Method for producing material for improving intestinal environment

Abstract

To provide an oral composition and a method for producing the same, enabling efficient generation of monomeric glutamic acid in a lower digestive tract.SOLUTION: A method for producing γ-oligoglutamic acid is provided, comprising: (1) hydrolyzing γ-polyglutamic acid in an acidic or alkaline aqueous solution to obtain an aqueous solution containing a hydrolysate; and (2) neutralizing the aqueous solution containing the hydrolysate obtained in step (1) and removing a precipitate of the formed salt. An oral composition, containing γ-oligoglutamic acid is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel method for producing γ-oligoglutamic acid, which can be used as a material for improving the intestinal environment. [Background technology]

[0002] Patent Documents 1 and 2 disclose foods that improve the intestinal environment by promoting the growth of lactic acid bacteria and bifidobacteria through the combined use of γ-polyglutamic acid and oligosaccharides. After ingestion, γ-polyglutamic acid and oligosaccharides reach the lower gastrointestinal tract without being broken down. There, monomeric glutamic acid is enzymatically and chemically cleaved from γ-polyglutamic acid by digestive juices or enzymes secreted by intestinal bacteria. The simultaneous presence of this glutamic acid and oligosaccharides in the intestine is thought to have a prebiotic effect. However, γ-polyglutamic acid has low enzymatic and chemical decomposition efficiency, and most of it is excreted in the feces, so there is a problem in that a large amount of γ-polyglutamic acid needs to be ingested to obtain sufficient effects. Another problem is that monomeric glutamic acid is metabolized after ingestion and does not reach the lower gastrointestinal tract where it can affect the intestinal flora.

[0003] Patent Document 3 discloses a method for producing low-molecular-weight γ-polyglutamic acid, which comprises heat-treating a culture medium of a γ-polyglutamic acid-producing bacterium that has accumulated γ-polyglutamic acid under acidic or alkaline conditions. However, in this production method, in order to recover degraded γ-polyglutamic acid from a reaction solution containing degraded γ-polyglutamic acid, precipitation is caused by leaving the reaction solution at pH 1.0 to 1.5 for 2 to 3 days, or by adding an organic solvent such as methanol, isopropyl alcohol, or acetone in an amount 2 to 10 times the reaction solution, followed by purification procedures such as desalting. Therefore, there are many practical problems with this method, as it takes a long time to achieve precipitation and requires a large amount of organic solvent, which has a high environmental impact. Therefore, it is desired to develop a method for producing γ-polyglutamic acid simply and easily by a filtration step and a powdering operation without using a large amount of organic solvent. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2019 / 070061 [Patent Document 2] US Patent Publication No. 2020 / 237790 [Patent Document 3] Japanese Patent Application Publication No. 7-316286 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an oral composition that can efficiently produce glutamic acid in the lower gastrointestinal tract and a method for producing the same. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems and have found that peptides obtained by breaking down γ-polyglutamic acid (i.e., γ-oligoglutamic acid) can efficiently produce glutamic acid in the lower gastrointestinal tract compared to the raw material γ-polyglutamic acid (see Test Example 4 below). On the other hand, γ-oligoglutamic acid has a molecular weight of several hundred to several thousand, and purification and desalting using a UF membrane removes even the desired molecular weight fraction, making it difficult to obtain γ-oligoglutamic acid in a high yield. Furthermore, precipitation using an organic solvent results in the precipitation of contaminating salts, making it impossible to carry out the desired purification procedure. Therefore, the production of γ-oligoglutamic acid requires a purification procedure different from that used for γ-polyglutamic acid, which has a molecular weight of tens of thousands or more. The present inventors have found that γ-oligoglutamic acid can be produced simply and with a high yield without using, for example, molecular weight fractionation and desalting using a UF membrane or organic solvents, by hydrolyzing γ-polyglutamic acid by treating it with an acid or alkali, and then neutralizing an aqueous solution containing the obtained hydrolysate to remove the resulting precipitate. Based on these findings, the present inventors conducted further studies and completed the present invention.

[0007] That is, the present invention is as follows. [1] (1) a step of hydrolyzing γ-polyglutamic acid in an acidic or alkaline aqueous solution to obtain an aqueous solution containing a hydrolysate; (2) A step of neutralizing the aqueous solution containing the hydrolyzate obtained in (1) and removing the resulting salt precipitate. A method for producing γ-oligoglutamic acid, comprising: [2] The method according to [1] above, wherein the hydrolysis in the step (1) is carried out in an aqueous solution of an inorganic acid. [3] The method according to [2] above, wherein the aqueous inorganic acid solution is an aqueous sulfuric acid solution. [4] The method according to [3] above, wherein the concentration of the aqueous sulfuric acid solution is 0.9 to 1 mol / L. [5] The method according to any one of the above [2] to [4], wherein the neutralization in the step (2) is carried out using an alkaline earth metal hydroxide. [6] The method according to any one of the above [2] to [4], wherein the neutralization in the step (2) is carried out using calcium hydroxide. [7] The method according to any one of the above [1] to [6], wherein the γ-oligoglutamic acid contains 2 to 15 glutamic acid residue molecules. [8] An oral composition containing gamma-oligoglutamic acid and oligosaccharides. [9] The composition according to the above [8], wherein the γ-oligoglutamic acid contains 2 to 15 molecules of glutamic acid residues.

[10] The composition described in [8] or [9] above, which is for improving the intestinal environment. [Effects of the Invention]

[0008] The method for producing γ-oligoglutamic acid of the present invention allows for the simple and efficient production of γ-oligoglutamic acid, and is advantageous in that it can be carried out without the use of organic solvents or without desalting or molecular weight fractionation using a UF membrane.

[0009] The oral composition containing γ-oligoglutamic acid of the present invention can efficiently (i.e., with high degradation efficiency) generate glutamic acid in the lower gastrointestinal tract of a subject who ingests it, compared to conventional oral compositions containing γ-polyglutamic acid. Therefore, the oral composition containing γ-oligoglutamic acid of the present invention can be expected to have the same effect as conventional oral compositions containing γ-polyglutamic acid, even with a smaller amount of the active ingredient (γ-oligoglutamic acid). As a result, cost reductions due to the reduced dosage and reduced impact on taste when incorporated into a composition can be expected.

[0010] Conventional oral compositions containing γ-polyglutamic acid have been limited in their product forms (dosage forms) due to the physical properties of γ-polyglutamic acid (high solution viscosity, high hygroscopicity, tendency to form lumps when dissolved) (for example, it has been difficult to make them into a beverage form). However, the oral compositions containing γ-oligoglutamic acid of the present invention are expected to be able to expand their product forms (dosage forms) (for example, they can be made into a beverage form) due to the physical properties of γ-oligoglutamic acid (low solution viscosity, low hygroscopicity, tendency to form lumps when dissolved).

[0011] When used in combination with an oligosaccharide, the oral composition containing γ-oligoglutamic acid of the present invention can be expected to have a prebiotic effect similar to that of the combined oral composition containing γ-polyglutamic acid and oligosaccharide described in the aforementioned Patent Documents 1 and 2. When the oral composition containing γ-oligoglutamic acid of the present invention is used in combination with an oligosaccharide, a small amount of the active ingredient (γ-oligoglutamic acid) can be used to achieve an effect similar to that of the combined oral composition containing γ-polyglutamic acid and oligosaccharide described in the aforementioned Patent Documents 1 and 2. As a result, cost reductions can be achieved by reducing the dosage, and the impact on taste when the composition is incorporated can be suppressed. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the relationship between the concentration of the aqueous sulfuric acid solution during hydrolysis and the sulfate ion and Ca ion contents in the obtained low-molecular-weight γ-PGA powder in Examples 1 to 4. [Figure 2] FIG. 2 shows reverse-phase HPLC chromatograms of the decomposed γ-PGAs obtained under different decomposition conditions in Examples 1 to 4, the synthetic standard having 2 to 15 residues obtained in Reference Example 1, and the UF membrane method sample obtained in Comparative Example 1. [Figure 3] FIG. 3 shows the relationship between the concentration of aqueous sulfuric acid solution during hydrolysis and the content of degraded γ-PGA with 15 or less residues in the degraded γ-PGA in Examples 1 to 4. [Figure 4] 4 shows the results of the evaluation of the decomposition efficiency of the reduced molecular weight γ-PGA powder of Example 1 using enzyme fractions derived from each strain in Test Example 3. Each plot shows the ratio of the decomposition efficiency of the reduced molecular weight γ-PGA powder of Example 1 to that of commercially available γ-PGA. [Figure 5] FIG. 5 shows the glutamic acid concentration in the cecal contents in Test Example 4 (mouse test). DETAILED DESCRIPTION OF THE INVENTION

[0013] The method for producing γ-oligoglutamic acid of the present invention includes the following steps (1) and (2). (1) A step of hydrolyzing γ-polyglutamic acid in an acidic or alkaline aqueous solution to obtain an aqueous solution containing a hydrolysate (2) A step of neutralizing the aqueous solution containing the hydrolyzate obtained in (1) and removing the resulting salt precipitate. In the production method of the present invention, γ-polyglutamic acid is hydrolyzed to reduce its molecular weight to obtain a reduced γ-polyglutamic acid (including γ-oligoglutamic acid). In this specification, γ-oligoglutamic acid is a polymer in which 2 to 29 glutamic acid units are bonded.

