Method for producing composition containing aromatic lactic acid

Abstract

To provide a technique of producing an aromatic lactic acid in higher production amounts than conventional ones.SOLUTION: The present invention discloses a method for producing a composition containing an aromatic lactic acid. The method includes: a first step in which cells of Bifidobacterium bacteria are cultured in a medium containing one or more selected from the group consisting of casein protein hydrolysate, whey protein hydrolysate, and a reductant; and a second step in which a fraction containing an aromatic lactic acid is recovered from the cultures resulting from the first step.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing a composition containing aromatic lactic acid.

Background Art

[0002] Aromatic lactic acid is metabolized from aromatic amino acids by various microorganisms. Aromatic lactic acids such as 3-phenyl lactic acid, 4-hydroxyphenyl lactic acid, and indole-3-lactic acid are ligands of the Aryl Hydrocarbon Receptor (AhR), and activate AhR by binding to AhR.

[0003] Activated AhR is known to have various effects. For example, activated AhR is known to have an effect of suppressing the over-proliferation of Intestinal Stem Cells (ISC), an effect of repairing tissue damage, and an effect of suppressing the formation of colorectal cancer (Non-Patent Document 1). In addition, activated AhR is known to have an effect of promoting the differentiation of goblet cells and an effect of promoting the production of mucin, and an effect of improving the intestinal barrier function (Non-Patent Document 2).

[0004] In addition, activated AhR is known to have an effect of regulating intestinal motility and an effect of maintaining homeostasis in the intestinal tract (Non-Patent Document 3).

[0005] In addition, since it is known that the ligand of AhR decreases in patients with Inflammatory Bowel Disease (IBD) (Non-Patent Document 4), activated AhR is considered to have an anti-inflammatory effect. In addition, indoleacrylic acid (IA) is a peripheral blood mononuclear cell activated by lipopolysaccharide (LPS) (Peripheral Blood Mononuclear Cells, Since it has the effect of suppressing the secretion of IL-6 and IL-1β in PBMCs (Non-Patent Document 2), activated AhR is considered to have an anti-inflammatory effect.

[0006] In addition, activated AhR has the effect of promoting the production of IL-22 and is known to be effective in preventing and improving infectious diseases (Non-Patent Document 5). In addition, since activated AhR senses the bacterial signal molecule (quorum-sensing (QS) molecule) of Pseudomonas aeruginosa, it is considered to be effective in preventing and improving infectious diseases (Non-Patent Document 6).

[0007] In addition, indole-3-lactic acid has antibacterial effects against fungi such as bacteria of the genus Penicillium and bacteria such as Escherichia coli and Bacillus cereus. Also, AhR activated by indole-3-lactic acid derived from Lactobacillus reuteri + is known to have the effect of reprogramming CD4 T helper cells into immunoregulatory T cells (Non-Patent Document 2).

[0008] Indole-3-lactic acid, which is one of the aromatic lactic acids, is known to be produced by bacteria of the genus Bifidobacterium in MRS medium containing L-cysteine, and it is known that the production amount is particularly large in bacteria of the genus Bifidobacterium of the human infant type. Such bacteria of the genus Bifidobacterium of the human infant type include Bifidobacterium longum subsp. longum BB536 (NITE BP-02621), Bifidobacterium longum subsp. longum ATCC 15707, Bifidobacterium longum subsp. Peasants Infantis M-63 (NITE BP-02623) and Bifidobacterium longum subsp. infantis ATCC 15697 have been reported (Non-Patent Document 8).

[0009] In addition, indole-3-lactic acid produced by Bifidobacterium longum subsp. infantis ATCC 15697 is known as an anti-inflammatory substance, and it has been suggested from cell experiments that it may be useful for the prevention of necrotizing enterocolitis (NEC) (Non-Patent Document 9). Also, it is known that the administration of the culture supernatant of Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623) changes the expression of genes related to anti-inflammation (Patent Document 1).

[0010] In addition, a method for producing a preparation is known in which a casein hydrolyzate or the like is incubated with Bifidobacterium bacteria to obtain a culture mixture, and then the Bifidobacterium bacteria in the mixture are inactivated by heating or the like, and the obtained mixture is combined with two or more non-digestible carbohydrates (Patent Document 2).

[0011] In addition, it has been reported that the addition of a reducing agent to the medium increases the amino acid production of Corynebacterium glutamicum (Non-Patent Document 10). Also, it is presumed that the addition of a reducing agent to the medium promotes the production of fermentation products of Bifidobacterium bacteria and lowers the pH (Non-Patent Document 11).

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0013] [Non-Patent Document 1] Immunity, 49(2):353-362 (2018) [Non-Patent Document 2] Nat. Com., 9(1):3294 (2018) [Non-Patent Document 3] Nature, 578(7794):284-289 (2020) [Non-Patent Document 4] Nat. Med., 22(6):598-605 (2016) [Non-Patent Document 5] Immunity, 22;39(2):372-85 (2013) [Non-Patent Document 6] Science, 20;366(6472) (2019) [Non-Patent Document 7] Microorganisms, 8(3):398 (2020) [Non-Patent Document 8] Microorganisms, 7(9):340 (2019) [Non-Patent Document 9] Pediatric Research, 88(2):209-217 (2020) [Non-Patent Document 10] Biotechnol. Bioeng., 5;40(7):851-7 (1992) [Non-Patent Document 11] AGRIS, vol.27, 230-235 (2001) [Summary of the Invention] [Problems to be Solved by the Invention]

[0014] An object of the present invention is to provide a technique for producing aromatic lactic acid with a higher production amount than conventional ones. [Means for Solving the Problems]

[0015] The inventors of the present invention have found that when culturing cells of a given Bifidobacterium bacterium in a medium containing a given protein hydrolyzate and / or a reducing agent, aromatic lactic acid can be produced in a higher production amount than before, and have thus completed the present invention.

[0016] That is, the present invention is a method for producing a composition containing aromatic lactic acid, comprising: culturing cells of a Bifidobacterium bacterium in a medium containing one or more selected from the group consisting of a casein protein hydrolyzate, a whey protein hydrolyzate, and a reducing agent step, and recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step and provides a production method.

[0017] In the production method, it is a preferred embodiment that the medium contains a casein protein hydrolyzate, and the casein protein hydrolyzate is a highly decomposed casein protein hydrolyzate.

[0018] In the production method, it is also preferred that the medium contains a whey protein hydrolyzate, and the whey protein hydrolyzate is a highly decomposed whey protein hydrolyzate.

[0019] In the production method, it is also a preferred embodiment that the reducing agent is one or more selected from the group consisting of citric acid, cysteine, ascorbic acid, and salts thereof.

[0020] In the production method, it is also a preferred embodiment that the aromatic lactic acid is one or more selected from the group consisting of 3-phenyllactic acid, 4-hydroxyphenyllactic acid, and indole-3-lactic acid.

[0021] In the production method, it is also a preferred embodiment that the recovering step includes a spray drying treatment or a freeze drying treatment.

[0022] The production method also preferably includes the Bifidobacterium bacterium being Bifidobacterium longum subsp. longum BB536 (NITE BP-02621) and / or Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623).

[0023] The present invention also provides a method for increasing the production amount of aromatic lactic acid in the cells of Bifidobacterium bacteria, including a step of culturing the cells in a medium containing one or more selected from the group consisting of a casein protein hydrolyzate, a whey protein hydrolyzate, and a reducing agent.

