. 6 shows HPLC chromatograms confirming the transglycosylation activity of the enzyme AtUGT71C1 (NUS-71C1 or NUS-71C1+Thrombin) on HTY. [Figure 7] FIG. 7 shows a chromatogram obtained when the Rs89B1 reaction solution obtained by the transglycosylation reaction of Rs89B1 with HTY was subjected to HPLC to purify the product. [Figure 8] FIG. 8 shows HPLC chromatograms of purified HTY, and the AtUGT71C1 and Rs89B1 reaction products. [Figure 9] FIG. 9 shows an HPLC chromatogram obtained when the AtUGT71C1 reaction product obtained by the transglycosylation reaction of AtUGT71C1 with HTY was subjected to HPLC and purified. [Figure 10] FIG. 10 shows the absorption spectrum of the HTY solution after incubation at 60° C. for 24 hours (lower graph) and the absorption spectrum before incubation (upper graph). [Figure 11] FIG. 11 shows an example of the results of a stability test on HTY, the Rs89B1 reaction product (HTY-4-O-Glc), and the AtUGT71C1 reaction product (HTY-3-O-Glc), showing the change in absorbance (500 nm) after incubation at 60°C for 22 hours. [Figure 12] FIG. 12 shows an example of the results of a stability test for HTY, HTY-4-O-Glc, and HTY-3-O-Glc, showing HPLC chromatograms after 22 hours of incubation at 60°C (22 h) and before incubation (0 h). [Figure 13] FIG. 13 shows HPLC chromatograms of the degradation experiment of HTY-4-O-Glc by yeast cell lysis. [Figure 14] Figure 14 shows a plot of the relative area values ​​obtained by dividing the area value of HTY-4-O-Glc at each sampling time by the area value of HTY-4-O-Glc at 0 h of incubation, which was taken as 100%, in an experiment on the degradation of HTY-4-O-Glc by yeast cell lysis. DETAILED DESCRIPTION OF THE INVENTION

[0016] [Composition] In one aspect, the present disclosure relates to a composition containing hydroxytyrosol 4-O-glucoside, wherein the content of hydroxytyrosol 4-O-glucoside in the composition is 85% by weight or more. Hydroxytyrosol 4-O-glucoside is a compound in which a glucoside is bonded to the hydroxyl group at the 4-position of hydroxytyrosol, and has a structure represented by the following formula: Hydroxytyrosol 4-O-glucoside can also be referred to as hydroxytyrosol 4-β-D-glucoside. [ka]

[0017] The composition of the present disclosure contains 85% by weight or more of hydroxytyrosol 4-O-glucoside. Hydroxytyrosol 4-O-glucoside has higher stability than hydroxytyrosol, and when applied to a living body, it may exhibit antioxidant activity similar to that of hydroxytyrosol. Therefore, in one or more embodiments, the composition of the present disclosure may be used as an ingredient in a food composition, beverage composition, health food, supplement, quasi-drug, pharmaceutical composition, cosmetic, polymerization inhibitor, and / or animal feed, thereby providing a quasi-drug, pharmaceutical composition, cosmetic, polymerization inhibitor, animal feed, or the like having excellent antioxidant activity.

[0018] The "content" in the present disclosure can be determined by liquid chromatography, and in one or more embodiments, can be obtained by measurement under HPLC condition C described in the Examples.

[0019] In one or more embodiments, the composition of the present disclosure contains a high content of hydroxytyrosol 4-O-glucoside. In one or more embodiments, the content of hydroxytyrosol 4-O-glucoside in the composition of the present disclosure is 86% by weight or more, 87% by weight or more, 88% by weight or more, 89% by weight or more, 90% by weight or more, 91% by weight or more, 92% by weight or more, 93% by weight or more, 94% by weight or more, 95% by weight or more, 96% by weight or more, 97% by weight or more, 98% by weight or more, or 99% by weight or more.

[0020] In one or more embodiments, the composition of the present disclosure may contain, or may be substantially free of, phenolic compounds other than hydroxytyrosol 4-O-glucoside. In one or more embodiments, examples of phenolic compounds other than hydroxytyrosol 4-O-glucoside include hydroxytyrosol, hydroxytyrosol 3-O-glucoside, and hydroxytyrosol 3,4-O-glucoside. In the present disclosure, "substantially free" means that the content in the composition of the present disclosure is 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less.

[0021] In one or more embodiments, the composition of the present disclosure may contain, or may be substantially free of, substances other than phenolic compounds. In one or more embodiments, examples of such substances include UDP-glucose and its degradation products. In one or more embodiments, examples of degradation products of UDP-glucose include UDP, uridine monophosphate (UMP), and uridine.

[0022] In one or more embodiments, the composition of the present disclosure may contain hydroxytyrosol in a content of 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less. In one or more embodiments, the content of hydroxytyrosol relative to hydroxytyrosol 4-O-glucoside is 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less. In one or more embodiments, the composition of the present disclosure may contain hydroxytyrosol 3-O-glucoside at a content of 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less. In one or more embodiments, the content of hydroxytyrosol 3-O-glucoside relative to hydroxytyrosol 4-O-glucoside is 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less. In one or more embodiments, the composition of the present disclosure may contain hydroxytyrosol 3,4-O-glucoside in an amount of 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less. In one or more embodiments, the amount of hydroxytyrosol 3,4-O-glucoside relative to hydroxytyrosol 4-O-glucoside is 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less. In one or more embodiments, the composition of the present disclosure may contain UDP-glucose and its decomposition products at a content of 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less. In one or more embodiments, the content of UDP-glucose and its decomposition products relative to hydroxytyrosol 4-O-glucoside is 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, or 0.5% by weight or less.

[0023] In one or more embodiments, the content of hydroxytyrosol 4-O-glucoside relative to the phenolic compounds is 90% by weight or more, 91% by weight or more, 92% by weight or more, 93% by weight or more, 94% by weight or more, 95% by weight or more, 96% by weight or more, 97% by weight or more, 98% by weight or more, or 99% by weight or more. In the present disclosure, the "content of hydroxytyrosol 4-O-glucoside relative to the phenolic compounds" refers to the ratio of the amount of hydroxytyrosol 4-O-glucoside to the total amount of phenolic compounds contained in the composition of the present disclosure, and the total amount of "phenolic compounds" refers to the sum of the amounts of hydroxytyrosol 4-O-glucoside, hydroxytyrosol, hydroxytyrosol 3-O-glucoside, and hydroxytyrosol 3,4-O-glucoside.