[0014] [Step (1): Hydrolysis step] In step (1), the γ-polyglutamic acid to be hydrolyzed (sometimes referred to herein as starting γ-polyglutamic acid or starting γ-PGA) is a polymer having 30 or more bonded glutamic acid units (for example, a polymer having 30 to 100,000 bonded glutamic acid units). γ-Polyglutamic acid is also called polyglutamic acid. In the present invention, the raw material γ-polyglutamic acid is preferably a γ-polyglutamic acid that is a polymer (high molecular weight substance) in which about 30 to about 5,000 D-glutamic acid and L-glutamic acid are bonded together in a ratio of about 8:2. The raw material γ-polyglutamic acid may be a commercially available product (for example, Caltake (trade name) manufactured by Ajinomoto Co., Inc.).

[0015] In step (1), the hydrolysis of the starting γ-polyglutamic acid is carried out in an acidic or alkaline aqueous solution. In step (1), when the hydrolysis of the starting γ-polyglutamic acid is carried out in an acidic aqueous solution, the acidic aqueous solution may be, for example, an inorganic acid aqueous solution (e.g., an aqueous sulfuric acid solution), and preferably an aqueous sulfuric acid solution. In this specification, the inorganic acid is also referred to as a mineral acid. In step (1), when hydrolysis of the starting γ-polyglutamic acid is carried out in an acidic aqueous solution (e.g., an inorganic acid aqueous solution (e.g., an aqueous sulfuric acid solution)), the concentration of the acid (e.g., an inorganic acid (e.g., sulfuric acid)) in the aqueous solution is, for example, 0.1 to 6 mol / L, preferably 0.5 to 2 mol / L, and more preferably 0.9 to 1 mol / L. In step (1), when hydrolysis of the starting γ-polyglutamic acid is carried out in an aqueous sulfuric acid solution, a γ-oligoglutamic acid containing 50% or more of γ-oligoglutamic acid having 15 or fewer residues can be obtained by using a 0.9 to 1 mol / L aqueous sulfuric acid solution.

[0016] In step (1), when the hydrolysis of the starting γ-polyglutamic acid is carried out in an alkaline aqueous solution, examples of the alkaline aqueous solution include alkaline earth metal hydroxides (e.g., calcium hydroxide, barium hydroxide), and an aqueous calcium hydroxide solution is preferred, with an aqueous calcium hydroxide solution being more preferred. In step (1), when hydrolysis of the starting γ-polyglutamic acid is carried out in an alkaline aqueous solution (e.g., an aqueous solution of an alkaline earth metal hydroxide), the concentration of the alkali (e.g., alkaline earth metal hydroxide) in the aqueous solution is, for example, 0.01 to 6 mol / L, 0.01 to 5 mol / L, 0.01 to 4 mol / L, or 0.01 to 3 mol / L, preferably 0.01 to 2 mol / L, more preferably 0.02 to 2 mol / L, and even more preferably 0.02 to 1 mol / L.

[0017] The temperature of the hydrolysis reaction in step (1) is, for example, 20 to 120°C, preferably 60 to 100°C, 65 to 100°C, more preferably 70 to 90°C, even more preferably 75 to 90°C, and still more preferably 80 to 90°C. The time for the hydrolysis reaction in step (1) is, for example, 0.1 to 24 hours, preferably 4 to 10 hours, and more preferably 5 to 7 hours.

[0018] In step (1), the starting γ-polyglutamic acid can be hydrolyzed in an acidic or alkaline aqueous solution by a known method. For example, an acidic or alkaline aqueous solution having the above-mentioned concentration can be added to the starting γ-polyglutamic acid powder and stirred to carry out the hydrolysis reaction, thereby obtaining an aqueous solution containing a hydrolysate.

[0019] [Step (2): Neutralization and sediment removal step] In step (2), the aqueous solution containing the hydrolysate obtained in step (1) is neutralized. When the starting γ-polyglutamic acid is hydrolyzed in an acidic aqueous solution in step (1), the aqueous solution containing the hydrolysate in step (2) can be neutralized using an alkali. In step (2), when an alkali is used to neutralize the aqueous solution containing the hydrolyzate obtained in step (1), examples of the alkali include hydroxides of alkaline earth metals (e.g., calcium hydroxide, barium hydroxide), and calcium hydroxide is preferred. In step (2), when an alkali is used to neutralize the aqueous solution containing the hydrolysate obtained in (1), the amount of alkali used is an amount that adjusts the aqueous solution containing the hydrolysate to, for example, pH 3 to 11 (preferably pH 4 to 7, more preferably pH 4 to 5).

[0020] When the starting γ-polyglutamic acid is hydrolyzed in an alkaline aqueous solution in step (1), the aqueous solution containing the hydrolysate in step (2) can be neutralized with an acid. In step (2), when an acid is used to neutralize the aqueous solution containing the hydrolysate obtained in step (1), examples of the acid include inorganic acids (such as sulfuric acid), and sulfuric acid is preferred. In step (2), when an acid (e.g., an inorganic acid (e.g., sulfuric acid)) is used to neutralize the aqueous solution containing the hydrolysate obtained in (1), the amount of acid used is an amount that adjusts the aqueous solution containing the hydrolysate to, for example, pH 3 to 11 (preferably pH 4 to 7, more preferably pH 4 to 5).

[0021] In step (2), after the addition of the acid or alkali for the neutralization, the aqueous solution containing the hydrolyzate is stirred to sufficiently form a precipitate (a salt of the acid and alkali). In step (2), the stirring temperature for forming a precipitate is, for example, 5 to 80°C, preferably 5 to 30°C, and more preferably 5 to 25°C. The stirring time for forming a precipitate in step (2) is, for example, 0.1 to 24 hours, preferably 0.5 to 6 hours, and more preferably 0.5 to 2 hours.

[0022] In step (2), after the stirring, the resulting precipitate is removed. The removal of the precipitate can be carried out by a known method, for example, by filtration through a 0.1 to 10 μm filter.

[0023] In step (2), the γ-oligoglutamic acid-containing solution obtained after removing the precipitate can be used as a food or food ingredient as is, but may be further purified, freeze-dried, or the like by known methods.

[0024] In the present invention, the combination of an "inorganic acid" and an "alkaline earth metal hydroxide" (a combination of an "inorganic acid" used in the hydrolysis in step (1) and an "alkaline earth metal hydroxide" used in the neutralization in step (2), or a combination of an "alkaline earth metal hydroxide" used in the hydrolysis in step (1) and an "inorganic acid" used in the neutralization in step (2)) is preferably a combination in which the solubility of a salt formed by neutralization of an acid (inorganic acid) with an alkali (alkaline earth metal hydroxide) in water at 25°C is 1 g / 100 g water or less. Examples of salts formed by neutralization of an acid and an alkali that have a solubility in water at 25°C of 1 g / 100 g water or less include calcium sulfate and barium sulfate. In the present invention, the combination of "inorganic acid" and "hydroxide of alkaline earth metal" is preferably a combination of sulfuric acid and calcium hydroxide, or a combination of sulfuric acid and barium hydroxide, and more preferably a combination of sulfuric acid and calcium hydroxide.

[0025] A preferred embodiment of the production method of the present invention is as follows. (1) hydrolyzing γ-polyglutamic acid in an aqueous sulfuric acid solution to obtain an aqueous solution containing a hydrolysate; and (2) A step of neutralizing the aqueous solution containing the hydrolyzate obtained in (1) with calcium hydroxide and removing the resulting calcium sulfate precipitate. A method for producing γ-oligoglutamic acid, comprising:

[0026] According to the production method of the present invention, γ-oligoglutamic acid (particularly γ-oligoglutamic acid containing 2 to 15 molecules of glutamic acid residues) can be efficiently produced. As used herein, "efficient production" refers to the ability to produce γ-oligoglutamic acid (particularly γ-oligoglutamic acid containing 2 to 15 glutamic acid residues) in good yield. Degraded γ-PGA refers to all γ-PGAs that have been subjected to a degrading treatment, and γ-oligoglutamic acid particularly refers to γ-glutamyl peptides containing 2 to 29 glutamine residues. According to the production method of the present invention, a low molecular weight γ-PGA powder can be obtained that contains γ-oligoglutamic acid having 2 to 29 glutamic acid residue molecules (particularly, γ-oligoglutamic acid having 2 to 15 glutamic acid residue molecules) in an area percentage determined by HPLC of preferably 19% or more, 20% or more, more preferably 30% or more, even more preferably 40% or more, even more preferably 50% or more, and particularly preferably 60% or more. In the present invention, the proportion of γ-oligoglutamic acid having a certain number of glutamic acid residues in a low molecular weight γ-PGA powder can be confirmed, for example, by the method described in Test Example 2 below or a method similar thereto.