[0024] The present invention also provides a processed product of Bifidobacterium bacteria cells containing indole-3-lactic acid, wherein the indole-3-lactic acid is 48 μg or more per 1 g on a solid content basis.

[0025] The present invention also provides a processed product of Bifidobacterium bacteria cells containing 3-phenyl lactic acid, wherein the 3-phenyl lactic acid is 60 μg or more per 1 g on a solid content basis.

[0026] The present invention also provides a processed product of Bifidobacterium bacteria cells containing indole-3-lactic acid and 3-phenyl lactic acid, wherein the total amount of the indole-3-lactic acid and the 3-phenyl lactic acid is 110 μg or more per 1 g on a solid content basis.

[0027] The present invention also includes a step of culturing Bifidobacterium bacteria cells in a medium containing one or more selected from the group consisting of a casein protein hydrolyzate, a whey protein hydrolyzate, and a reducing agent, a step of recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step, and A step of formulating the fraction recovered in the step of recovery as an active ingredient A method for producing a composition for preventing or improving inflammation, which comprises , can be provided.

[0028] In a preferred embodiment, the inflammation is necrotic inflammation in the production method.

[0029] In a more preferred embodiment, the necrotic inflammation is necrotizing enterocolitis in the production method.

[0030] In a preferred embodiment, the composition is a food or drink composition in the production method.

[0031] In a preferred embodiment, the composition is a pharmaceutical composition in the production method.

Advantages of the Invention

[0032] According to the present invention, aromatic lactic acid can be produced in a higher production amount than before. For example, when the same bacterial cells are administered to a neonate with few Bifidobacterium bacteria in the intestine, individual differences occur in the colonization and metabolism of the same bacterial cells in the intestine. However, according to the present invention, if a composition rich in aromatic lactic acid can be produced, a sufficient amount of aromatic lactic acid can be ingested by the neonate.

Brief Description of the Drawings

[0033]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0034] Next, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention. In this specification, percentages are by weight (mass) unless otherwise specified.

[0035] The method for producing a composition containing aromatic lactic acid according to the present invention comprises culturing cells of Bifidobacterium bacteria in a medium containing one or more selected from the group consisting of a casein protein hydrolyzate, a whey protein hydrolyzate, and a reducing agent, and recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step and includes.

[0036] The aromatic lactic acid of the present invention may be any one produced from aromatic amino acids by cells of Bifidobacterium bacteria. One or more than two kinds of aromatic lactic acid of the present invention may be used. For example, 3-phenyl lactic acid, 4-hydroxyphenyl lactic acid, indole-3-lactic acid can be mentioned. 3-Phenyl lactic acid is converted from tyrosine, 4-hydroxyphenyl lactic acid is converted from phenylalanine, and indole-3-lactic acid is converted from tryptophan.

[0037] The casein protein hydrolyzate of the present invention is not particularly limited as long as it increases the production amount of aromatic lactic acid produced from aromatic amino acids by culturing cells of Bifidobacterium bacteria is not particularly limited.

[0038] The average molecular weight of the casein protein hydrolyzate in the present invention is determined by the following concept of number average molecular weight. The number average molecular weight is, as described in, for example, the literature (edited by the Society of Polymer Science, Japan, "Fundamentals of Polymer Science," pages 116-119, Tokyo Kagaku Dojin Co., Ltd., 1978), an average value of the molecular weights of polymer compounds shown based on the following different indices. That is, polymer compounds such as protein hydrolysates are heterogeneous substances and have a molecular weight distribution. Therefore, in order to physically and chemically handle the molecular weight of protein hydrolysates, it is necessary to indicate it by the average molecular weight. The number average molecular weight (hereinafter sometimes abbreviated as Mn) is an average with respect to the number of molecules.

[0039] In this specification, the average molecular weight of the casein protein hydrolysate refers to that measured and calculated by the following method. That is, using high performance liquid chromatography, a polyhydroxyethyl aspartamide column (manufactured by Poly LC; diameter 4.6 × 200 mm) is used and eluted at an elution rate of 0.4 mL / min with 20 mM sodium chloride and 50 mM formic acid (edited by Nobuo Urai et al., "High Performance Liquid Chromatography of Proteins and Peptides," Chemical Supplement No. 102, page 241, Chemical Dojin Co., Ltd., 1984). Detection is performed using a UV detector (manufactured by Shimadzu Corporation), and data analysis is carried out by a GPC analysis system (manufactured by Shimadzu Corporation) to calculate the number average molecular weight. For the standard for molecular weight calculation, proteins and / or peptides with known molecular weights can be appropriately used.

[0040] Protein hydrolysates generally contain free amino acids in the manufacturing process. In this specification, the amino acid liberation rate is the ratio of the amount of free amino acids to the whole casein protein hydrolysate and can be measured and calculated by the following method. For amino acids other than tryptophan, cysteine, and methionine, the sample is hydrolyzed with 6 N hydrochloric acid at 110 °C for 24 hours. For tryptophan, it is alkali-decomposed with barium hydroxide at 110 °C for 22 hours. For cysteine and methionine, after formic acid treatment, they are hydrolyzed with 6 N hydrochloric acid at 110 °C for 18 hours, and then analyzed by an amino acid analyzer (model 835, manufactured by Hitachi, Ltd.) to measure the mass of the amino acids. The composition of each amino acid in the sample is measured by the above method, and these are summed up to calculate the mass of all amino acids in the sample. Next, the sample is deproteinized with sulfosalicylic acid, and the mass of each remaining free amino acid is measured by the above method, and these are summed up to calculate the mass of all free amino acids in the sample. From these values, the free amino acid content rate in the sample is calculated by the following formula. Amino acid liberation rate (mass %) = (mass of all free amino acids / mass of all amino acids) × 100

[0041] In obtaining a casein protein hydrolyzate, the casein protein as the starting material may be, for example, various commercially available caseins, or those isolated from milk of humans, cows, horses, sheep, goats, etc. by a conventional method (for example, isoelectric precipitation method), or those produced by genetic recombination technology or the like. Casein is classified into α-casein, β-casein, and κ-casein, and any casein can be used in the present invention.

[0042] A general method for obtaining a casein protein hydrolyzate will be described below, but it is not particularly limited thereto. The raw material casein protein is dispersed and dissolved in water or warm water. The concentration of the solution is not particularly limited, but usually, a concentration range of about 5 to 15% in terms of protein is desirable from the viewpoints of efficiency and operability. It is desirable to heat-sterilize the obtained solution containing the casein protein at 70 to 90 °C for about 10 minutes to 15 seconds from the viewpoint of preventing spoilage due to contamination by miscellaneous bacteria. Next, an alkali agent or an acid agent is added to the solution containing the casein protein, and p It is preferable to adjust the pH to the optimum pH of the hydrolase using H or near it. The alkali agent or acid agent can be any alkali agent or acid agent as long as it is acceptable for food or pharmaceuticals. Specifically, examples of the alkali agent include sodium hydroxide, potassium hydroxide, potassium carbonate, etc., and examples of the acid agent include hydrochloric acid, citric acid, phosphoric acid, acetic acid, etc. Next, a hydrolase solution is added to the casein protein solution. The hydrolase is not particularly limited as long as it is an enzyme that hydrolyzes proteins, and it is preferably an enzyme derived from animals or microorganisms. Also, the enzyme is preferably an endopeptidase. As the endopeptidase, various enzymes such as pancreatin, pepsin, trypsin, elastase, etc. can be used. Note that "derived from" means originally possessed by the above organisms and does not mean the source of collection. For example, a protease produced by introducing a gene encoding a protease produced by Bacillus subtilis into Escherichia coli and expressing the gene is "derived from" Bacillus subtilis.