[0024] In one or more embodiments, the composition of the present disclosure consists essentially of hydroxytyrosol 4-O-glucoside, hydroxytyrosol, and hydroxytyrosol 3-O-glucoside, because a powdered composition containing hydroxytyrosol 4-O-glucoside at a high concentration can be easily obtained. In one or more embodiments, the composition of the present disclosure consists essentially of 90% by weight or more of hydroxytyrosol 4-O-glucoside, hydroxytyrosol, and hydroxytyrosol 3-O-glucoside, or consists essentially of 90% by weight or more of hydroxytyrosol 4-O-glucoside, 5% by weight or less of hydroxytyrosol, and 5% by weight or less of hydroxytyrosol 3-O-glucoside, and more preferably consists essentially of 98% by weight or more of hydroxytyrosol 4-O-glucoside, 1% by weight or less of hydroxytyrosol, and 1% by weight or less of hydroxytyrosol 3-O-glucoside.

[0025] In one or more embodiments, the composition of the present disclosure has high storage stability. In one or more embodiments, the composition of the present disclosure has high storage stability. In one or more embodiments, the composition of the present disclosure has an absorbance at a wavelength of 500 nm of 0.1 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less after standing in a Tris-HCl (pH 8.0) solution at 60°C for 6 hours. In this disclosure, "standing still" refers to leaving the solution in a stationary state without stirring or mixing. For example, even if a container containing the solution is moved, this falls under "standing still" as long as no artificial physical stimuli are applied from the outside. Absorbance can be measured by the method described in the stability test in the Examples.

[0026] The form of the composition of the present disclosure is not particularly limited, and in one or more embodiments, examples include solids, dry products such as granules and powders, pastes, liquids, etc. In one or more embodiments, the composition of the present disclosure may have high storage stability even in a liquid state.

[0027] In one or more embodiments, the composition of the present disclosure may have a total weight of 1 mg or more, 10 mg or more, or 100 mg or more.

[0028] In one or more embodiments, the composition of the present disclosure may be obtained by an in vitro method using the enzyme described below, or may be obtained by a method using a microorganism expressing the enzyme described below.

[0029] When hydroxytyrosol 4-O-glucoside is applied to a living body, it is converted to free hydroxytyrosol by glycosidases and the like present in the body. Therefore, in one or more embodiments, the composition of the present disclosure may have antioxidant activity comparable to that of hydroxytyrosol. Hydroxytyrosol is known to have high antioxidant activity and is known to have various effects, such as whitening effect, antiviral, antibacterial, anti-inflammatory, anticarcinogenic, antihypertensive, antithrombotic, and anti-arteriosclerosis. Therefore, in one or more embodiments, the composition of the present disclosure may have effects such as whitening effect, antiviral, antibacterial, anti-inflammatory, anticarcinogenic, antihypertensive, antithrombotic, and anti-arteriosclerosis.

[0030] In one or more embodiments, the composition of the present disclosure can be used as an ingredient for foods and beverages such as food compositions, beverage compositions, and health foods, as a cosmetic ingredient, and as an ingredient for animal feed, quasi-drugs, supplements, pharmaceuticals, and the like. In one or more embodiments, the composition of the present disclosure can be used as a polymerization inhibitor for electronic substrate materials, etc. In another aspect, the present disclosure relates to foods and beverages such as food compositions, beverage compositions, and health foods, as well as cosmetics, animal feed, supplements, quasi-drugs, or pharmaceutical compositions that contain the composition of the present disclosure as an active ingredient. In one or more embodiments, health foods include foods with health claims, such as foods for specified health uses, foods with nutrient functions, and foods with functional claims, whose food functions are labeled in accordance with national standards for safety and efficacy.

[0031] [Manufacturing method] In another aspect, the present disclosure relates to a method for producing hydroxytyrosol 4-O-glucoside. The method of the present disclosure includes mixing hydroxytyrosol, glucose, and UDP-glucosyltransferase 89B1 or a homolog thereof to obtain hydroxytyrosol 4-O-glucoside. In one or more embodiments, the mixing is performed under conditions that allow the UDP-glucosyltransferase to transglycosylate from UDP-glucose to hydroxytyrosol. This mixing can produce hydroxytyrosol 4-O-glucoside. In one or more embodiments, the method of the present disclosure can efficiently produce hydroxytyrosol 4-O-glucoside, preferably a composition having a hydroxytyrosol 4-O-glucoside content of 85% by weight or more relative to the phenolic compounds. The method of the present disclosure can produce the composition of the present disclosure. In one or more embodiments, phenolic compounds include hydroxytyrosol, hydroxytyrosol 3-O-glucoside, and hydroxytyrosol 3,4-O-glucoside.

[0032] In the present disclosure, "UDP-glucosyltransferase 89B1" refers to an enzyme having the activity of transferring glucose to a substrate, preferably an enzyme having the activity of transferring the glucose of UDP-glucose to hydroxytyrosol, more preferably an enzyme having the activity of transferring the glucose of UDP-glucose to the hydroxyl group at position 4 of hydroxytyrosol, and even more preferably an enzyme having the activity of specifically transferring the glucose of UDP-glucose to the hydroxyl group at position 4 of hydroxytyrosol. In the present disclosure, "having the activity of specifically transferring the glucose of UDP-glucose to the hydroxyl group at position 4 of hydroxytyrosol" means that the transfer activity to the hydroxyl group at position 4 is 5-fold, 10-fold, 15-fold, 17-fold, or 19-fold or more compared to transfer to the hydroxyl group at position 3 or 1 of hydroxytyrosol. UDP glucosyltransferases in the present disclosure can include flavonol 3-O-glucosyltransferases.

[0033] In the present disclosure, a "homolog of UDP-glucosyltransferase 89B1" refers to an enzyme that has an amino acid sequence or nucleotide sequence similar to that of UDP-glucosyltransferase and has the activity of transferring glucose to a substrate, preferably an enzyme that has the activity of transferring the glucose of UDP-glucose to hydroxytyrosol, more preferably an enzyme that has the activity of transferring the glucose of UDP-glucose to the hydroxyl group at the 4th position of hydroxytyrosol, and even more preferably an enzyme that has the activity of transferring the glucose of UDP-glucose specifically to the hydroxyl group at the 4th position of hydroxytyrosol. In the present disclosure, "similar amino acid sequences or nucleotide sequences" in one or more embodiments means that the sequence identity is 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more.