[0027] The present invention also relates to an oral composition containing γ-oligoglutamic acid and oligosaccharides as active ingredients (sometimes referred to herein as the oral composition of the present invention). In the oral composition of the present invention, the γ-oligoglutamic acid may be produced not only by the production method of the present invention described above, but also by a known method (for example, the method described in Production Example 1 below). In the oral composition of the present invention, the γ-oligoglutamic acid preferably contains 2 to 29 molecules, more preferably 5 to 20 molecules, and even more preferably 2 to 15 molecules of glutamic acid residues. In the oral composition of the present invention, examples of oligosaccharides include coffee bean mannooligosaccharides, lactoferrin oligosaccharides, galactooligosaccharides, fructooligosaccharides, soybean oligosaccharides, xylooligosaccharides, and isomaltooligosaccharides, with fructooligosaccharides, coffee bean mannooligosaccharides, isomaltooligosaccharides, and galactooligosaccharides being preferred. Oligosaccharides can be used alone or in combination of two or more.

[0028] In the present invention, the oral composition may be in any form, and examples thereof include powders, granules (including fine granules), tablets, hard capsules, soft capsules, liquids (e.g., solutions, suspensions, emulsions, liquid seasonings (e.g., liquid sweeteners)), beverages, jellies, puddings, yogurt, candies, chewing gums, etc. The oral composition of the present invention is preferably a food product. In this specification, the term "food" is a concept that broadly encompasses anything that can be taken orally (excluding pharmaceuticals), and includes not only so-called "foods" but also health supplements, health functional foods (e.g., foods for specified health uses, foods with functional claims), supplements, etc.

[0029] In the oral composition of the present invention, the content of γ-oligoglutamic acid is preferably 0.01 to 50% by weight, more preferably 0.05 to 30% by weight, and even more preferably 0.1 to 10% by weight. In the oral composition of the present invention, the weight ratio of oligosaccharide to γ-oligoglutamic acid (oligosaccharide:γ-oligoglutamic acid) is preferably 1:0.05-20, more preferably 1:0.1-10, even more preferably 1:0.1-5, still more preferably 1:0.1-3, and particularly preferably 1:0.2-2.

[0030] The oral composition of the present invention is expected to be effective in improving the intestinal environment when used in combination with the other active ingredient, oligosaccharide, because the active ingredient, gamma-oligoglutamic acid (particularly gamma-oligoglutamic acid containing 2 to 15 glutamic acid residues), reaches the lower gastrointestinal tract without being broken down after ingestion and can efficiently produce glutamic acid in the lower gastrointestinal tract.

[0031] As used herein, "improving the intestinal environment" refers to a relative increase in the number of lactic acid bacteria and / or bifidobacteria present in the lower gastrointestinal tract. A relative increase refers to promoting the dominance of lactic acid bacteria and / or bifidobacteria in the intestinal flora. The dominance of lactic acid bacteria and bifidobacteria in the intestinal flora can be confirmed by measuring the amount of lactic acid bacteria and bifidobacteria in feces using quantitative PCR or the like. As used herein, "lower gastrointestinal tract" means the ileum, cecum, colon, and rectum. [Example]

[0032] The present invention will be explained in more detail below with reference to examples and test examples, but the present invention is not limited to these examples and test examples. The instruments and measurement conditions used in the analytical methods (reverse phase HPLC, ion chromatography, amino acid analysis) in the following Examples, Test Examples, and Reference Examples are shown below. In the following Examples, Test Examples and Reference Examples, M represents mol / L. The following abbreviations may be used in this specification. Glu: glutamic acid γ-OGA: γ-oligoglutamic acid γ-PGA: γ-polyglutamic acid

[0033] [Reversed-phase HPLC (instrument, measurement conditions)] Analysis was performed under the following conditions and with the sequence shown in Table 1. Equipment: SHIMADZU PROMINENCE series Column: Symmetry300 C4 5μm, 4.6×50 mm, Waters Column temperature: 30℃ Flow rate: 0.8 mL / min Detection: 220 nm Injection volume: 10 μL Buffer A: 0.1% trifluoroacetic acid in water Buffer B: 0.1% trifluoroacetic acid acetonitrile

[0034] [Table 1]

[0035] [Ion chromatography (device, measurement conditions)] Equipment: Tosoh IC-2010 Measurement conditions: Equipment standard conditions [Anion Chromatography] Column: TSKgel (registered trademark) superIC-Anion HSII Buffer: 3.8 mM NaHCO3 + 3.0 mM NcCO Flow rate: 1.5 mL / min Time: 30 minutes Temperature: 40℃ Detector: Electrical conductivity detector [Cation Chromatography] Column: TSKgel® superIC-Cation HS II Buffer: 4 mM methanesulfonic acid + 1.1 mM 18-crown-6-ether Flow rate: 1.2 mL / min Time: 8 minutes Temperature: 40℃ Detector: Electrical conductivity detector

[0036] [Amino acid analysis (instrument, measurement conditions)] Equipment: HITACHI LA8080 Amino Acid Analyzer "Total Glu content" A 100 mg sample powder was placed in a glass pressure tube, 20 mL of 6 M hydrochloric acid was added, and the tube was then capped and heated in a heating oven at 110 °C for 22 hours. The tube was removed from the oven and allowed to cool, and the sample was then rinsed into a recovery flask. The solvent was removed using an evaporator, and the residue was dissolved in 0.02 M hydrochloric acid and diluted to 100 mL. The sample was used for analysis, and the total Glu content was calculated using a Hitachi LA8080 Amino Acid Analyzer. "Monomer Glu content" The target sample powder was dissolved in ultrapure water and quantified using a HITACHI LA8080 Amino Acid Analyzer to calculate the monomer Glu content. "γ-PGA content", "Low molecular γ-PGA content" For the target sample, the Glu content was calculated by subtracting the monomeric Glu content from the total Glu content, and multiplying the result by 0.8775. "Moisture content" Approximately 1 g of the target sample was heated at 105°C for 6 hours, and the weight was calculated from the ratio of the weight before and after heating. "Sulfate ion content" and "Ca ion content" The target sample was dissolved in ultrapure water, and the amount was determined by anion chromatography or cation chromatography, and the amount was calculated.

[0037] Table 2 shows the reagents used in the following Examples, Test Examples, and Reference Examples.

[0038] [Table 2]

[0039] [Production Example 1] Synthesis of a 4-residue peptide (Step 1) Synthesis of Compound 1 To a solution of Cbz-glutamic acid α-t-butyl ester (0.675 g, 2.00 mmol) in acetonitrile (20 mL), HOAt (0.28 g, 2.06 mmol) and WSC·HCl (0.39 g, 2.03 mmol) were added and stirred at room temperature for 1 hour. To the reaction mixture, glutamic acid α-,γ-di-t-butyl ester hydrochloride (0.62 g, 2.10 mmol) and triethylamine (0.40 mL, 2.87 mmol) were added and stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure, followed by addition of ethyl acetate and subsequent partition washing with 10% aqueous citric acid and saturated aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate and then concentrated to dryness under reduced pressure to obtain compound 1. Yield 98%; ESI MS m / z 579.4 (M+H) + ; 1H NMR (400 MHz, CD3OD)δ1.45 (9H, s), 1.47 (9H, s), 1.48 (9H, s), 1.82-2.00 (2H, m), 2.06-2.21 (2H, m), 2.33 (2H, t, J=7.6 Hz), 2.49 (2H, t, J=7.6 Hz), 3.99 (1H, dd, J=4.8, 9.6 Hz), 4.33 (1H, dd, J=5.2, 9.2 Hz), 5.12 (1H, d, J=12.4 Hz), 5.16 (1H, d, J=12.4 Hz), 7.32-7.39 (5H, m).

[0040] [ka]

[0041] (Step 2) Synthesis of Compound 2 To a solution (20 mL) of compound 1 (1.12 g, 1.94 mmol) in ethanol, 10% palladium on carbon (0.10 g) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 18 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated to dryness under reduced pressure to give compound 2. Yield 96%; ESI MS m / z 445.3 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.47 (9H, s), 1.49 (9H, s), 1.51 (9H, s), 1.80-1.93 (2H, m), 1.99-2.15 (2H, m), 2.32-2.38 (4H, m), 3.36 (1H, dd, J=5.2, 7.6 Hz), 4.31 (1H, dd, J=5.2, 9.2 Hz).