[0043] One type of hydrolase may be used, or two or more types may be used. When using two or more types of enzymes, the respective enzyme reactions may be carried out simultaneously or separately. The solution added with the enzyme is maintained at an appropriate temperature according to the type of enzyme, for example, 30 to 60 °C, preferably 45 to 55 °C, to start the hydrolysis of the casein protein. The hydrolysis reaction time is continued until a preferable decomposition rate is reached while monitoring the decomposition rate of the enzyme reaction.

[0044] The hydrolysis reaction is stopped by inactivating the enzyme in the hydrolysis solution, and it can be carried out by a heat inactivation treatment according to a conventional method. The heating temperature and holding time of the heat inactivation treatment can be appropriately set considering the thermal stability of the enzyme used so that it can be sufficiently inactivated. For example, it can be carried out at a holding time of 30 minutes to 2 seconds in the temperature range of 80 to 130 °C. Note that the obtained reaction solution may be adjusted to a pH range of 5.5 to 7 with an acid such as citric acid as needed.

[0045] The solution containing the obtained casein protein hydrolyzate can be used as it is, and if necessary, it can also be used as a concentrated solution obtained by concentrating this solution by a known method, and further, as a powder obtained by drying this concentrated solution by a known method.

[0046] Examples of the casein protein hydrolyzate of the present invention include highly hydrolyzed casein protein hydrolyzate, moderately hydrolyzed casein protein hydrolyzate, and lowly hydrolyzed casein protein hydrolyzate according to the degree of hydrolysis. Any one of them may be used, or a plurality of them may be used. Further, one or more selected from the group consisting of highly hydrolyzed casein protein hydrolyzate, moderately hydrolyzed casein protein hydrolyzate, and lowly hydrolyzed casein protein hydrolyzate may be used.

[0047] As the casein protein hydrolyzate of the present invention, since the production amount of aromatic lactic acid produced from aromatic amino acids by culturing cells of Bifidobacterium bacteria increases, the higher the degree of hydrolysis, the more preferable. That is, moderately hydrolyzed casein protein hydrolyzate is more preferable than lowly hydrolyzed casein protein hydrolyzate, and highly hydrolyzed casein protein hydrolyzate is more preferable than moderately hydrolyzed casein protein hydrolyzate.

[0048] In this specification, the highly hydrolyzed casein protein hydrolyzate refers to a casein protein hydrolyzate in which the proportion of peptides having a molecular weight of less than 500 is 50% or more based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC). Specifically, CU5000, C3500Ca, MCH-30 (all manufactured by Morinaga Milk Industry Co., Ltd.), etc. can be mentioned. In addition to these, highly hydrolyzed casein protein hydrolyzate X can also be mentioned.

[0049] The highly hydrolyzed casein protein hydrolyzate X can be prepared, for example, as follows. Disperse 10 kg of edible casein (Alacid 720, manufactured by Fonterra, edible casein content: 88%) in deionized water at a concentration of 10%, adjust the pH to 7.0 with a 10% sodium hydroxide solution and dissolve it. Sterilize the resulting solution by heating at 80°C for 10 minutes, maintain it at 55°C, adjust the pH to 9.0 with a 10% sodium hydroxide solution, add 40 g of an enzyme derived from Bacillus licheniformis (manufactured by Amano Enzyme Inc.) and 400 g of Aspergillus oryzae (manufactured by Shin Nippon Chemical Industry Co., Ltd.) and perform hydrolysis for 14 hours. Then, inactivate the enzyme by performing a heat treatment at 90°C for 10 minutes. Thereafter, remove the precipitate with diatomaceous earth silica 300SA (manufactured by Chuo Silica), concentrate it, and then spray-dry it to obtain a powdery highly decomposed casein protein hydrolyzate X.

[0050] In this specification, the moderately decomposed casein protein hydrolyzate refers to a casein protein hydrolyzate in which the proportion of peptides with a molecular weight of less than 500 is 30% or more and less than 50% based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC).

[0051] In this specification, the lowly decomposed casein protein hydrolyzate refers to a casein protein hydrolyzate in which the proportion of peptides with a molecular weight of less than 500 is 0 or more and less than 30% based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC).

[0052] The whey protein hydrolyzate of the present invention is not particularly limited as long as the production amount of aromatic lactic acid produced from aromatic amino acids is increased by culturing cells of bacteria belonging to the genus Bifidobacterium.

[0053] In the present invention, the average molecular weight of the whey protein hydrolyzate is determined by the concept of the number average molecular weight, and the amino acid liberation rate is the ratio of the amount of free amino acids to the whole whey protein hydrolyzate. For the measurement and calculation thereof, the descriptions of the average molecular weight and amino acid liberation rate of the casein protein hydrolyzate described above are respectively incorporated by reference.

[0054] In obtaining the whey protein hydrolyzate, the whey protein used as the starting material may be, for example, a commercially available product or whey separated from milk, skim milk, etc. by a known method (for example, cheese whey, acid whey, membrane-separated whey, whey powder, desalted whey powder, etc.), or a separated and purified whey protein concentrate (WPC), whey protein isolate (WPI), etc. One kind or a mixture of two or more kinds selected therefrom may be mentioned. The mixture may be mixed at any ratio. In addition to the whey protein derived from general milk, the whey protein may be derived from plants such as soybeans (so-called "soy whey", etc.), but it is more preferably derived from milk, and even more preferably derived from bovine milk.

[0055] A general method for obtaining the whey protein hydrolyzate will be described below, but it is not particularly limited thereto. Disperse and dissolve the raw material whey protein in water or warm water to prepare a whey protein aqueous solution. Furthermore, it is preferable to desalt the whey protein aqueous solution by an ion exchange method using a sodium-type or potassium-type cation exchange resin (preferably a strongly acidic cation exchange resin), electrodialysis, ultrafiltration membrane method, loose reverse osmosis membrane method, etc., and appropriately adjust the pH and calcium concentration. Either a column type or a batch type may be adopted during desalting. Also, the whey protein aqueous solution may be appropriately heat-sterilized before desalting or the like.

[0056] Next, the whey protein aqueous solution is subjected to a hydrolysis treatment. Examples of the hydrolysis treatment include enzyme treatment, acid treatment, alkali treatment, heat treatment, etc., and two or more of these treatments may be appropriately combined. The protein hydrolase of the present disclosure is, for example, derived from plants, animals, microorganisms, etc., and one kind or a combination of two or more kinds thereof can be used. As the protein hydrolase, endoprotease is preferable. Examples of the endoprotease include serine protease, metalloprotease, cysteine protease, and aspartic protease, and one or more of these can be selected and used. Among these, it is preferable to use serine protease and / or metalloprotease. In addition, proteases are classified into alkaline protease, neutral protease, and acidic protease. Among these, it is preferable to use neutral protease.