[0034] In one or more embodiments, UDP-glucosyltransferase 89B1 may be derived from the genera Raphanus, Arabidopsis, Brassica, Hirschfeldia, Sinapis, Camelina, Eutrema, Capsella, Microthlaspi, Arabis, and Tarenaya, etc. In one or more embodiments, UDP-glucosyltransferase 89B1 may be derived from the genera Raphanus sativus, Arabidopsis thaliana, Arabidopsis suecica, Arabidopsis lyrata subsp. Lyrata, Brassica carinata, Brassica cretica, Brassica rapa, Brassica napus, Hirschfeldia incana, Sinapis alba, Camelina sativa, Eutrema salsugineum, Capsella rubella, Microthlaspi erraticum, Arabis alpina, and Tarenaya hasslerian, etc.

[0035] In one or more embodiments, examples of UDP-glucosyltransferase 89B1 include flavonol 3-O-glucosyltransferase UGT89B1 (Raphanus sativus), flavonol 3-O-glucosyltransferase UGT89B1 (also referred to as UDP-glucosyl transferase 89B1) (Arabidopsis thaliana), hypothetical protein Bca52824_015732 (Brassica carinata), flavonol 3-O-glucosyltransferase UGT89B1 (Brassica rapa), flavonol 3-O-glucosyltransferase UGT89B1 (Brassica napus), flavonol 3-O-glucosyltransferase UGT89B1 (Hirschfeldia incana), and UDP-glucuronosyl / UDP-glucosyltransferase (Arabidopsis suecica), UDP-glycosyltransferase 89B1 (Arabidopsis lyrata subsp. lyrata), UDP-glycosyltransferase 89B1-like (Camelina sativa), UDP-glycosyltransferase 89B1 (Eutrema salsugineum), UDP-glycosyltransferase 89B1 (Capsella rubella), and UDP-glycosyltransferase 89B1 (Tarenaya hassleriana).

[0036] The amino acid sequence of flavonol 3-O-glucosyltransferase UGT89B1 (derived from Raphanus sativus) is set forth as SEQ ID NO: 1. The amino acid sequence of flavonol 3-O-glucosyltransferase UGT89B1 (derived from Arabidopsis thaliana, also known as UDP-glucosyl transferase 89B1) is set forth as SEQ ID NO: 2. The amino acid sequence of flavonol 3-O-glucosyltransferase UGT89B1 (derived from Brassica rapa) is set forth as SEQ ID NO: 3. The amino acid sequence of flavonol 3-O-glucosyltransferase UGT89B1 (derived from Brassica napus) is set forth as SEQ ID NO: 4 or 6. The amino acid sequence of flavonol 3-O-glucosyltransferase UGT89B1 (derived from Hirschfeldia incana) is set forth as SEQ ID NO: 5. The amino acid sequence of UDP-glucuronosyl / UDP-glucosyltransferase (derived from Arabidopsis suecica) is set forth as SEQ ID NO: 7 or 8. The amino acid sequence of UDP-glycosyltransferase 89B1 (derived from Arabidopsis lyrata subsp. lyrata) is set forth as SEQ ID NO: 9. The amino acid sequence of UDP-glycosyltransferase 89B1-like (derived from Camelina sativa) is set forth as SEQ ID NO: 10. The amino acid sequence of UDP-glycosyltransferase 89B1 (derived from Eutrema salsugineum) is set forth as SEQ ID NO: 11. The amino acid sequence of UDP-glycosyltransferase 89B1 (derived from Capsella rubella) is set forth as SEQ ID NO: 12. The amino acid sequence of UDP-glycosyltransferase 89B1 (derived from Tarenaya hassleriana) is set forth as SEQ ID NO: 13.

[0037] In one or more embodiments, other amino acid sequences of UDP-glucosyltransferase 89B1 include the amino acid sequences represented by SEQ ID NOs: 14 to 21. The amino acid sequence of SEQ ID NO: 14 is the amino acid sequence of a protein (derived from Brassica napus) that has the activity of transferring glucose to a substrate. The amino acid sequence of SEQ ID NO: 15 is the amino acid sequence of a protein (derived from Brassica carinata) that has the activity of transferring glucose to a substrate. The amino acid sequence of SEQ ID NO: 16 is the amino acid sequence of a protein (derived from Sinapis alba) that has the activity of transferring glucose to a substrate. The amino acid sequence of SEQ ID NO: 17 is the amino acid sequence of a protein (derived from Sinapis alba) that has the activity of transferring glucose to a substrate. The amino acid sequence of SEQ ID NO: 18 is the amino acid sequence of a protein (derived from Brassica cretica) that has the activity of transferring glucose to a substrate. The amino acid sequence of SEQ ID NO: 19 is the amino acid sequence of a protein (derived from Brassica carinata) that has the activity of transferring glucose to a substrate. The amino acid sequence of SEQ ID NO: 20 is the amino acid sequence of a protein (derived from Microthlaspi erraticum) that has the activity of transferring glucose to a substrate. The amino acid sequence of SEQ ID NO: 21 is the amino acid sequence of a protein (derived from Arabis alpina) that has the activity of transferring glucose to a substrate.

[0038] In one or more embodiments, examples of homologs of UDP-glucosyltransferase 89B1 include enzymes that have 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 21 and have the activity of transferring glucose to a substrate. In the present disclosure, "amino acid sequence identity" can be performed using readily available sequence comparison computer programs, such as the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387), the BLAST package (Ausubel et al. (1999) ibid-Ch. 18), and FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410).