[0042] [ka]

[0043] (Step 3) Synthesis of Compound 3 Compound 3 was obtained using Cbz-glutamic acid-α-t-butyl ester and compound 2 as starting materials in the same manner as in step 1 above. Yield 91%; ESI MS m / z 764.4 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.47-1.48 (36H, m), 1.82-1.92 (3H, m), 2.04-2.26 (3H, m), 2.30-2.41 (6H, m), 4.09 (1H, dd, J=4.4, 10.0 Hz), 4.28-4.34 (2H, m), 5.10 (1H, d, J=12.4 Hz), 5.16 (1H, d, J=12.4 Hz), 7.31-7.41 (5H, m).

[0044] [ka]

[0045] (Step 4) Synthesis of Compound 4 Compound 4 was obtained from compound 3 in the same manner as in step 2 above. Yield 95%; ESI MS m / z 630.4 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.47-1.51 (36H, m), 1.84-1.98 (3H, m), 1.99-2.19 (3H, m), 2.32-2.40 (6H, m), 3.42 (1H, dd, J=5.6, 7.2 Hz), 4.26-4.33 (2H, m).

[0046] [ka]

[0047] (Step 5) Synthesis of Compound 5 Compound 5 was obtained using Cbz-glutamic acid-α-t-butyl ester and compound 4 as starting materials in the same manner as in step 1 above. Yield 103%; ESI MS m / z 983.5 (M+Cl) - ; 1 H NMR (400 MHz, CD3OD)δ1.47-1.49 (36H, m), 1.78-2.00 (3H, m), 2.05-2.26 (3H, m), 2.30-2.46 (6H, m), 4.09 (1H, m), 4.27-4.35 (2H, m), 5.11 (1H, d, J= 12.4 Hz), 5.16 (1H, d, J=12.4 Hz), 7.30-7.41 (5H, m).

[0048] [ka]

[0049] (Step 6) Synthesis of Compound 6 Compound 6 was obtained from compound 5 in the same manner as in step 2 above. Yield 94%; ESI MS m / z 815.5 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.47 (9H, s), 1.49 (27H, brs), 1.51 (9H, s), 1.81-19.3 (4H, m), 2.00-2.25 (4H, m), 2.33-2.40 (8H, m), 3.38*, 4.28-4.34 (3H, m). *Partially overlaps with the solvent peak

[0050] [ka]

[0051] (Step 7) Synthesis of Compound 7 To a solution of compound 6 (0.30 g, 0.37 mmol) in dioxane (1 mL), 4N hydrogen chloride in 1,4-dioxane (3 mL) was added under ice cooling, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated to dryness under reduced pressure to give compound 7 (hydrochloride). Yield 99%; ESI MS m / z 535.3 (M+H) + ; 1 H NMR (400 MHz, D2O)δ1.85-1.95 (3H, m), 2.07-2.22 (5H, m), 2.31-2.51 (8H, m), 4.01 (1H, t, J=6.8 Hz), 4.25-4.35 (3H, m).

[0052] [ka]

[0053] [Production Example 2] Synthesis of 10-residue peptide (Step 1) Synthesis of Compound 8 Compound 8 was obtained using Boc-glutamic acid α-t-butyl ester and glutamic acid α-t-butyl, γ-benzyl ester hydrochloride as starting materials in the same manner as in Step 1 of Production Example 1. Yield 96%; ESI MS m / z 579.4 (M+H) + ; 1 H NMR (400 MHz, CD3OD) 1.47 (18H, s), 1.48 (9H, s), 1.82-1.95 (2H, m), 2.04-2.20 (2H, m), 2.32-2.38 (4H, m), 4.08 (1H, dd, J=4.8, 8.8 Hz), 4.30 (1H, dd, J=5.2, 9.2 Hz), 5.09 (1H, d, J=12.4 Hz), 5.13 (1H, d, J=12.4 Hz), 7.31-7.40 (5H, m).

[0054] [ka]

[0055] (Step 2) Synthesis of Compound 9 To a solution (80 mL) of compound 8 (11.15 g, 19.27 mmol) in ethanol, 5% palladium on carbon (1.00 g) was added and the mixture was stirred under a hydrogen atmosphere at room temperature for 6 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated to dryness under reduced pressure to give compound 9. Yield 96%; ESI MS m / z 489.3 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.47 (9H, s), 1.49 (18H, s), 1.85-2.00 (2H, m), 2.08-2.16 (2H, m), 2.34-2.41 (4H, m), 3.99 (1H, dd, J=4.8, 9.2 Hz), 4.30 (1H, dd, J=4.2, 8.8 Hz).

[0056] [ka]

[0057] (Step 3) Synthesis of Compound 10 Compound 10 was obtained using compound 9 and glutamic acid α-t-butyl, γ-benzyl ester hydrochloride as starting materials in the same manner as in step 1 of Production Example 1. Yield 91%; ESI MS m / z 764.4 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.47-1.49 (36H, m), 1.80-2.00 (3H, m), 2.12-2.22 (3H, m), 2.42-2.52 (6H, m), 3.99 (1H, dd, J=4.4, 9.6 Hz), 4.27-4.36 (2H, m), 5.13 (1H, d, J=12.4 Hz), 5.17 (1H, d, J=12.4 Hz), 7.32-7.40 (5H, m).

[0058] [ka]

[0059] (Step 4) Synthesis of Compound 11 Compound 10 was used as a starting material in the same manner as in step 2 above to obtain compound 11. Yield 93%; ESI MS m / z 674.4 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.46 (3H, s), 1.47 (6H, s), 1.49 (27H, s), 1.79-1.97 (3H, m), 2.09-2.22 (3H, m), 2.33-2.44 (6H, m), 4.00 (1H, m), 4.26-4.34 (2H, m).

[0060] [ka]

[0061] (Step 5) Synthesis of Compound 12 Compound 12 was obtained using compound 11 and glutamic acid α-t-butyl, γ-benzyl ester hydrochloride as starting materials in the same manner as in Step 1 of Production Example 1. Yield 87%; ESI MS m / z 949.5 (M−H) - ; 1 H NMR (400 MHz, CD3OD)δ1.47 (45H, s), 1.78-2.01 (3H, m), 2.13-2.23 (3H, m), 2.28-2.44 (4H, m), 2.49-2.54 (2H, m), 4.01 (1H, m), 4.27-4.39 (3H, m), 5.13 (1H, d, J=12.0 Hz), 5.16 (1H, d, J=12.0 Hz), 7.32-7.40 (5H, m).

[0062] [ka]

[0063] (Step 6) Synthesis of Compound 13 Compound 12 was used as a starting material in the same manner as in step 2 above to obtain compound 13. Yield 92%; ESI MS m / z 859.7 (M+H)+; 1H NMR (400 MHz, CD3OD)δ1.48-1.49 (45H, m), 1.79-1.98 (4H, m), 2.08-2.25 (4H, m), 2.34-2.44 (8H, m), 4.02 (1H, dd, J=4.4, 10.0 Hz), 4.30-4.36 (3H, m).

[0064] [ka]

[0065] (Step 7) Synthesis of Compound 14 Compound 14 was obtained using Cbz-glutamic acid-α-t-butyl ester and Compound 6 as starting materials in the same manner as in Step 1 of Production Example 1. Yield 93%; ESI MS m / z 1134.7 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.47-1.49 (54H, m), 1.76-1.94 (6H, m), 2.05-2.13 (2H, m), 2.16-2.46 (12H, m), 4.11 (1H, m), 4.32-4.36 (4H, m), 5.12 (1H, d, J=12.4 Hz), 5.17 (1H, d, J=12.4 Hz), 7.31-7.42 (5H, m).

[0066] [ka]

[0067] (Step 8) Synthesis of Compound 15 Compound 15 was obtained from compound 14 in the same manner as in Step 2 of Production Example 1. Yield 97%; ESI MS m / z 1000.7 (M+H) + ; 1H NMR (400 MHz, CD3OD)δ1.476 (9H, s), 1.484 (9H, s), 1.49 (27H, s), 1.51 (9H, s), 1.80-1.94 (6H, m), 2.01-2.12 (3H, m), 2.16-2.30 (3H, m), 2.32-2.44 (12H, m), 3.39 (1H, dd, J=5.6, 7.6 Hz), 4.30-4.35 (4H, m).

[0068] [ka]

[0069] (Step 9) Synthesis of Compound 16 Compound 16 was obtained using Cbz-glutamic acid-α-t-butyl ester and compound 15 as starting materials in the same manner as in Step 1 of Production Example 1. Yield 91%; ESI MS m / z 1319.8 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ1.47-1.49 (63H, m), 1.76-1.94 (8H, m), 2.06-2.43 (16H, m), 4.12 (1H, dd, J=4.8, 10.0 Hz), 4.34-4.37 (5H, m), 5.11-5.16 (2H, m), 7.31-7.42 (5H, m).