[0057] Commercially available products can be used as the protein hydrolase. Examples of the protein hydrolase include Bioprase (manufactured by Nagase Biochemics Co., Ltd.), Proleather (manufactured by Amano Enzyme Inc.), Protease S (manufactured by Amano Enzyme Inc.), PTN6.0S (manufactured by Novozymes A / S), Savinase (manufactured by Novozymes A / S), GODO B.A.P (manufactured by Kato Alcohol Co., Ltd.), Protease N (manufactured by Amano Enzyme Inc.), GODO B.N.P (manufactured by Kato Alcohol Co., Ltd.), Neutrase (manufactured by Novozymes A / S), Alcalase (manufactured by Novozymes A / S), Trypsin (manufactured by Novozymes A / S), Chymotrypsin (manufactured by Novozymes A / S), Subtilisin (manufactured by Novozymes A / S), Papain (manufactured by Amano Enzyme Inc.), Bromelain (manufactured by Amano Enzyme Inc.), etc., and one or more enzymes can be selected from these and used. Among these, one or more neutral proteases selected from subtilisin (e.g., Bioprase), trypsin (e.g., PTN6.0S), and bachillolysin (e.g., Protease N) are preferable, and more preferably, these three are used in combination.

[0058] The amount of the endoprotease used for the whey protein is not particularly limited and may be appropriately adjusted according to the substrate concentration, enzyme titer, reaction temperature, reaction time, etc. By appropriately adjusting the hydrolysis conditions with the protein hydrolase, a whey protein hydrolyzate within a specific average molecular weight range can be obtained. Before hydrolysis by the protein hydrolase, the pH of the raw milk protein solution can also be adjusted to the optimum pH of the enzyme used by using salts that can be used in foods, such as potassium carbonate and sodium hydroxide. The reaction temperature of the protein hydrolase is preferably within the range of the optimum temperature of the enzyme used. The reaction holding time of the protein hydrolase may be appropriately adjusted so as to obtain the specific non-protein nitrogen ratio. The hydrolysis by the protein hydrolase may be terminated by heating the enzyme to inactivate it. For example, when inactivating at 100 °C or higher (preferably 110 - 130 °C), it is preferably carried out for 1 - 3 seconds, and when inactivating at less than 100 °C and 60 °C or higher, it is preferably carried out for 3 - 40 minutes.

[0059] In the production of the whey protein hydrolyzate according to the present invention, when hydrolyzing a solution with unadjusted calcium concentration, the obtained decomposition solution may be desalted as described above and the calcium concentration may be adjusted. Then, the enzyme is inactivated by heating according to a conventional method. The reaction heating temperature and the reaction holding time can be appropriately set considering the thermal stability of the enzyme used so that it can be sufficiently inactivated. After heat inactivation, it can be cooled by a conventional method and used as it is, and if necessary, it can be concentrated to obtain a concentrated solution, and further the concentrated solution can be dried to obtain a powder product.

[0060] When hydrolyzing the whey protein aqueous solution by acid treatment or alkali treatment, the pH of the whey protein aqueous solution may be adjusted for the treatment. In the case of the treatment by such pH adjustment, the pH of the whey protein aqueous solution is preferably pH 5 or less or pH 9 or more, and more preferably pH 4 or less or pH 10 or more. The aqueous solution thus treated with pH can be left or stirred at room temperature for several minutes or more, preferably 5 minutes to 1 hour, to obtain a hydrolyzate by acid treatment or alkali treatment. Here, "room temperature" is about 4 - 40 °C, but 10 - 30 °C is preferable. Alternatively, the whey protein aqueous solution may be hydrolyzed by heat treatment. This whey protein aqueous solution may not be pH-adjusted, or may be pH-adjusted (specifically, acidic (pH 5 or lower), neutral (pH 6 - 8), alkaline (pH 8 or higher)). The heat treatment may be carried out at about 4 - 100°C under conditions similar to the above acid-base treatment.

[0061] The obtained whey protein hydrolyzate can exhibit its efficacy even when used in an unpurified state, but further, known separation and purification may be appropriately performed. For example, molecular weight fractionation can be performed on the obtained whey protein hydrolyzate to obtain a whey protein hydrolyzate that satisfies a specific range of average molecular weight and amino acid liberation rate. As the molecular weight fractionation, for example, methods such as ultrafiltration and gel filtration can be adopted, thereby increasing the removal rate of peptides and free amino acids with unnecessary molecular weights. In the case of ultrafiltration, a desired ultrafiltration membrane may be used, and in the case of gel filtration, a gel filtration agent used for the desired size exclusion chromatography may be used. Furthermore, known separation and purification methods (for example, ion exchange resins, etc.) may be used to remove salts and impurities or increase the purity.

[0062] Examples of the whey protein hydrolyzate of the present invention include highly hydrolyzed whey protein hydrolyzate, moderately hydrolyzed whey protein hydrolyzate, and lowly hydrolyzed whey protein hydrolyzate according to the degree of hydrolysis. Any one of them may be used, or a plurality of them may be used. Also, one or more selected from the group consisting of highly hydrolyzed whey protein hydrolyzate, moderately hydrolyzed whey protein hydrolyzate, and lowly hydrolyzed whey protein hydrolyzate may be used.

[0063] As the whey protein hydrolyzate of the present invention, since the production amount of aromatic lactic acid produced from aromatic amino acids by culturing cells of Bifidobacterium bacteria increases, the higher the degree of hydrolysis, the more preferable. That is, a moderately hydrolyzed whey protein hydrolyzate is more preferable than a lowly hydrolyzed whey protein hydrolyzate, and a highly hydrolyzed whey protein hydrolyzate is more preferable than a moderately hydrolyzed whey protein hydrolyzate.

[0064] In this specification, the highly hydrolyzed whey protein hydrolyzate refers to a whey protein hydrolyzate in which the proportion of peptides having a molecular weight of less than 500 is 50% or more based on the result of measuring the molecular weight distribution by high performance liquid chromatography (HPLC). Specifically, WU2900 (manufactured by Morinaga Milk Industry Co., Ltd.) and the like can be mentioned.

[0065] In this specification, the moderately hydrolyzed whey protein hydrolyzate refers to a whey protein hydrolyzate in which the proportion of peptides having a molecular weight of less than 500 is 30% or more and less than 50% based on the result of measuring the molecular weight distribution by high performance liquid chromatography (HPLC). Specifically, W1600 (manufactured by Morinaga Milk Industry Co., Ltd.) and the like can be mentioned.

[0066] In this specification, the lowly hydrolyzed whey protein hydrolyzate refers to a whey protein hydrolyzate in which the proportion of peptides having a molecular weight of less than 500 is 0 or more and less than 30% based on the result of measuring the molecular weight distribution by high performance liquid chromatography (HPLC).

[0067] The reducing agent of the present invention is not particularly limited as long as the production amount of aromatic lactic acid produced from aromatic amino acids by culturing cells of Bifidobacterium bacteria increases. The reducing agent of the present invention may be used alone or in combination of two or more. For example, citric acid, cysteine, ascorbic acid, and salts thereof can be mentioned. Regarding the salts thereof, as the citrate, trisodium citrate can be mentioned. As the cysteine salt, L-cysteine hydrochloride can be mentioned. As the ascorbate, sodium ascorbate can be mentioned.