[0039] In one or more embodiments, the method of the present disclosure may include, after mixing, incubating the hydroxytyrosol, glucose, and UDP-glucosyltransferase 89B1 or a homolog thereof under conditions that activate UDP-glucosyltransferase 89B1 and allow production of hydroxytyrosol 4-O-glucoside. Reaction conditions when using UDP-glucosyltransferase 89B1 derived from Raphanus sativus are not particularly limited, and in one or more embodiments, can include 10 to 100 mmol / L Tris-HCl (pH 6 to 7), 50 to 200 mmol / L UDP-glucose, 10 to 100 mmol / L hydroxytyrosol, and 0.1 to 1 mg / mL UDP-glucosyltransferase 89B1. In one or more embodiments, this mixture can be incubated at 30°C for a predetermined time (e.g., 10 to 48 hours) to obtain hydroxytyrosol 4-O-glucoside.

[0040] In one or more embodiments, the methods of the present disclosure may use UDP-glucosyltransferase 71B1, 78D2, or 73B3 in place of or in combination with UDP-glucosyltransferase 89B1.

[0041] In one or more embodiments of the method of the present disclosure, the obtained hydroxytyrosol 4-O-glucoside may be recovered and used as is, or may be purified as necessary to obtain a hydroxytyrosol 4-O-glucoside-containing composition with higher purity. In one or more embodiments, purification may be performed using a column packed with an adsorption resin, gel filtration, or the like.

[0042] [Production method using microorganisms] In one or more embodiments, the composition of the present disclosure may contain hydroxytyrosol 4-O-glucoside obtained from a microorganism in which a gene encoding UDP-glucosyltransferase 89B1 or a homolog thereof has been expressibly introduced into a microbial host capable of producing hydroxytyrosol. In yet another aspect, the present disclosure relates to a transformant capable of producing hydroxytyrosol 4-O-glucoside, in which a gene encoding UDP-glucosyltransferase 89B1 or a homolog thereof has been expressibly introduced into a microbial host capable of producing hydroxytyrosol. In one or more embodiments, microorganisms capable of producing hydroxytyrosol can be obtained based on Japanese Patent Application Laid-Open Nos. 2022-130128 and 2022-79337, etc.

[0043] The transformant disclosed in JP 2022-130128 A is capable of producing hydroxytyrosol using carbohydrates as a raw material. The microorganism disclosed in JP 2022-79337 A is capable of producing hydroxytyrosol using tyrosol as a raw material. In yet another aspect, the present disclosure relates to a method for producing hydroxytyrosol 4-O-glucoside, comprising reacting a carbohydrate raw material with a transformant capable of producing hydroxytyrosol 4-O-glucoside of the present disclosure. In yet another aspect, the present disclosure relates to a method for producing hydroxytyrosol 4-O-glucoside, comprising reacting tyrosol with a transformant capable of producing hydroxytyrosol 4-O-glucoside of the present disclosure.

[0044] The present disclosure further relates to one or more of the following non-limiting embodiments. [1] A composition containing hydroxytyrosol 4-O-glucoside, A composition, wherein the content of hydroxytyrosol 4-O-glucoside in the composition is 85% by weight or more. [2] The composition described in [1], which has an absorbance of 0.1 or less at a wavelength of 500 nm after being left standing in a Tris-HCl (pH 8.0) solution at 60°C for 6 hours. [3] The composition described in [1] or [2], wherein the content of hydroxytyrosol relative to the hydroxytyrosol 4-O-glucoside is 5% by weight or less. [4] A method for producing hydroxytyrosol 4-O-glucoside, comprising: A method comprising mixing hydroxytyrosol, UDP-glucose, and UDP-glucosyltransferase 89B1 or a homolog thereof to obtain hydroxytyrosol 4-O-glucoside. [5] The method according to [4], further comprising recovering the obtained hydroxytyrosol 4-O-glucoside. [6] A composition according to any one of [1] to [3], containing hydroxytyrosol 4-O-glucoside obtained by the method according to [4] or [5]. [7] A composition according to any one of [1] to [3] for use in at least one selected from the group consisting of ingredients for food and beverages, ingredients for supplements, cosmetic materials, polymerization inhibitors, and animal feed. [8] A food composition, a beverage composition, a health food, a supplement, a quasi-drug, a pharmaceutical composition, a cosmetic, a polymerization inhibitor, or an animal feed containing the composition according to any one of [1] to [3]. [Example]

[0045] Hereinafter, the present disclosure will be described in more detail with reference to examples, but these are merely illustrative examples and the present disclosure is not limited to these examples.

[0046] [List of Abbreviations] Rs89B1: UDP-glucosyltransferase 89B1 from Raphanus sativus (radish) AtUGT71C1: UDP-glucosyltransferase 71C1 from Arabidopsis thaliana UDP-Glc: uridine diphosphate glucose HTY: 3-hydroxytyrosol HTY-3-O-Glc: Hydroxytyrosol 3-O-glucoside HTY-4-O-Glc: Hydroxytyrosol 4-O-glucoside Tris: Trishydroxymethylaminomethane TFA: Trifluoroacetic acid

[0047] [Primer List] The following primers A to F were used to obtain the Rs89B1 enzyme. [Table 1]