[0070] [ka]

[0071] (Step 10) Synthesis of Compound 17 Compound 17 was obtained from compound 16 in the same manner as in Step 2 of Production Example 1. Yield 89%; ESI MS m / z 1185.8 (M+H) + ; 1H NMR (400 MHz, CD3OD)δ1.476 (9H, s), 1.478 (9H, s), 1.485-1.488 (36H, m), 1.51 (9H, s), 1.76-1.94 (6H, m), 2.00-2.12 (3H, m), 2.27-2.47 (15H, m), 3.39 (1H, dd, J=5.6, 7.6 Hz), 4.33-4.36 (5H, m).

[0072] [ka]

[0073] (Step 11) Synthesis of Compound 18 Compound 18 was obtained using Compound 13 and Compound 17 as starting materials in the same manner as in Step 1 of Production Example 1. Yield 88%; 1 H NMR (400 MHz, CD3OD)δ1.47-1.49 (108H, m), 1.74-1.93 (12H, m), 2.05-2.14 (2H, m), 2.26-2.59 (26H, m), 4.06 (1H, dd, J=4.0, 10.8 Hz), 4.30-4.48 (9H, m).

[0074] [ka]

[0075] (Step 12) Synthesis of Compound 19 Compound 18 (13.2 g, 0.65 mmol) was dissolved in 4N hydrogen chloride-1,4-dioxane solution (100 mL) under ice cooling and stirred at room temperature for 6 hours. The precipitate was washed with acetonitrile to give compound 19 (hydrochloride salt). Yield 97%; ESI MS m / z 1309.5 (M+H) + ; 1H NMR (400 MHz, D2O)δ1.73-1.85 (9H, m), 1.96-2.12 (12H, m), 2.24-2.32 (17H, m), 2.38-2.43 (2H, m), 3.94 (1H, t, J=6.4 Hz), 4.16-4.25 (9H, m).

[0076] [ka]

[0077] (Peptide preparation synthesis) [Reference Example 1] Synthesis of 5-residue peptide 22 (Step 1) Synthesis of Compound 20 Compound 20 was obtained using compound 13 and glutamic acid α-t-butyl, γ-benzyl ester hydrochloride as starting materials in the same manner as in Step 1 of Production Example 1. Yield 88%; ESI MS m / z 1134.8 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ7.40-7.32 (5H, m), 5.17 (1H, d, J=12.4 Hz), 5.13 (1H, d, J=12.4 Hz), 4.39-4.33 (4H, m), 4.02 (1H, dd, J=4.4, 10.4 Hz), 2.54-2.32 (10H, m), 2.26-2.12 (5H, m), 2.08-1.77 (5H, m), 1.49 (9H, s), 1.483 (9H, s), 1.475 (9H, s), 1.472 (9H, s), 1.466 (9H, s), 1.46 (9H, s).

[0078] [ka]

[0079] (Step 2) Synthesis of Compound 21 Compound 21 was obtained from compound 20 in the same manner as in Step 2 of Production Example 2. Yield 93%; ESI MS m / z 1044.7 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ4.37-4.33 (4H, m), 4.03 (1H, m), 2.40-2.36 (10H, m), 2.28-2.10, (5H, m), 1.97-1.83 (5H, m), 1.48 (54H, brs).

[0080] [ka]

[0081] (Step 3) Synthesis of Compound 22 Compound 21 (29 mg, 0.028 mmol) was dissolved in TFA / water (95:5) (2 mL) under ice-cooling and stirred at room temperature for 3 days. The solvent and excess reagents were removed under reduced pressure to give compound 22 (TFA salt). Yield 99%; ESI MS m / z 664.2 (M+H) + ; 1 H NMR (400 MHz, D2O)δ4.35 (1H, dd, J=5.2, 9.2 Hz), 4.31-4.27 (3H, m), 3.96 (1H, t, J=6.4 Hz), 2.51-2.47 (2H, m), 2.42-2.33 (8H, m), 2.18-2.10 (6H, m), 1.95-1.87 (4H, m).

[0082] [ka]

[0083] [Reference Example 2] Synthesis of 10-residue peptide 31 (Step 1) Synthesis of Compound 23 Compound 23 was obtained using Cbz-glutamic acid α-t-butyl ester and compound 17 as starting materials in the same manner as in Step 1 of Production Example 1. Yield 92%; ESI MS m / z 1504.9 (M+H) + ;1 H NMR (400 MHz, CD3OD)δ7.43-7.32 (5H, m), 5.18 (1H, d, J=12.0 Hz), 5.13 (1H, d, J=12.0 Hz), 4.45-4.34 (6H, m), 4.13 (1H, dd, J=4.0, 6.8 Hz), 2.46-2.31 (16H, m), 2.11-2.06 (2H, m), 1.93-1.73 (10H, m), 1.49-1.47 (72H, m).

[0084] [ka]

[0085] (Step 2) Synthesis of Compound 24 Compound 24 was obtained from compound 23 in the same manner as in Step 2 of Production Example 1. Yield 91%; ESI MS m / z 1370.9 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ4.42-4.33 (6H, m), 3.40 (1H, dd, J=5.2, 7.6 Hz), 2.42-2.33 (18H, m), 2.13-2.02 (2H, m), 1.94-1.75 (8H, m), 1.51 (9H, s), 1.487 (36H, brs), 1.476 (27H, brs).

[0086] [ka]

[0087] (Step 3) Synthesis of Compound 25 Compound 25 was obtained using Cbz-glutamic acid α-t-butyl ester and compound 24 as starting materials in the same manner as in Step 1 of Production Example 1. Yield 93%; ESI MS m / z 1691.0 (M+H) + ; 1H NMR (400 MHz, CD3OD)δ7.43-7.32 (5H, m), 5.18 (1H, d, J=12.0 Hz), 5.13 (1H, d, J=12.0 Hz), 4.46-4.43 (7H, m), 4.12 (1H, m), 2.51-2.22 (22H, m), 2.13-2.04 (2H, m), 1.94-1.70 (8H, m), 1.49-1.47 (81H, m).

[0088] [ka]

[0089] (Step 4) Synthesis of Compound 26 Compound 26 was obtained from compound 25 in the same manner as in Step 2 of Production Example 1. Yield 97%; ESI MS m / z 1556.0 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ4.45-4.34 (7H, m), 3.40 (1H, dd, J=5.6, 7.6 Hz), 2.53-2.31 (22H, m), 2.13-2.02 (2H, m), 1.93-1.75 (8H, m), 1.51-1.47 (81H, m).

[0090] [ka]

[0091] (Step 5) Synthesis of Compound 27 Compound 27 was obtained using Cbz-glutamic acid α-t-butyl ester and compound 26 as starting materials in the same manner as in Step 1 of Production Example 1. Yield 98%; ESI MS m / z 1875.2 (M+H) + ; 1H NMR (400 MHz, CD3OD)δ7.43-7.32 (5H, m), 5.18 (1H, d, J=12.4 Hz), 5.14 (1H, d, J=12.4 Hz), 4.47-4.35 (8H, m), 4.12 (1H, dd, J=7.2, 10.0 Hz), 2.57-2.29 (24H, m), 2.13-2.06 (2H, m), 1.94-1.69 (10H, m), 1.49-1.47 (90H, m).

[0092] [ka]

[0093] (Step 6) Synthesis of Compound 28 Compound 27 was used as a starting material in the same manner as in Step 2 of Production Example 1 to obtain Compound 28. Yield 96%; ESI MS m / z 1741.1 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ4.34-4.22 (8H, m), 3.29 (1H, dd, J=5.6, 7.6 Hz), 2.41-2.09 (24H, m), 2.01-1.90 (2H, m), 1.81-1.62 (10H, m), 1.39-1.35 (90H, m).

[0094] [ka]

[0095] (Step 7) Synthesis of Compound 29 Compound 29 was obtained using Cbz-glutamic acid α-t-butyl ester and Compound 28 as starting materials in the same manner as in Step 1 of Production Example 1. Yield 98%; ESI MS m / z 1031.2 (M) 2+ ; 1H NMR (400 MHz, CD3OD)δ7.43-7.32 (5H, m), 5.18 (1H, d, J=12.4 Hz), 5.14 (1H, d, J=12.4 Hz), 4.47-4.34 (9H, m), 4.14 (1H, dd, J=4.0, 10.8 Hz), 2.55-2.23 (26H, m), 2.09 (2H, m), 1.93-1.69 (12H, m), 1.49-1.47 (99H, m).

[0096] [ka]

[0097] (Step 8) Synthesis of Compound 30 Compound 30 was obtained from compound 29 in the same manner as in step 2 of Production Example 1. Yield 94%; ESI MS m / z 1927.3 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ4.46-4.36 (9H, m), 3.40 (1H, dd, J=5.2, 8.0 Hz), 2.54-2.22 (26H, m), 2.13-2.02 (2H, m), 1.93-1.74 (12H, m), 1.51-1.47 (99H, m).