[0068] The Bifidobacterium bacteria of the present invention are not particularly limited as long as they can produce aromatic lactic acid from aromatic amino acids. For example, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium psudocatenulatum, Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. animalis, Bifidobacterium pseudolongum subsp. globosum, Bifidobacterium pseudolongum subsp. pseudolongm, Bifidobacterium thermophilum, etc. may be mentioned. In addition, Bifidobacterium longum subsp. longum may sometimes be simply referred to as Bifidobacterium longum. Also, Bifidobacterium longum subsp. infantis may sometimes be simply referred to as Bifidobacterium infantis.

[0069] Specifically, Bifidobacterium longum subsp. longum ATCC15707, Bifidobacterium longum subsp. longum BB536 (NITE BP-02621), Bifidobacterium longum subsp. infantis ATCC15697, Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623)Bifidobacterium infantis M-63 (NITE BP-02623), Bifidobacterium breve ATCC15700, Bifidobacterium breve FERM BP-11175, Bifidobacterium breve M-16V (NITE BP-02622), Bifidobacterium bifidum ATCC29521, Bifidobacterium adolescentis ATCC15703, Bifidobacterium angulatum ATCC27535, Bifidobacterium dentium DSM20436, Bifidobacterium psudocatenulatum ATCC27919, Bifidobacterium animalis subsp. lactis DSM10140, Bifidobacterium animalis subsp. animalis AT DSM10140), Bifidobacterium animalis subsp. animalis AT CC25527 (Bifidobacterium animalis subsp. animalis ATCC25527), Bifidobacterium pseudolongum subsp. globosum JCM5820, Bifidobacterium pseudolongum subsp. pseudolongm ATCC25526, Bifidobacterium thermophilum ATCC25525, and the like can be mentioned. As for the bacteria belonging to the genus Bifidobacterium, one kind or two or more kinds can be used. Also, one strain or two or more strains can be used.

[0070] The bacteria assigned with ATCC numbers can be obtained from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America). The bacteria assigned with DSM numbers can be obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (address: Inhoffenstraβe 7B, 38124 Braunschweig, Germany). The bacteria assigned with JCM numbers can be obtained from the Japan Collection of Microorganisms (Microbial Material Development Office, RIKEN BioResource Center, National Institute of Advanced Industrial Science and Technology, postal code: 305 - 0074, address: 3 - 1 - 1 Takano - dai, Tsukuba City, Ibaraki Prefecture).

[0071] Bifidobacterium longum subsp. longum BB536 (NITE BP-02621 was internationally deposited under the Budapest Treaty on January 26, 2018, with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu-shi, Chiba 292-0818, Japan) under the accession number NITE BP-02621. Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623) was internationally deposited under the Budapest Treaty on January 26, 2018, with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu-shi, Chiba 292-0818, Japan) under the accession number NITE BP-02623. Bifidobacterium breve FERM BP-11175 was internationally deposited under the Budapest Treaty on August 25, 2009, with the Patent Organisms Depositary, National Institute of Advanced Industrial Science and Technology (currently the Patent Microorganisms Depositary, National Institute of Technology and Evaluation; Room 120, 2-5-8 Kazusa Kamashima, Kisarazu-shi, Chiba 292-0818, Japan) under the accession number FERM BP-11175. Bifidobacterium breve M-16V (NITE BP-02622) was internationally deposited under the Budapest Treaty on January 26, 2018, with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu-shi, Chiba 292-0818, Japan) under the accession number NITE BP-02622.

[0072] Note that the Bifidobacterium longum subsp. longum BB536 (NITE BP-02621) of the present invention includes not only the strain itself that has been deposited or registered with a predetermined institution under the name of the bacterium (hereinafter, also referred to as the "deposited strain" for convenience of explanation), but also strains that are substantially equivalent thereto (also referred to as "derived strains" or "induced strains"). That is, "Bifidobacterium longum subsp. longum BB536 (NITE BP-02621)" includes not only the strain itself deposited with the above depository institution under the accession number NITE BP-02621, but also strains that are substantially equivalent thereto. The "strain substantially equivalent to the above deposited strain" belongs to the same species as the above deposited strain, the effects of the present invention can be obtained, and the nucleotide sequence of its 16S rRNA gene preferably has an identity of 98% or more, more preferably 99% or more, still more preferably 100% with respect to the nucleotide sequence of the 16S rRNA gene of the above deposited strain, and preferably has the same mycological properties as the above deposited strain. The strain substantially equivalent to the above deposited strain may be, for example, a derived strain having the above deposited strain as a parent strain. Examples of derived strains include strains bred from the deposited strain and strains naturally occurring from the deposited strain. Examples of breeding methods include modification by genetic engineering techniques and modification by mutagenesis treatment. Examples of mutagenesis treatment include irradiation with X-rays, irradiation with ultraviolet rays, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. Examples of strains naturally occurring from the deposited strain include strains that naturally occur during the use of the deposited strain. Examples of such strains include mutant strains that naturally occur by culturing the deposited strain (for example, subculture). Derived strains may be constructed by one kind of modification or by two or more kinds of modifications. This also applies to the above deposited strain to which an accession number has been assigned.

[0073] In the present invention, in the step of culturing, the cells of the Bifidobacterium bacterium to be cultured may be those previously cultured as follows. The culturing method is not particularly limited as long as the Bifidobacterium bacterium can grow. For example, the method usually used for culturing the Bifidobacterium bacterium can be used as it is or with appropriate modifications. The culturing temperature may be, for example, 25 to 50°C, preferably 35 to 42°C. The culturing can preferably be carried out under anaerobic conditions, for example, while aerating an anaerobic gas such as carbon dioxide gas. Also, the culturing can be carried out under microaerobic conditions such as liquid stationary culture. The culturing can be carried out, for example, until the Bifidobacterium bacterium grows to a desired extent.

[0074] The medium used for the cultivation at this time is not particularly limited as long as Bifidobacterium bacteria can grow. For example, a medium commonly used for culturing Bifidobacterium bacteria can be used as it is or with appropriate modifications. That is, as the carbon source, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and molasses can be used according to their assimilability. As the nitrogen source, for example, ammonium salts and nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate can be used. Also, as inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, etc. can be used. In addition, organic components such as peptone, soybean powder, defatted soybean meal, meat extract, and yeast extract may be used. Also, as a medium commonly used for culturing Bifidobacterium breve, specifically, Reinforced Clostridial medium, MRS medium (de Man, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium), TOSP medium (TOS propionate medium), and TOSP Mup medium (TOS propionate mupirocin medium) can be mentioned.

[0075] The medium used in the culturing step is not particularly limited as long as the same bacterial cells can produce aromatic lactic acid from aromatic amino acids and the fraction containing aromatic lactic acid recovered from the culture after culturing can be formulated into a food or beverage composition or a pharmaceutical composition and can be safely ingested by the subject, etc. For example, like that used in the examples described later, 5% Sweet whey powder, Examples of the milk medium include, but are not limited to, a milk medium containing SWP, a milk medium containing 5% skim milk (SM). As the carbon source, for example, saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and molasses can be used according to their assimilability. As the nitrogen source, for example, ammonium salts and nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate can be used. As the inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, etc. can be used. Also, organic components such as peptone, soybean powder, defatted soybean meal, meat extract, and yeast extract can be used as well.