[0048] [Obtaining purified Rs89B1 enzyme] The Rs89B1 gene was amplified by PCR (primers A and B) using PrimeSTAR® Max DNA polymerase (Takara Bio) with cDNA (SEQ ID NO: 28) prepared from commercially available radish as a template. The PCR-amplified Rs89B1 gene was inserted into the multicloning site of the expression plasmid pREP1 vector, so that a 6x histidine tag sequence was added to the C-terminus. First, the 6x histidine tag sequence was inserted into the multicloning site of the pREP1 vector by PCR (primers C and D) using PrimeSTAR® Max DNA polymerase to create the pREP1-His vector. The pREP1-His vector was linearized by PCR (primers E and F) using PrimeSTAR® Max DNA polymerase, and then ligated to the Rs89B1 gene fragment using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs). The Escherichia coli DH5α strain was then transformed and selected on a solid lysogeny broth medium (LB medium: 10.0 g / L hypopeptone, 5.0 g / L yeast extract, 10 g / L NaCl, pH 6.8) containing 100 mg / L ampicillin. The selected DH5α strain was then grown by overnight shaking culture at 37°C in LB liquid medium containing 100 mg / mL ampicillin. MagExtractor was used to extract the collected cells. TMThe pREP1-Rs89B1 plasmid was extracted using Plasmid- (Toyobo Co., Ltd.) and purified. The obtained pREP1-Rs89B1 plasmid was transformed into Schizosaccharomyces pombe TN4 strain (genotype: h-leu1-32) by the lithium acetate method (Nucleic Acids Res. 1990 Nov 25; 18(22): 6485-6489.). The lithium acetate method is outlined below. The TN4 strain was cultured in YES liquid medium (5 g / L yeast extract, 20 g / L glucose, 0.225 g / L uracil, 0.225 g / L L-leucine, 0.225 g / L L-histidine, 0.225 g / L L-lysine, 0.225 g / L adenine) until OD600 = 0.5, harvested, and washed several times with sterilized water. The OD600 was then increased to 0.5 by adding 0.1 mol / L lithium acetate solution (pH 4.9). 600The cells were suspended at a concentration of 2–5 and incubated at 30°C for 60 minutes. 100 μL of the cell suspension was mixed with 1 μg of pREP1-Rs89B1 (15 μL) in TE buffer (pH 7.5, 10 mmol / L Tris-HCl with 1.0 mmol / L EDTA-2Na) and gently mixed. 290 μL of 50% (w / v) PEG 4000 preheated to 30°C was then added, mixed gently, and incubated at 30°C for 60 minutes. The cells were collected by centrifugation at 5000 × g for 2 minutes, the supernatant was removed, and the cells were suspended in 1.0 mL of 1 / 2 YE medium (0.25% yeast extract, 1.5% glucose, 30 μg / mL L-leucine) and resuspended at 30°C for 60 minutes. Then, 300μL was added to EMM-leu solid medium (20g / L Glucose, 5.0g / L NH4Cl, 2.2g / L Na2HPO4, 3.0g / L Potassium hydrogen phthalate, 0.225g / L Uracil, 0.225g / L L-Histidine, 0.225g / L L-Lysine, 0.225g / L Adenine, 1.1g / L MnCl2·6H2O, 147mg / L CaCl2·2H2O, 1.0g / L KCl, 40mg / L Na2SO4, 0.5mg / mL Boric acid, 0.4mg / L ZnSO4, 0.2mg / L FeCl3, 0.04mg / L Molybdic acid, 0.1mg / L KI, 0.04mg / L CuSO4·5H2O, 1mg / L Citric The transformants carrying pREP1-Rs89B1 (Rs89B1-expressing strains) were cultured and selected in a medium containing 1 mg / L of 1000 kJ / ml ... The resulting Rs89B1-expressing strain was cultured in EMM-leu liquid medium at 30°C for 72 or 90 hours with shaking for induction of expression. Cells were harvested by centrifugation at 5,000 × g for 5 minutes and washed with 50 mmol / L Tris-HCl buffer (pH 8.0). An equal volume of 5 mm glass beads and 5 mL of 50 mmol / L Tris-HCl buffer (pH 8.0) with 0.1 mmol / L phenylmethylsulfonyl fluoride (PMSF) were added to the washed cells. The cells were disrupted by vortexing for 30 seconds and then placed on ice for 30 seconds, 30 times, and centrifuged at 5,000 × g for 15 minutes to obtain a cell-free extract. 3.0 mol / L NaCl solution was added to the resulting cell-free extract to a final concentration of 500 mmol / L, and the extract was then loaded onto a Ni-IMAC column (BioRad). After elution with elution buffer (50 mmol / L Tris-HCl buffer (pH 8.0), 500 mmol / L imidazole, 500 mmol / L NaCl), the purified Rs89B1 protein was obtained by desalting on a PD-10 column (GE Healthcare) equilibrated with storage buffer (50 mmol / L Tris-HCl buffer (pH 6.5), 50% (v / v) glycerol).

[0049] To confirm the degree of protein purification, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the fraction purified using the Ni-IMAC column and the sample desalted using the PD-10 column. After electrophoresis, the protein was stained with CBB staining solution. The results are shown in Figures 1 and 2. Fractions 1 to 3 in Figure 1 are the fractions from the Ni-IMAC column. In Figure 2, Rs89B1 is the sample desalted using the PD-10 column, and M is the marker (SMobio PM1500). Fractions 1 and 2 were used for desalting using the PD-10 column. As shown in Figures 1 and 2, a band of the expected size (approximately 52 kDa) was confirmed in the fraction eluted with 500 mmol / L imidazole.

[0050] [Obtaining purified AtUGT71C1 enzyme] A gene (OptUGT71C1, SEQ ID NO: 29) codon-optimized for E. coli was prepared using the gene synthesis service of Eurofins Genomics. The OptUGT71C1 gene was inserted into the multicloning site (between Sac I and Hind III) of pET43.1b to create the pET43-AtUGT71C1 expression vector. The resulting expression vector was transformed into SHuffle T7 lysY Competent E. coli (New England Biolabs). The vector was then plated on LB solid medium containing 100 mg / L ampicillin and cultured at 30°C for 24 hours to select and obtain a strain expressing N-terminally NUS-tagged AtUGT71C1 (NUS-AtUGT71C1-expressing strain). The amino acid sequence of NUS-tagged AtUGT71C1 is shown in SEQ ID NO: 30. The obtained Nus-AtUGT71C1 expression strain was cultured overnight at 30°C in LB liquid medium containing 100 mg / L ampicillin to prepare a preculture solution. The preculture solution was inoculated at 10 mL / L into Terrific Broth (TB medium: 12 g / L hypopolypeptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4) containing 100 mg / L ampicillin, and incubated at 30°C until OD 660It was cultured with shaking until OD = 0.5. After adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.4 mmol / L, it was cultured with shaking at 16 °C for 16 hours. The obtained culture broth was centrifuged at 5,000×g for 10 minutes to collect the bacteria, and the obtained bacterial cells were washed with 50 mmol / L Tris-HCl buffer (pH 8.0). 50 mmol / L Tris-HCl buffer (pH 8.0) with 0.1 mmol / L PMSF was added to the washed bacterial cells, and cell disruption was performed by ultrasonic wave, followed by centrifugation at 5,000×g for 15 minutes to obtain a cell-free extract. A 3.0 mol / L NaCl solution was added to the obtained cell-free extract to a final concentration of 500 mmol / L, and it was applied to a Ni-IMAC column. After eluting with the above elution buffer, desalting was performed using a PD-10 column equilibrated with a storage buffer to obtain a purified NUS-AtUGT71C1 protein.