[0098] [ka]

[0099] (Step 9) Synthesis of Compound 31 Compound 30 was used as a starting material in the same manner as in Step 3 of Reference Example 1 to give Compound 31 (TFA salt). Yield 99%; ESI MS m / z 655.5 (M) 2+ ; 1H NMR (400 MHz,D2O)δ4.36-4.26 (9H, m), 4.00 (1H, t, J=6.8 Hz), 2.50-2.46 (2H, m), 2.41-2.32 (16H, m), 2.19-2.11 (12H, m), 1.93-1.88 (10H, m).

[0100] [ka]

[0101] [Reference Example 3] Synthesis of 15-residue peptide 41 (Step 1) Synthesis of Compound 32 Compound 32 was obtained using Boc-D-glutamic acid α-t-butyl ester and D-glutamic acid α-t-butyl, γ-benzyl ester hydrochloride as starting materials in the same manner as in Step 1 of Production Example 1. Yield 99%; ESI MS m / z 579.3 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ7.39-7.31 (5H, m), 5.16 (1H, d, J=12.4 Hz), 5.13 (1H, d, J=12.4 Hz), 4.33 (1H, dd, J=5.2. 9.2 Hz), 3.99 (1H, dd, J=4.8, 9.6 Hz), 2.49 (2H, t, J=7.6 Hz), 2.33 (2H, t, J=7.6 Hz), 2.21-2.06 (2H, m), 1.99-1.81 (2H, m), 1.47 (18H, s), 1.45 (9H, s).

[0102] [ka]

[0103] (Step 2) Synthesis of Compound 33 Compound 33 was obtained from compound 32 in the same manner as in Step 2 of Production Example 2. Yield 91%; ESI MS m / z 489.3 (M+H)+ ; 1 H NMR (400 MHz, CD3OD)δ4.31 (1H, dd, J=5.2, 8.8 Hz), 4.00 (1H, dd, J=4.0, 8.8 Hz), 2.43-2.34 (4H, m), 2.17-2.08 (2H, m), 1.50 (18H, s), 1.46 (9H, s).

[0104] [ka]

[0105] (Step 3) Synthesis of Compound 34 Compound 34 was obtained using compound 33 and D-glutamic acid α-t-butyl, γ-benzyl ester hydrochloride as starting materials in the same manner as in Step 1 of Production Example 1. Yield 95%; ESI MS m / z 764.4 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ7.40-7.32 (5H, m), 5.17 (1H, d, J=12.4, 5.13 (1H, d, J=12.4 Hz), 4.34 (1H, dd, J=5.2, 8.8 Hz), 4.28 (1H, dd, J=4.4, 9.6 Hz), 3.99 (1H, dd, J=4.4, 9.6 Hz), 2.52-2.48 (2H, m), 2.38-2.31 (4H, m), 2.22-2.11 (2H, m), 2.00-1.80 (2H, m), 1.48 (27H, brs), 1.47 (9H, s).

[0106] [ka]

[0107] (Step 4) Synthesis of Compound 35 Compound 35 was obtained from compound 34 in the same manner as in Step 2 of Production Example 2. Yield 92%; ESI MS m / z 674.4 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ4.34-4.27 (2H, m), 4.00 (1H, m), 2.43-2.33 (6H, m), 2.19-2.10 (3H, m), 1.96-1.85 (3H, m), 1.49 (27H, s), 1.47 (9H, s).

[0108] [ka]

[0109] (Step 5) Synthesis of Compound 36 Compound 36 was obtained using compound 35 and glutamic acid α-t-butyl, γ-benzyl ester hydrochloride as starting materials in the same manner as in Step 1 of Production Example 1. Yield 97%; ESI MS m / z 949.6 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ7.38-7.33 (5H, m), 5.12 (1H, d, J=12.4 Hz), 5.12 (1H, d, J=12.4 Hz), 4.34-4.30 (3H, m), 4.02 (1H, dd, J=4.0, 10.0 Hz), 2.51 (2H, t, J=7.6 Hz), 2.43-2.27 (6H, m), 2.24-2.21 (2H, m), 2.02-1.73 (6H, m), 1.48-1.46 (45H, s).

[0110] [ka]

[0111] (Step 6) Synthesis of Compound 37 Compound 37 was obtained from compound 36 in the same manner as in Step 2 of Production Example 2. Yield 92%; ESI MS m / z 859.5 (M+H) + ;1 H NMR (400 MHz, CD3OD)δ4.35-4.29 (3H, m), 4.02 (1H, dd, J=4.4, 10.4 Hz), 2.44-2.34 (8H, m), 2.23-2.09 (4H, m), 1.96-1.84 (4H, m), 1.49-1.47 (45H, m).

[0112] [ka]

[0113] (Step 7) Synthesis of Compound 38 Compound 37 and glutamic acid α-t-butyl, γ-benzyl ester hydrochloride were used as starting materials in the same manner as in Step 1 of Production Example 1 to obtain Compound 38. Yield 92%; ESI MS m / z 1134.7 (M+H) + ; 1 H NMR (400 MHz, CD3OD)δ7.38-7.33 (5H, m), 5.17 (1H, d, J=12.4 Hz), 5.13 (1H, d, J=12.4 Hz), 4.36-4.32 (3H, m), 4.25 (1H, dd, J=4.8, 8.8 Hz), 4.01 (1H, dd, J=4.0, 10.0 Hz), 2.49 (2H, t, J=7.6 Hz), 2.40-2.33 (6H, m), 2.25-2.11 (6H, m), 2.00-1.76 (6H, m), 1.48 (27H, s), 1.47 (27H, s).

[0114] [ka]

[0115] (Step 8) Synthesis of Compound 39 Compound 39 was obtained from compound 38 in the same manner as in Step 2 of Production Example 2. Yield 97%; ESI MS m / z 1044.7 (M+H) + ;1 H NMR (400 MHz, CD3OD)δ8.33-8.19 (4H, m), 6.92 (1H, d, J=8.4 Hz), 4.36-4.25 (4H, m), 4.02 (1H, m), 2.42-2.34 (10H, m), 2.27-2.08, (5H, m), 2.01-1.79 (5H, m), 1.49-1.48 (54H, brs).

[0116] [ka]

[0117] (Step 9) Synthesis of Compound 40 Compound 40 was obtained using compounds 30 and 39 as starting materials in the same manner as in Step 1 of Production Example 1. Yield 98%; ESI MS m / z 985.2 (M) 3+ ; 1 H NMR (400 MHz, CD3OD)δ4.37-4.24 (14H, m), 3.91 (1H, dd, J=4.8, 9.2 Hz), 2.45-2.21 (44H, m), 2.13-2.09 (4H, m), 1.80-1.61 (12H, m), 1.38-1.35 (153H, m)

[0118] [ka]

[0119] (Step 10) Synthesis of Compound 41 Compound 41 (TFA salt) was obtained using compound 40 as a starting material in the same manner as in Step 3 of Reference Example 1. Yield 99%; ESI MS m / z 1955.8 (M+H) + ; 1H NMR (400 MHz, D2O)δ4.29-4.21 (14H, m), 3.96 (1H, t, J=6.4 Hz), 2.45-2.43 (2H, m), 2.37-2.26 (28H, m), 2.13-2.09 (16H, m), 1.90-1.79 (14H, m).

[0120] [ka]

[0121] [Example 1] Production of low molecular weight γ-polyglutamic acid (including γ-oligoglutamic acid) by hydrolysis with 1.0 M sulfuric acid (1) 2.5 g of commercially available γ-polyglutamic acid powder (product name: Caltake) was placed in a glass pressure tube, and 10 mL of 1.0 M sulfuric acid was added. (2) After stirring the sample in the glass tube with a Teflon (registered trademark) rod, the lid was closed and the glass pressure tube was immersed in a water bath and the temperature was adjusted so that the internal liquid temperature was 84-86°C. (3) After heating for 6 hours while maintaining the temperature, the glass tube was removed from the water bath and allowed to cool in ice water. The sample was then transferred to a 100 mL beaker, and the residue in the glass pressure tube was washed with milliQ water. (4) While stirring the solution in the 100 mL beaker with a magnetic stirrer, Ca(OH)2 was added to neutralize the solution, adjusting the pH to 4.1 to 4.2. (5) Stirring was continued at room temperature for approximately 30-60 minutes until sufficient calcium sulfate precipitate was formed. (6) The sample slurry was filtered through a 0.45 μm filter, and the resulting permeate was freeze-dried in vacuo to obtain a low molecular weight γ-PGA powder (1.9 g).