[0076] The method for culturing the cells of the genus Bifidobacterium in the culturing step is not particularly limited as long as the cells can produce aromatic lactic acid from aromatic amino acids. Also, it is preferable that the cells can grow. As the culturing method, for example, the method usually used for culturing the genus Bifidobacterium can be used as it is or with appropriate modification. The culturing temperature can be, for example, 25 to 50 °C, preferably 35 to 42 °C. The culturing can preferably be carried out under anaerobic conditions, for example, by aerating an anaerobic gas such as carbon dioxide gas. Also, the culturing can be carried out under microaerobic conditions such as liquid static culture. The culturing can be carried out, for example, until the cells of the genus Bifidobacterium grow to a desired extent.

[0077] The method for producing the composition containing aromatic lactic acid according to the present invention includes a step of recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step.

[0078] The step of recovering is not particularly limited as long as a fraction containing aromatic lactic acid can be recovered from the culture obtained in the culturing step, and may include, for example, spray drying treatment, freeze drying treatment, heat treatment, centrifugation treatment, microfiltration treatment, ultrafiltration treatment, etc. Further, since aromatic lactic acid is contained in the culture supernatant of the culture obtained in the culturing step, the above treatment performed on the culture supernatant after the culturing step may be included.

[0079] The step of recovering may or may not include a step of removing the cells of the genus Bifidobacterium cultured in the culturing step, but it is preferably not included. Therefore, the fraction containing aromatic lactic acid recovered in the recovering step may or may not contain the cells of the genus Bifidobacterium cultured in the culturing step, but preferably contains them.

[0080] When the step of recovering does not include a step of removing the cells of the genus Bifidobacterium cultured in the culturing step, it may or may not include a treatment for killing the cells, but it is preferably not included, and a treatment with high viability of the cells is preferred. Therefore, when the fraction containing aromatic lactic acid recovered in the recovering step contains the cells of the genus Bifidobacterium cultured in the culturing step, the cells may be live cells, dead cells, or a mixture of both, but live cells are preferred and preferably contain live cells. This is because an effect as a probiotic can be expected when the cells are live cells and when live cells are included.

[0081] In addition, the method for measuring the amount of aromatic lactic acid contained in the culture obtained in the culturing step can follow a conventional method. For example, as in the examples described later, aromatic lactic acid can be extracted from the supernatant of the culture, derivatized, and the amount of aromatic lactic acid can be measured using GC-MS or the like.

[0082] Another aspect of the present invention is A method for increasing the production amount of aromatic lactic acid by cells of the genus Bifidobacterium, comprising A method comprising the step of culturing the cells in a medium containing one or more selected from the group consisting of a casein protein hydrolyzate, a whey protein hydrolyzate, and a reducing agent is provided.

[0083] By culturing cells of Bifidobacterium bacteria in a medium containing one or more selected from the group consisting of a casein protein hydrolyzate, a whey protein hydrolyzate, and a reducing agent, the production amount of aromatic lactic acid by the cells can be increased.

[0084] Bifidobacterium bacteria, aromatic lactic acid, casein protein hydrolyzate, whey For the step of culturing cells of whey protein hydrolyzate, reducing agent, and Bifidobacterium bacteria, the contents described above are incorporated by reference.

[0085] The production amount of aromatic lactic acid when the cells are cultured in a medium containing a casein protein hydrolyzate is preferably 1.2 times or more, more preferably 1.5 times or more, still more preferably 1.7 times or more, and even more preferably 2 times or more, relative to the production amount of aromatic lactic acid when the cells are cultured in a medium not containing a casein protein hydrolyzate. On the other hand, the higher the upper limit, the more preferable it is, but for example, it is 20 times or less.

[0086] The production amount of aromatic lactic acid when the cells are cultured in a medium containing a whey protein hydrolyzate is preferably 1.2 times or more, more preferably 1.5 times or more, still more preferably 1.7 times or more, and even more preferably 2 times or more, relative to the production amount of aromatic lactic acid when the cells are cultured in a medium not containing a whey protein hydrolyzate. On the other hand, the higher the upper limit, the more preferable it is, but for example, it is 20 times or less.

[0087] When the bacterial cells are cultured in a medium containing a reducing agent, the production amount of aromatic lactic acid is preferably 1.2 times or more, more preferably 1.5 times or more, still more preferably 1.8 times or more, relative to the production amount of aromatic lactic acid when the bacterial cells are cultured in a medium not containing a reducing agent. On the other hand, the higher the upper limit is, the more preferable it is. For example, it is 20 times or less.

[0088] When the bacterial cells are cultured in a medium containing a casein protein hydrolyzate and a reducing agent, the production amount of aromatic lactic acid is preferably 2 times or more, more preferably 2.5 times or more, still more preferably 3 times or more, even more preferably 3.5 times or more, relative to the production amount of aromatic lactic acid when the bacterial cells are cultured in a medium not containing a casein protein hydrolyzate and a reducing agent. On the other hand, the higher the upper limit is, the more preferable it is. For example, it is 30 times or less.

[0089] When the bacterial cells are cultured in a medium containing a whey protein hydrolyzate and a reducing agent, the production amount of aromatic lactic acid is preferably 2 times or more, more preferably 2.5 times or more, still more preferably 3 times or more, even more preferably 3.5 times or more, relative to the production amount of aromatic lactic acid when the bacterial cells are cultured in a medium not containing a whey protein hydrolyzate and a reducing agent. On the other hand, the higher the upper limit is, the more preferable it is. For example, it is 30 times or less.

[0090] Another aspect of the present invention is a processed product of bacterial cells containing indole-3-lactic acid, wherein the indole-3-lactic acid is 48 μg or more per 1 g on a solid content basis, and it is a processed product of bacterial cells of the genus Bifidobacterium. It is preferable that the indole-3-lactic acid is 50 μg or more per 1 g on a solid content basis, more preferably 60 μg or more, still more preferably 100 μg or more, and particularly preferably 150 μg or more.

[0091] Another aspect of the present invention is a processed product of bacterial cells containing 3-phenyl lactic acid, wherein the 3-phenyl lactic acid is 60 μg or more per 1 g on a solid content basis, and it is a processed product of bacterial cells of the genus Bifidobacterium. Preferably, the 3-phenyl lactic acid is 140 μg or more per 1 g on a solid content basis, more preferably 150 μg or more, still more preferably 200 μg or more, and particularly preferably 250 μg or more.

[0092] Another aspect of the present invention is a processed product of bacterial cells containing indole-3-lactic acid and 3-phenyl lactic acid, wherein the total amount of the indole-3-lactic acid and the 3-phenyl lactic acid is 110 μg or more per 1 g on a solid content basis, and it is a processed product of bacterial cells of the genus Bifidobacterium. Preferably, the total amount of the indole-3-lactic acid and the 3-phenyl lactic acid is 190 μg or more per 1 g on a solid content basis, more preferably 200 μg or more, still more preferably 300 μg or more, and particularly preferably 400 μg or more. Yes.

[0093] Another aspect of the present invention is a step of culturing bacterial cells of the genus Bifidobacterium in a medium containing one or more selected from the group consisting of a casein protein hydrolyzate, a whey protein hydrolyzate, and a reducing agent, a step of recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step, and a step of formulating the fraction recovered in the recovering step into a preparation using the fraction as an active ingredient, which is a method for producing a composition for preventing or improving inflammation.