[0051] Using the fractions of the Ni-IMAC column, SDS-PAGE and CBB staining were performed in the same manner as for the acquisition of the Rs89B1 purified enzyme. The results are shown in Figure 3. As shown in Figure 3, bands of the expected size (about 120 kDa) could be confirmed in fractions 2 and 3. Desalting with the PD-10 column was performed using fraction 3.

[0052] <Cleavage of the NUS tag> To remove the NUS tag from the NUS-AtUGT71C1 protein, digestion with Thrombin was performed. Thrombin (derived from bovine serum, manufactured by Nacalai Tesque) equivalent to 1 U / mL was added to the purified NUS-AtUGT71C1 protein solution and incubated at 16 °C for 30 minutes. The results are shown in Figure 4. As shown in Figure 4, the NUS tag (about 55 kDa) could be removed from the NUS-AtUGT71C1 protein by treatment with Thrombin.

[0053] [Measurement of Rs89B1 activity] <Measurement of enzyme activity against catechol compounds> Using the enzyme reaction solution shown below, the presence or absence of the glycosyltransferase activity of Rs89B1 towards compounds having a catechol structure (hydroxytyrosol (HTY), dihydroxyphenyl lactic acid (DHPLA), and caffeic acid (HCA)) was confirmed. Enzyme reaction solution composition (50 μL): 242 μg / mL Rs89B1, 2.5 mmol / L UDP-Glc, 50 mmol / L Tris-HCl (pH 6.5), 1.0 mmol / L acceptor substrate (DHPLA, HCA, or HTY) The enzyme reaction was carried out at 35 °C for 30 minutes and the reaction was stopped by heating at 100 °C for 5 minutes. Thereafter, insoluble matters were removed by centrifugation at 15,000×g for 10 minutes. The supernatant was passed through a 0.22-μm nylon membrane filter (manufactured by Shimadzu GLC), and the filtrate was analyzed under the following <HPLC analysis conditions (A)>. The results of HPLC are shown in Fig. 5 (+Enzyme). As shown in Fig. 5, since the peaks of the product and the substrate (DHPLA or HTY) were very close to each other, after the above enzyme reaction, the substrate (DHPLA or HTY) was spiked into the reaction-stopped sample (+Enzyme), and HPLC analysis was performed. The results of HPLC are shown in Fig. 5 (STD + reaction mix). <HPLC analysis conditions (A)> HPLC: Nexera 40 series (manufactured by Shimadzu Corporation) HPLC column: Sunniest C18, inner diameter 2.0 mm × 150 mm (manufactured by Chromanik Technologies Co., Ltd.) Column temperature: 40 °C Flow rate: 0.200 mL / min Eluent A: 0.1% (v / v) trifluoroacetic acid (TFA) Eluent B: Acetonitrile with 0.1% (v / v) TFA Gradient (Time, %B): (0, 5) → (15, 50) Detection: Photodiode array (Nexera SPD-40M, absorbance 280 nm (bandwidth 4 nm; reference OFF)) Amount of analysis sample: 5 μL <Result> As shown in FIG. 5, Rs89B1 transferred glucose to DHPLA, HCA, and HTY.

[0054] [Measurement of AtUGT71C1 Activity] <Measurement of Enzyme Activity against HTY> Next, the enzyme activity of AtUGT71C1 against hydroxytyrosol was confirmed using the enzyme reaction solution composition shown below. The AtUGT71C1 protein solution prepared in the above <Cleavage of NUS Tag> was used as the enzyme solution. Enzyme reaction solution composition (50 μL): 1 mg / mL AtUGT71C1 enzyme solution, 2.5 mmol / L UDP-Glc, 50 mmol / L Tris-HCl (pH 6.5), 1.0 mmol / L HTY The enzyme reaction was carried out at 30° C. for 1 hour and stopped by heating at 100° C. for 5 minutes. Then, insoluble matters were removed by centrifugation at 15,000×g for 10 minutes. The supernatant was passed through a 0.22-μm nylon membrane filter (manufactured by Shimadzu GLC), and the filtrate was analyzed under the above <HPLC Analysis Conditions (A)>. The results are shown in FIG. 6. <Result> As shown in FIG. 6, AtUGT71C1 transferred glucose to HTY.

[0055] [NMR Measurement of Rs89B1 Reaction Product] The sugar transfer reaction to HTY by Rs89B1 was carried out to obtain the reaction product (HTY-Glc), and its structure was determined by NMR measurement. <Obtaining of Rs89B1 Reaction Product> The enzyme reaction was carried out with the following enzyme solution composition. The enzyme reaction was carried out at 30° C. overnight. Rs89B1 reaction solution (1.0 mL): 50 mmol / L Tris-HCl (pH 6.5), 0.53 mg / mL (0.54 mU / mL) Rs89B1, 100 mmol / L UDP-Glc, 50 mmol / L HTY The enzymatic reaction was stopped by adding 0.1% HCl, which was 1 / 10 the volume of the enzyme solution. A portion was diluted 100-fold and analyzed under the above <HPLC analysis conditions (A)>. As a result, the conversion rate was estimated to be 66.9% of the theoretical value (when all HTY was converted to the enzymatic reaction product). Subsequently, it was concentrated by freeze-drying, 1 mL of ethanol was added, and protein removal was performed by centrifugation at 15,000×g for 15 minutes. The supernatant was concentrated using a centrifugal evaporator, and the re-dissolved solution was separated under <HPLC analysis conditions (B)>. The peak near an elution time of 6.5 min (the peak indicated by the arrow in Fig. 7) was fractionated. The fractionated peak was concentrated using a centrifugal evaporator, re-dissolved in ultrapure water, and then freeze-dried to obtain 11.2 mg of the Rs89B1 reaction product. The results of the HPLC analysis are shown in Fig. 7. Note that the peak near an elution time of 7.5 min to 8.0 min is HTY, and the peak near 2.5 min is assumed to be a mixture of UDP / UDP-Glc / protein. <HPLC analysis conditions (B)> HPLC: Nexera 40 series (manufactured by Shimadzu Corporation) HPLC column: COSMOSIL 5C18 AR-II, inner diameter 4.6 mm × 250 mm (manufactured by Nacalai Tesque, Inc.) Column temperature: 40 °C Flow rate: 1.000 mL / min Eluent A: 0.1% (v / v) formic acid Eluent B: Acetonitrile with 0.1% (v / v) formic acid Isocratic: 7.5% eluent B Detection: Photodiode array (Nexera SPD-40M, absorbance 280 nm (bandwidth 4 nm; reference OFF)) Analysis sample volume: 40 μL