[0122] [Example 2] Production of low molecular weight γ-polyglutamic acid (including γ-oligoglutamic acid) by hydrolysis with 0.9 M sulfuric acid (1) 2.5 g of commercially available γ-polyglutamic acid powder (product name: Caltake) was placed in a glass pressure tube, and 10 mL of 0.9 M sulfuric acid was added. (2) After stirring the sample in the glass tube with a Teflon (registered trademark) rod, the lid was closed and the glass pressure tube was immersed in a water bath and the temperature was adjusted so that the internal liquid temperature was 84-86°C. (3) After heating for 6 hours while maintaining the temperature, the glass tube was removed from the water bath and allowed to cool in ice water. The sample was then transferred to a 100 mL beaker, and the residue in the glass pressure tube was washed with milliQ water. (4) While stirring the solution in the 100 mL beaker with a magnetic stirrer, Ca(OH)2 was added to neutralize the solution, adjusting the pH to 4.1 to 4.2. (5) Stirring was continued at room temperature for approximately 30-60 minutes until sufficient calcium sulfate precipitate was formed. (6) The sample slurry was filtered through a 0.45 μm filter, and the resulting permeate was freeze-dried in vacuo to obtain a low molecular weight γ-PGA powder (2.1 g).

[0123] [Example 3] Production of low molecular weight γ-polyglutamic acid (including γ-oligoglutamic acid) by hydrolysis with 0.7 M sulfuric acid (1) 2.5 g of commercially available γ-polyglutamic acid powder (product name: Caltake) was placed in a glass pressure tube, and 10 mL of 0.7 M sulfuric acid was added. (2) After stirring the sample in the glass tube with a Teflon (registered trademark) rod, the lid was closed and the glass pressure tube was immersed in a water bath and the temperature was adjusted so that the internal liquid temperature was 84-86°C. (3) After heating for 6 hours while maintaining the temperature, the glass tube was removed from the water bath and allowed to cool in ice water. The sample was then transferred to a 100 mL beaker, and the residue in the glass pressure tube was washed with milliQ water. (4) While stirring the solution in the 100 mL beaker with a magnetic stirrer, Ca(OH)2 was added to neutralize the solution, adjusting the pH to 4.1 to 4.2. (5) Stirring was continued at room temperature for approximately 30-60 minutes until sufficient calcium sulfate precipitate was formed. (6) The sample slurry was filtered through a 0.45 μm filter, and the resulting permeate was freeze-dried in vacuo to obtain a low molecular weight γ-PGA powder (1.9 g).

[0124] [Example 4] Production of low molecular weight γ-polyglutamic acid (including γ-oligoglutamic acid) by hydrolysis with 0.5 M sulfuric acid (1) 2.5 g of commercially available γ-polyglutamic acid powder (product name: Caltake) was placed in a glass pressure tube, and 10 mL of 0.5 M sulfuric acid was added. (2) After stirring the sample in the glass tube with a Teflon (registered trademark) rod, the lid was closed and the glass pressure tube was immersed in a water bath and the temperature was adjusted so that the internal liquid temperature was 84-86°C. (3) After heating for 6 hours while maintaining the temperature, the glass tube was removed from the water bath and allowed to cool in ice water. The sample was then transferred to a 100 mL beaker, and the residue in the glass pressure tube was washed with milliQ water. (4) While stirring the solution in the 100 mL beaker with a magnetic stirrer, Ca(OH)2 was added to neutralize the solution, adjusting the pH to 4.1 to 4.2. (5) Stirring was continued at room temperature for approximately 30-60 minutes until sufficient calcium sulfate precipitate was formed. (6) The sample slurry was filtered through a 0.45 μm filter, and the resulting permeate was freeze-dried in vacuo to obtain a low molecular weight γ-PGA powder (2.0 g).

[0125] Comparative Example 1: Production of low molecular weight γ-polyglutamic acid (including γ-oligoglutamic acid) by hydrolysis with 0.5 M sulfuric acid (UF membrane purification) (1) 2.5 g of commercially available γ-polyglutamic acid powder (product name: Caltake) was placed in a glass pressure tube, and 20 mL of 0.5 M sulfuric acid was added. A total of six glass pressure tubes containing samples were prepared in the same manner. (2) After stirring the sample in the glass tube with a Teflon (registered trademark) rod, the lid was closed and the glass pressure tube was immersed in a water bath and the temperature was adjusted so that the internal liquid temperature was 84-86°C. (3) After heating for 6 hours while maintaining the temperature, the glass tube was removed from the water bath and allowed to cool in ice water. The sample was then transferred to a 500 mL beaker, and the residue in the glass pressure tube was washed with milliQ water. (4) While stirring the solution in the 500 mL beaker with a magnetic stirrer, NaOH was added to neutralize the solution, adjusting the pH to 4.1 to 4.2. (5) The resulting solution was subjected to a centrifugal membrane module (Amicon (registered trademark) Ultra-15 (3K) and centrifuged at 5000 G. Ultrapure water was added to the retentate on the membrane and centrifuged again. This procedure was repeated three times, and the filtrate was recovered. Subsequently, the recovered filtrate was subjected to a centrifugal membrane module (PALL Macrosep Advance Centrifugal Device 1K) and centrifuged at 5000 G. Ultrapure water was added to the retentate on the membrane and centrifuged again. This procedure was repeated four times. The resulting retentate was freeze-dried to obtain a low molecular weight γ-PGA powder (6.0 g).

[0126] [Test Example 1] The depolymerized γ-PGA powders obtained in Examples 1 to 4 and the γ-polyglutamic acid powder (Caltake) used as the raw material (hereinafter referred to as "raw material γ-PGA") were analyzed or calculated for "total Glu content," "γ-PGA content," "depolymerized γ-PGA content," "monomer Glu content," "water content," "sulfate ion content," and "Ca ion content" according to the "Amino Acid Analysis (Apparatus, Measurement Conditions)" described above. The results are shown in Table 3.

[0127] [Table 3]

[0128] FIG. 1 shows the relationship between the sulfuric acid concentration during hydrolysis and the sulfate ion and Ca ion contents in the low-molecular-weight γ-PGA powder obtained after hydrolysis, neutralization, and filtration of calcium sulfate precipitate (slurry) in Examples 1 to 4. The results in Figure 1 confirm that the neutralization and precipitate (calcium sulfate) removal in the manufacturing method of the present invention successfully removed sulfate ions and Ca ions in the obtained low molecular weight γ-PGA (including γ-OGA) sample to a level that was not problematic for the expected intake amount (e.g., 300 mg).

[0129] [Test Example 2] The following analyses were carried out on the low molecular weight γ-PGA powders obtained in Examples 1 to 4. The results are shown in Table 4. (Degraded γ-PGA yield) Based on the yield of each of the reduced molecular weight γ-PGA powders obtained in Examples 1 to 4, the reduced molecular weight γ-PGA content in each of the reduced molecular weight γ-PGA powders obtained in Examples 1 to 4 calculated in Test Example 1, the amount of raw material γ-PGA used (i.e., 2.5 g), and the γ-PGA content in the raw material γ-PGA calculated in Test Example 1 (i.e., 79.6 wt%), the yield of reduced molecular weight γ-PGA in the reduced molecular weight γ-PGA powders obtained in Examples 1 to 4 was calculated according to the following calculation formula 1.

[0130]

number

[0131] (PGA percentage of 15 residues or less) Degraded γ-PGA powder obtained in Example 1 (referred to as "1.0 M Ca sulfate precipitate"), degraded γ-PGA powder obtained in Example 2 (referred to as "0.9 M Ca sulfate precipitate"), degraded γ-PGA powder obtained in Example 3 (referred to as "1.0 M Ca sulfate precipitate"), degraded γ-PGA powder obtained in Example 4 (referred to as "0.5 M Ca sulfate precipitate"), degraded γ-PGA powder obtained in Comparative Example 1 (referred to as "UF membrane method sample"), Cultake (γ-PGA powder before degradation), Compound 41 (15-residue peptide preparation 41) obtained in Reference Example 3 (referred to as "15-residue synthetic preparation"), Compound 31 (10-residue peptide preparation 31) obtained in Reference Example 2 (referred to as "10-residue synthetic preparation"), Compound 22 (5-residue peptide preparation 22) obtained in Reference Example 1 (referred to as "5-residue The following compounds were subjected to reversed-phase HPLC: γ-L-Glutamyl L-Glutamic Acid (trade name) (CAS No. 1116-22-9, Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as a "two-residue synthetic preparation"), γ-L-Glutamyl L-Glutamic Acid (trade name) (CAS No. 1116-22-9, Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as a "two-residue synthetic preparation"), and water. The resulting reverse phase HPLC chromatogram is shown in FIG. The results in Figure 2 show that the "UF membrane method sample" that underwent purification and desalting using a UF membrane did not yield the desired molecular weight fraction (γ-oligoglutamic acid with 2 to 15 glutamic acid residues).

[0132] The proportion of PGA with 15 or less residues in the low molecular weight γ-PGA powders obtained in Examples 1 to 4 was calculated by dividing the area of ​​the region sandwiched between the peaks of the 2-residue standard and the 15-residue synthetic standard in Figure 2 by the total area and multiplying the result by 100.