[0094] Regarding the step of culturing bacterial cells of the genus Bifidobacterium in a medium containing one or more selected from the group consisting of a casein protein hydrolyzate, a whey protein hydrolyzate, and a reducing agent, and the step of recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step, the foregoing content is incorporated by reference.

[0095] The manufacturing method according to this aspect includes a step of formulating a fraction containing the aromatic lactic acid recovered in the step of recovering as an active ingredient.

[0096] In the formulation, according to a conventional method, the fraction containing the aromatic lactic acid recovered in the step of recovering can be formulated as an active ingredient.

[0097] By formulating a fraction containing the aromatic lactic acid recovered in the step of recovering as an active ingredient, a composition for preventing or improving inflammation can be obtained. The inflammation is preferably necrotic inflammation, and the necrotic inflammation is preferably necrotizing enteritis.

[0098] Also, as described in the background art, since aromatic lactic acid can activate AhR, the composition can be used not only for preventing or improving inflammation but also for other actions of activated AhR. That is, as uses, for suppressing excessive proliferation of intestinal stem cells, for repairing tissue damage, for suppressing the formation of colorectal cancer, for promoting the differentiation of goblet cells, for promoting the production of mucin, for improving intestinal barrier function, for regulating intestinal motility, for maintaining homeostasis in the intestine, for anti - inflammation, for promoting the production of L - 22, for preventing and improving infectious diseases, for antibacterial use, for reprogramming intraepithelial CD4 + T helper cells into immunoregulatory T cells, for enhancing the effect of nerve growth factor (NGF), for promoting nerve development, etc. can be mentioned.

[0099] It is also possible to include a fraction containing the aromatic lactic acid recovered in the step of recovering as an active ingredient in a food or drink composition and process it as a food or drink composition for preventing or improving inflammation as one aspect of the composition.

[0100] Such food and drink compositions may be in the form of liquids, pastes, gel-like solids, powders, etc., and may be food and drink products, including tablet confections, liquid foods, etc. In addition, for example, wheat flour products such as bread, macaroni, spaghetti, noodles, cake mixes, tempura batter, breadcrumbs, etc.; instant foods such as instant noodles, cup noodles, retort foods, canned cooked foods, microwave foods, instant soups and stews, instant miso soup and drinks, canned soups, freeze-dried foods, and other instant foods; agricultural processed products such as canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (grain processed products), etc.; fishery processed products such as canned fishery products, fish ham and sausage, fish paste products, seafood delicacies, tsukudani, etc.; livestock processed products such as canned livestock products and pastes, livestock meat ham and sausage, etc.; milk and dairy products such as processed milk, milk beverages, yogurts, lactic acid bacteria beverages, cheeses, ice creams, prepared powdered milk, creams, and other dairy products; fats and oils such as butter, margarines, vegetable oils, etc.; basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirins, vinegars, etc.; compound seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other compound seasonings; frozen foods such as raw material frozen foods, semi-cooked frozen foods, and cooked frozen foods; confectioneries such as caramels, candies, gummies, chewing gums, chocolates, cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectioneries, rice confectioneries, bean confectioneries, dessert confectioneries, jellies, and other confectioneries; preferred beverages such as carbonated beverages, natural fruit juices, fruit juice beverages, soft drinks with fruit juice, fruit pulp beverages, fruit drinks with fruit pieces, vegetable-based beverages, soy milk, soy milk beverages, coffee beverages, tea beverages, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages, and other preferred beverages; other commercially available foods such as baby food, furikake, nori for ochazuke, etc.; prepared powdered milk for baby care; enteral nutrition foods; special-purpose foods, health functional foods (foods for specified health use, nutritional functional foods, foods with functional claims); dietary supplements, etc.

[0101] In addition, it is also possible to include the fraction containing the aromatic lactic acid recovered in the above-mentioned recovery step as an active ingredient in a pharmaceutical composition and process it as a pharmaceutical composition for preventing or improving inflammation as one aspect of the composition.

[0102] The dosage form of the pharmaceutical composition is not particularly limited, and specifically, tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, ointments, patches, eye drops, nasal drops, etc. can be exemplified. Further, in formulating, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, diluents, surfactants, or solvents for injections, which are usually used as pharmaceutical carriers, can be used.

[0103] In addition, as the pharmaceutical carrier, various organic or inorganic carriers can be used according to the dosage form. Examples of the carrier in the case of solid preparations include excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, etc.

[0104] Examples of the excipient include sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium metasilicate aluminate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.

[0105] Examples of the binder include gelatin; polyvinylpyrrolidone; macrogol, etc. in addition to the above excipients.

[0106] Examples of the disintegrant include chemically modified starches or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, etc. in addition to the above excipients.

[0107] Examples of the lubricant include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as beeswax and carnauba wax; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and hydrated silicic acid; starch derivatives and the like.

[0108] Examples of the stabilizer include paraoxybenzoic acid esters such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like.

[0109] Examples of the flavoring and odor-correcting agent include sweeteners, acidulants, fragrances and the like. In addition, examples of the carrier used in the case of the liquid preparation for oral administration include solvents such as water, flavoring and odor-correcting agents and the like.

Examples

[0110] Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited to these examples.

[0111] [Example 1] Evaluation of the production amount of aromatic lactic acid In this example, the cells of Bifidobacterium longum subsp. longum BB536, the cells of Bifidobacterium longum subsp. infantis M-63, and the cells of Bifidobacterium lactis DSM10140 were cultured in a milk medium having the following composition, and 3-phenyl lactic acid (3-PLA), 4-hydroxyphenyl lactic acid (4-OH PLA), and indole-3-lactic acid (ILA) in the culture supernatant were measured to evaluate the production amount of aromatic lactic acid. T

[0112] (1) Preparation of Bacterial Culture Supernatant Each strain was anaerobically cultured at 37°C for 15 hours using Anaeropack in a medium containing 5%, 1%, and 1% of sweet whey powder (SWP), highly hydrolyzed casein protein, and moderately hydrolyzed whey protein, respectively.

[0113] (2) Extraction of Aromatic Lactic Acid from Culture Supernatant The supernatant was dispensed in 50 μl aliquots into 1.5 ml screw-cap tubes on ice, and 10 μl of 0.5 mg / ml 2-isopropylmalic acid (2-IS) was added to each tube as an internal standard. A mixed solvent of water:methanol:chloroform = 1:2.5:1 was prepared and 250 μl of it was added to each tube using a glass pipette. After thorough mixing by vortexing, the mixture was incubated at 37°C and 1200 rpm for 5 minutes using a block incubator with a shaking function. Centrifugation was performed at 4°C and 16000×g for 3 minutes, and 200 μl of the supernatant was recovered from each tube into 1.5 ml Eppendorf tubes. 178 μl of MilliQ water was added to the recovered supernatant, and after thorough mixing by vortexing, centrifugation was performed at 4°C and 16000×g for 3 minutes. 250 μl of the supernatant without absorbing the pellet was recovered into a 1.5 ml screw-cap tube and dried by treatment with a centrifugal evaporator for 3 hours or more.