[0056] <NMR measurement> In order to determine the structure of the compound generated by Rs89B1, NMR measurement was performed. The Rs89B1 reaction product obtained in <Obtaining the Rs89B1 reaction product> was dissolved in heavy water, freeze-dried several times, and then dissolved in 0.75 mL of Methanol-D4 + 0.03% TMS (manufactured by Euriso-top).1 1H-NMR was acquired at 400 MHz using a JEOL ECZ400. The chemical shift analysis was performed using JEOL Delta6.2.0. The obtained 1 1H-NMR chemical shifts are as follows. Chemical shift (with TMS at 0 ppm) 1H-NMR (400 MHz, METHANOL-D4) δ 7.10 (d, J = 8.2 Hz, 1H-5), 6.73 (d, J = 1.8 Hz, 1H-2), 6.64 (dd, J = 8.2, 2.3 Hz, 1H-6), 4.69 (t, J = 3.7 Hz, 1H-1'), 3.88 (t, J = 5.5 Hz, 1Ha-6'), 3.73 - 3.67 (m, 1Hb-6', 2H-8), 3.51 - 3.34 (m, 4H-2'-5'), 2.71 (t, J = 7.1 Hz, 2H-7)

[0057] By NMR measurement, the Rs89B1 reaction product was hydroxytyrosol 4-O-glucoside (HTY-4-O-Glc) in which glucose was bound to the hydroxyl group at the 4-position (para-position) of hydroxytyrosol. Also, from the HPLC analysis results in Fig. 7, it was found that Rs89B1 selectively introduced glucose at the 4-position of HTY. By the sugar transfer reaction of HTY by Rs89B1, HTY-4-O-Glc could be selectively produced.

[0058] The lyophilized product of the Rs89B1 reaction product obtained in <Obtaining the Rs89B1 Reaction Product> was redissolved in ultrapure water and analyzed under <HPLC Analysis Conditions (C)>. The HPLC results are shown in Fig. 8 (lower part). As shown in Fig. 8, the content of HTY-4-O-Glc in the Rs89B1 reaction product was 98 wt% or more. The Rs89B1 reaction product contained almost no phenolic compounds other than HTY-4-O-Glc (e.g., HTY and HTY-3-O-Glc), and the content was 1 wt% or less. Also, the solution obtained by redissolving the lyophilized product of the Rs89B1 reaction product in ultrapure water was colorless and transparent. <HPLC Analysis Conditions (C)> HPLC: Nexera 40 series (manufactured by Shimadzu Corporation) HPLC column: Sunniest C18, inner diameter 2.0 mm × 150 mm (manufactured by Chromanik Technologies Co., Ltd.) Column temperature: 40 °C Flow rate: 0.200 mL / min Eluent A: 0.1% (v / v) TFA Eluent B: Acetonitrile with 0.1% (v / v) TFA Isocratic: 7.5% Eluent B Detection: Photodiode array (Nexera SPD-40M, absorbance 280 nm (bandwidth 4 nm; reference OFF)) Analytical sample volume: 2 μL

[0059] [Obtaining and Structure Determination of AtUGT71C1 Reaction Product] The sugar transfer reaction of HTY by AtUGT71C1 was carried out to obtain the reaction product (HTY-Glc). <Obtaining of AtUGT71C1 Reaction Product> The enzyme reaction was carried out with the following enzyme solution composition. The enzyme reaction was carried out at 30 °C overnight (O / N). Rs89B1 reaction solution (0.5 mL): 50 mmol / L Tris-HCl (pH 6.5), 1.22 mg / mL AtUGT71C1 enzyme solution, 100 mmol / L UDP-Glc, 25 mmol / L HTY The enzymatic reaction was stopped by adding 0.1% HCl, which was 1 / 10 the volume of the enzyme solution. A portion was diluted 100-fold and analyzed under the above <HPLC analysis conditions (A)>. As a result, the conversion rate was estimated to be 38.1% of the theoretical value (when all HTY was converted to the enzymatic reaction product). Subsequently, it was concentrated by freeze-drying, 1 mL of ethanol was added, and protein removal was performed by centrifugation at 15,000×g for 15 minutes. The supernatant was concentrated using a centrifugal evaporator, and the re-dissolved solution was separated under the above <HPLC analysis conditions (B)>. The peak near an elution time of 7.0 min (the peak indicated by the arrow in Fig. 9) was fractionated. The fractionated peak was concentrated using a centrifugal evaporator, re-dissolved in ultrapure water, and then freeze-dried to obtain 1.4 mg of the AtUGT71C1 reaction product.

[0060] Based on the HPLC elution position and Eng-Kiat Lim et al., (Biochem J. 2003 Aug 1; 373(Pt 3): 987-992), the AtUGT71C1 reaction product was estimated to be HTY-3-O-Glc in which glucose was bound to the hydroxyl group at the 3-position (meta-position) of hydroxytyrosol. Also, from the HPLC analysis results in Fig. 9, it was found that AtUGT71C1 selectively introduced glucose at the 3-position of HTY.

[0061] The freeze-dried product of the AtUGT71C1 reaction product obtained in <Obtaining the AtUGT71C1 Reaction Product> was re-dissolved in ultrapure water and analyzed under the above <HPLC analysis conditions (C)>. The HPLC results are shown in Fig. 8 (lower part).

[0062] [Stability Test of HTY-4-O-Glc and HTY-3-O-Glc] A test was conducted to confirm the stability of HTY-4-O-Glc (Rs89B1 reaction product) and HTY-3-O-Glc (AtUGT71C1 reaction product) obtained as described above. [Measurement of Absorption Spectrum of Discolored HTY Solution] A 20 mmol / L HTY solution was incubated (left standing) at 60 °C for 24 hours in a 50 mmol / L Tris-HCl (pH 8.0) solution. The absorption spectra of the HTY solution that turned reddish-brown upon incubation and the HTY solution before incubation were measured using an Eppendorf BioSpectrometer (manufactured by Eppendorf) and a μCuvette (manufactured by Eppendorf) (Figure 10). As a result, since it was found that a compound having an absorption wavelength around 500 nm was generated in the HTY solution that turned reddish-brown, the subsequent compound stability was evaluated by monitoring this absorption wavelength.