[0133] [Table 4]

[0134] The relationship between the sulfuric acid concentration during hydrolysis and the content of γ-PGA degraded to 15 or less residues in Examples 1 to 4 is shown in FIG. The results in Figure 3 show that the degree of decomposition can be controlled by the sulfuric acid concentration, depending on the results of the γ-OGA function evaluation.

[0135] [Test Example 3] (Evaluation of degradability of low molecular weight γ-PGA using enzyme fraction derived from culture medium) The degradability of the degraded γ-PGA powder obtained in Example 1 was evaluated using the enzyme fraction obtained in the "Method for obtaining enzyme fractions for evaluating enzymatic degradation" below, in accordance with the "Method for evaluating degradability of degraded γ-PGA using culture medium-derived enzyme fraction" below. (Method for obtaining enzyme fractions for enzymatic degradation evaluation) (1) To prepare seed cultures for the six strains shown in Table 5, 50 μl of glycerol stock culture stored at -180°C was cultured in LB agar medium at 37°C for 24 hours. The formed colonies were cultured in 5 mL of LB liquid medium for 24 hours (test tube, 120 rpm), and a glycerol stock culture was prepared from the culture and stored at -180°C as seed cultures. (2) As a preculture, 50 μL of the seed culture was cultured in 5 mL of LB liquid medium for 24 hours (test tube, 120 rpm) to prepare a preculture solution. (3) For enzyme production, 100 mL of E9 medium was placed in a 300 mL Erlenmeyer flask. 2 mL of the preculture solution (2%) was inoculated into the E9 medium and cultured at 37°C for 6 days (180 rpm). The E9 medium composition was citric acid 12.0, glycerol 80.0, NH4Cl 7.0, K2HPO4 0.5, MgSO4 7H2O 0.5, FeCl3 6H2O 0.04, CaCl2 2H2O 0.15, and MnSO4 H2O 0.104 (all units are g / L). (4) The culture medium obtained in (3) was centrifuged at 8,000 × g and 4°C for 10 minutes to obtain the supernatant. (5) Approximately 40 mL of the resulting supernatant was filtered using a centrifugal membrane module (Amicon Ultra-15 PLHK Ultracel, 100 kDa) to obtain approximately 30 mL of permeate. (5) The resulting permeate was concentrated using an Amicon membrane module (Amicon Ultra-15 PLBC Ultracel, 3 kDa) and washed four times with 20 mM Tris-HCl buffer (pH 8.0), ultimately concentrating it approximately 15-fold to obtain approximately 2 mL of enzyme fractions derived from the culture medium of each of the six strains.

[0136] [Table 5]

[0137] (Method for evaluating the degradability of low molecular weight γ-PGA using enzyme fractions derived from culture medium) (1) Commercially available γ-PGA (product name: Caltake) and the freeze-dried powder of reduced molecular weight PGA produced in Example 1 were dissolved in 20 mM Tris-HCl (pH 8.0) to prepare a 0.1% aqueous solution, which was then dispensed in 4,470 μL aliquots into 5 mL sample tubes and cooled on ice. (2) The enzyme fraction solution prepared above from each strain culture was diluted 5-fold with 20 mM Tris-HCl (pH 8.0). (3) Under ice cooling, 30 μL of the diluted enzyme fraction solution prepared in (2) was added to the sample tube in (1) and stirred, and then 432 μL was quickly dispensed into 1.5 mL sample tubes and immersed in a 37°C water bath to initiate the reaction. (4) At 0, 30, 60, 105, 240, and 360 minutes after the start of the reaction, the 1.5 mL tube was removed from the water bath and quickly cooled on ice. 24 μL of a 1.5 mol / L aqueous acetic acid solution was added and mixed thoroughly. (5) 400 μL of the solution obtained in (4) was added to a 2 mL sample tube containing 1,000 μL of saline, and after thorough mixing, 700 μL was dispensed into another 2 mL sample tube. (6) To the 700 μL dispensed in (5), 700 μL of 10% trichloroacetic acid aqueous solution was added and mixed thoroughly, and then the mixture was left to stand on ice for 10 minutes. The solution (7)(6) was centrifuged at 8,000 × g for 4 minutes, and 500 μL of the supernatant was dispensed into a 2 mL sample tube, to which 750 μL of 300 mM phosphate buffer (pH 8.0) was added to prepare an analytical sample. (8) The analytical samples were analyzed using a HITACHI LA8080 Amino Acid Analyzer to quantify the concentration of monomeric Glu. (9) The quantitative values ​​obtained above were plotted as the ratio of the decomposition efficiency of the decomposed γ-PGA to that of commercially available γ-PGA using the following equation (1) (Figure 4).

[0138]

number

[0139] In equation (1), t0 represents the sample at reaction time 0 minutes, and t i indicates the sample time at which the reaction was stopped.

[0140] The results in Figure 4 show that the amount of monomeric glutamic acid released from γ-OGA by the enzymatic reaction derived from Bacillus was 6 to 28 times that of γ-PGA. This indicates that, depending on the conditions and environment, γ-OGA can release larger amounts of monomeric glutamic acid in a shorter time than γ-PGA during enzymatic degradation.

[0141] Test Example 4: Evaluation of Chemically Synthesized 4- and 10-Glu Peptides in Mice (1) 10-20 week old C57BL / 6J mice (Charles River Japan) were used, with one mouse per cage. Glutamic acid, the 4-residue Glu peptide obtained in Production Example 1, the 10-residue Glu peptide obtained in Production Example 2, and γ-PGA (product name: Cultake) were each dissolved in distilled water to a 1% w / v concentration, and the mice were allowed to drink the water ad libitum for one week. The control group was allowed to drink distilled water ad libitum for one week. The mice were given a diet of CRF-1 (Oriental Yeast Co., Ltd.) for conventional breeding ad libitum. (2) After the test, the cecum was removed and the contents were collected. The Glu concentration in the cecal contents was measured by centrifuging the pre-weighed cecal contents at 10,000 × g and using the supernatant. The supernatant was deproteinized using methanol, and the Glu concentration was measured using a liquid chromatograph mass spectrometer (Agilent Technologies LC-MS / MS mass spectrometry system). (3) Separation was performed by injecting 2 μL of sample onto an Inertsil ODS-3 (2.1 × 100 mm, 2 μm, GL Sciences) column at 40°C. The mobile phase consisted of 0.2% acetic acid (A) and 0.2% acetic acid / acetonitrile (B) solutions, with a constant flow rate of 0.2 mL / min. The mobile phase B concentrations were 5% (0–1 min), 30% (3 min), 48% (7 min), 95% (7.1–11 min), and 5% (11.1–16 min). Mass measurements were performed under the following conditions: Gas Temp: 200°C, Gas Flow: 20 L / min, Nebulizer: 50 psi, Sheath Gas Temp: 400°C, Sheath Gas Flow: 12 L / min, Capillary: 3000 V, and ESI (+) ionization. The peak area ratios of aqueous Glu solutions with known concentrations (eight aqueous Glu solutions with different concentrations ranging from 0.001 to 2.7 μM) were calculated, and the concentrations were determined based on a standard curve prepared from the calculated peak area ratios.

[0142] The results are shown in Figure 5. The results in FIG. 5 show that the glutamate level in the cecum of mice administered γ-OGA was increased compared to the glutamate level in the cecum of mice administered γ-PGA. [Industrial Applicability]

[0143] According to the present invention, it is possible to provide an oral composition that can efficiently produce glutamic acid in the lower gastrointestinal tract and a method for producing the same.

Claims

1. (1) hydrolyzing γ-polyglutamic acid in an acidic or alkaline aqueous solution to obtain an aqueous solution containing a hydrolysate; and (2) A step of neutralizing the aqueous solution containing the hydrolyzate obtained in (1) and removing the resulting salt precipitate. A method for producing γ-oligoglutamic acid, comprising:

2. 2. The method according to claim 1, wherein the hydrolysis in step (1) is carried out in an aqueous solution of an inorganic acid.

3. 3. The method of claim 2, wherein the aqueous inorganic acid solution is an aqueous sulfuric acid solution.

4. 4. The method according to claim 3, wherein the concentration of the aqueous sulfuric acid solution is 0.9 to 1 mol / L.

5. The method according to any one of claims 2 to 4, wherein the neutralization in step (2) is carried out using an alkaline earth metal hydroxide.

6. The method according to any one of claims 2 to 4, wherein the neutralization in step (2) is carried out using calcium hydroxide.

7. The method according to claim 1 or 2, wherein the γ-oligoglutamic acid contains 2 to 15 glutamic acid residues.

8. An oral composition containing gamma-oligoglutamic acid and an oligosaccharide.

9. 9. The composition according to claim 8, wherein the γ-oligoglutamic acid contains 2 to 15 glutamic acid residues.

10. The composition according to claim 8 or 9, which is for improving the intestinal environment.

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