[0063] <Stability test> The stability test was performed on HTY, HTY-4-O-Glc, and HTY-3-O-Glc. 100 μL of a 50 mM Tris-HCl (pH 8.0) solution containing 15.9 mmol / L HTY, 19.9 mmol / L HTY-3-O-Glc, or 20.1 mmol / L HTY-4-O-Glc was prepared, and these solutions were incubated at 60 °C. After 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 22 hours, 2.5 μL samples were taken, and the absorbance at 500 nm was measured using an Eppendorf BioSpectrometer and a μCuvette. In addition, the samples after 22 hours were analyzed under the above <HPLC analysis conditions (C)>. The results are shown in Figures 11 and 12. <Results> Figure 11 shows the measurement results of the absorbance at 500 nm, and Figure 12 shows the results of the HPLC analysis. As shown in Figure 11, for HTY, a rapid increase in absorbance indicating rapid polymerization was observed immediately after the start of incubation. In a solution containing a high concentration of HTY-3-O-Glc, an increase in absorbance and reddish-brown coloring were also confirmed upon incubation. That is, HTY-3-O-Glc (the reaction product of AtUGT71C1) was slowly hydrolyzed by incubation. This was also confirmed from the HPLC analysis results in Figure 12. On the other hand, no coloring was confirmed in the solution containing a high concentration of HTY-4-O-Glc, and hydrolysis of the glycoside was not confirmed either. Therefore, HTY-4-O-Glc was very stable compared to HTY and HTY-3-O-Glc.

[0064] [Degradation of HTY-4-O-Glc by yeast cell lysate supernatant] As evidence that HTY-4-O-Glc is expected to exhibit the same function as HYT, a model experiment using yeast cell lysate supernatant was conducted. HTY-4-O-Glc was prepared by performing an enzymatic reaction on 0.2 mL of an enzymatic reaction solution (150 mM HTY, 150 mM UDP-Glc, 0.28 μg / μL (0.31 μU / μL) Rs89B1) at 30 °C overnight, fractionating it under the above <HPLC analysis conditions (B)>, and freeze-drying (yield: 38.2%). The obtained HTY-4-O-Glc was dissolved in phosphate-buffered saline (PBS(-)) to a concentration of 2 mmol / L. The mock sample used was the lysate of Schizosaccharomyces pombe TN4 strain cultured overnight in 5 mL of YES medium. The cultured cells were collected by centrifugation at 5,000×g for 10 minutes, washed with PBS(-), and then cell disruption was carried out by adding glass beads of φ5.0 mm equivalent to the amount of cells and vortexing in 1 mL of PBS(-). Subsequently, centrifugation was performed at 15,000×g for 10 minutes, and the supernatant was collected as cell lysis. The obtained cell lysis (cell-free extract) and the HTY-4-O-Glc solution were mixed in equal amounts and incubated at 35 °C (cell lysis-added sample). Also, a sample with PBS(-) added instead of cell lysis as a control (control sample) was incubated in the same manner. After 0, 2.5, 5, 10, and 15 minutes, 10 μL aliquots were taken and the proteins in the cell lysis were denatured by boiling at 10 °C for 10 minutes. Then, centrifugation was performed and the supernatant was analyzed under the above <HPLC analysis conditions (A)>. A part of the results is shown in Figure 13. [Results] The area value of HTY-4-O-Glc at 0 h incubation for the control sample and the cell lysis-added sample was set at 100%, and the relative area values ​​were plotted by dividing the area value at each sampling time. The results are shown in Figure 14. As shown in Figure 14, in the cell lysis-added sample (+ cell lysis), HTY-4-O-Glc was rapidly degraded upon addition of cell lysis, decreasing by half after 2.5 min and reaching the limit of detection (LOD) after 10 min. Furthermore, HPLC analysis (Figure 13) revealed that HTY-4-O-Glc was produced by the degradation of HTY-4-O-Glc upon addition of cell lysis. This degradation is thought to be due to the action of a glycosyl hydrolase (β-glucosidase) contained in the cell lysis. β-glucosidase is found in a wide range of organisms. When HTY-4-O-Glc is used by humans, animals, plants, and microorganisms, glucose is released from HTY-4-O-Glc by the action of glycosyl hydrolases, presumably resulting in a function similar to that of HTY (Karkovic Markovic A, Toric J, Barbaric M, Jakobusic Brala C. Hydroxytyrosol, Tyrosol and Derivatives and Their Potential Effects on Human Health. Molecules. 2019 May 24;24(10):2001. doi: 10.3390 / molecules24102001. PMID: 31137753; PMCID: PMC6571782.).

Claims

1. A composition containing hydroxytyrosol 4-O-glucoside, The composition has a hydroxytyrosol 4-O-glucoside content of 85% by weight or more.

2. 2. The composition according to claim 1, which has an absorbance at a wavelength of 500 nm of 0.1 or less after being left to stand in a Tris-HCl (pH 8.0) solution at 60° C. for 6 hours.

3. The composition according to claim 1 or 2, wherein the content of hydroxytyrosol relative to the hydroxytyrosol 4-O-glucoside is 5% by weight or less.

4. 1. A method for producing hydroxytyrosol 4-O-glucoside, comprising: A method comprising mixing hydroxytyrosol, UDP-glucose, and UDP-glucosyltransferase 89B1 or a homolog thereof to obtain hydroxytyrosol 4-O-glucoside.

5. 5. The method of claim 4, further comprising recovering the resulting hydroxytyrosol 4-O-glucoside.

6. The composition according to claim 1 or 2, comprising hydroxytyrosol 4-O-glucoside obtained by the method according to claim 4.

7. The composition according to claim 1 or 2, which is used for at least one selected from the group consisting of ingredients for foods and beverages, ingredients for supplements, cosmetic materials, polymerization inhibitors, and animal feed.

8. 3. A food composition, a beverage composition, a health food, a supplement, a quasi-drug, a pharmaceutical composition, a cosmetic, a polymerization inhibitor, or an animal feed, which contains the composition according to claim 1 or 2.

Images