[0114] (3) Derivatization of Aromatic Lactic Acid Methoxyamine hydrochloride prepared at 20 mg / ml using pyridine as a solvent was added to each of the dried samples in an amount of 80 μl. Treatment was performed using a sonicator for 10 minutes or more until no residue in the tube could be confirmed. After treatment at 30°C and 1200 rpm for 90 minutes or more using a block incubator with a shaking function, 40 μl of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added. Treatment was performed at 37°C and 1200 rpm for 30 minutes or more using a block incubator with a shaking function, followed by centrifugation at 16000×g for 3 minutes, and 70 μl of the supernatant was subjected to GC-MS analysis.

[0115] (4) Measurement of the amount of aromatic lactic acid using GC-MS The sample of (3) above was subjected to GC-MS. GC-MS used GC-MS QP2010 Ultra and Smart metabolite database (Shimadzu Corporation) and was quantified by the internal standard method. The measurement of the amount of aromatic lactic acid in GC-MS was performed under the following analysis conditions.

[0116]

Table 1

[0117] (5) Results B. longum BB536, B. infantis M-63, B. lactis DSM10140 T The production amounts of 3-PLA, 4-OH PLA, and ILA produced by each of the bacterial cells were measured (Figs. 1, 2, and 3). The bacterial cells of B. longum BB536 and the bacterial cells of B. infantis M-63 were found to produce a larger amount of aromatic lactic acid than the bacterial cells of B. lactis DSM10140. T

[0118] [Example 2] Examination of the culture medium In this example, an examination was conducted on the culture medium for high production of aromatic lactic acid by bacterial cells of the genus Bifidobacterium.

[0119] (1) Examination of the components in the culture medium For 5% SWP, 27 protein hydrolysates with different degrees of hydrolysis and origins were added, and the amount of aromatic lactic acid when B. longum BB536 was cultured in each medium for 9 hours was measured by GC-MS as in Example 1. As the 27 protein hydrolysates, casein protein hydrolysate, whey protein hydrolysate, peptides derived from rice protein (rice protein hydrolysate), or peptides derived from pea protein (pea protein hydrolysate) were used, and those with lower degrees of hydrolysis were used first and then those with higher degrees of hydrolysis. As a result of comparing the production amounts of aromatic lactic acid when each protein hydrolysate was used, it was found that the production amounts of 3-PLA and ILA increased when highly hydrolyzed casein protein hydrolysate was used. The top 5 protein hydrolysates with high production amounts of ILA and 3-PLA are shown in Table 2 and Table 3, respectively. In the subsequent studies, highly hydrolyzed casein protein hydrolysate A and moderately hydrolyzed whey protein hydrolysate B were used.

[0120]

Table 2

[0121]

Table 3

[0122] (2) Verification of the effect of reducing agents on the production amount of aromatic lactic acid In addition to the control medium containing 5% SWP and 2% highly hydrolyzed casein protein hydrolysate A, a medium obtained by further adding 0.6% trisodium citrate to the control medium, a medium obtained by further adding 0.05% L-cysteine hydrochloride to the control medium, and a medium obtained by further adding 1.0% sodium ascorbate to the control medium were each prepared. In each medium, B. longum BB536 was cultured for 9 hours, and the total amount of aromatic lactic acid (total amount of 3-PLA, 4-OH PLA, and ILA) in the medium was measured by GC-MS in the same manner as in Example 1. As a result, an increasing trend in the total amount of aromatic lactic acid was observed by adding a reducing agent to the medium (Figure 4). From the above, it is considered that the addition of the reducing agent is effective in increasing the total amount of aromatic lactic acid.

[0123] [Example 3] Verification of the production amount of aromatic lactic acid in a medium containing skim milk In this example, the effect on the production amount of aromatic lactic acid when using a medium containing skim milk was verified. Using 5% skim milk powder (Skim Milk, SM) as a control medium, a medium obtained by adding 2% highly decomposed casein protein hydrolyzate A to the control medium, a medium obtained by adding 2% highly decomposed casein protein hydrolyzate A and 0.6% trisodium citrate (cit) to the control medium, a medium obtained by adding 2% moderately decomposed whey protein hydrolyzate B to the control medium, and a medium obtained by adding 2% moderately decomposed whey protein hydrolyzate B and 0.6% trisodium citrate to the control medium were each prepared. B. longum BB536 was cultured in each medium for 9 hours, and the amounts of 3-PLA and ILA in the medium were measured by GC-MS in the same manner as in Example 1. As a result, similar to the examination results in SWP (Example 1, Example 2), it was suggested that the amounts of 3-PLA and ILA increased by the addition of protein hydrolyzate and / or reducing agent, and particularly the production amounts of 3-PLA and ILA increased when a highly decomposed casein protein hydrolyzate and a reducing agent were combined (Figure 5, Figure 6).

[0124] [Example 4] Recovery of the fraction containing aromatic lactic acid In the production of PCM (probiotics conditioned medium), as a step of recovering the fraction containing aromatic lactic acid from the culture supernatant, a step of dehydrating and drying by freeze-drying or spray-drying to make it powdered can be considered. Therefore, the amount of ILA and 3-PLA per 1 g of solid matter, and their total amounts were calculated (Table 4).

[0125] [Table 4]

[0126] From the above, in order to obtain a large amount of aromatic lactic acid produced by Bifidobacterium bacteria, with respect to the medium of milk components, (i) adding a highly decomposed protein hydrolyzate (ii) adding a reducing agent was considered preferable.

Claims

1. A method for producing a composition comprising aromatic lactic acid, comprising the steps of: Cultivating Bifidobacterium bacteria in a medium containing whey protein hydrolysate; recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step; Including, Manufacturing method.

2. The whey protein hydrolysate is A whey protein hydrolysate having a ratio of peptides having a molecular weight of less than 500 of 50% or more based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC), and / or Based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC), the whey protein hydrolysate has a ratio of peptides having a molecular weight of less than 500 of 30% or more and less than 50%. The method of claim 1 .

3. The method according to claim 1 or 2, wherein the medium contains casein protein hydrolysate.

4. The method according to any one of claims 1 to 3, wherein the culture medium contains a reducing agent.

5. The method according to any one of claims 1 to 4, wherein the aromatic lactic acid is one or more selected from the group consisting of 3-phenyl lactic acid, 4-hydroxyphenyl lactic acid, and indole-3-lactic acid.

6. The method according to any one of claims 1 to 5, wherein the recovering step comprises a spray-drying process or a freeze-drying process.

7. The method according to any one of claims 1 to 6, wherein the Bifidobacterium bacterium is Bifidobacterium longum subsp. longum BB536 (NITE BP-02621) and / or Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623).

8. A method for increasing the amount of aromatic lactic acid produced by Bifidobacterium bacteria, comprising: Cultivating the bacterial cells in a medium containing whey protein hydrolysate; method.

9. The whey protein hydrolysate is A whey protein hydrolysate having a ratio of peptides having a molecular weight of less than 500 of 50% or more based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC), and / or Based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC), the whey protein hydrolysate has a ratio of peptides having a molecular weight of less than 500 of 30% or more and less than 50%. The method according to claim 8.

10. Cultivating Bifidobacterium bacteria in a medium containing whey protein hydrolysate; recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step; and a step of formulating the fraction recovered in the recovering step as an active ingredient; Including, A method for producing a composition for preventing or ameliorating inflammation.

11. The whey protein hydrolysate is A whey protein hydrolysate having a ratio of peptides having a molecular weight of less than 500 of 50% or more based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC), and / or Based on the results of molecular weight distribution measurement by high performance liquid chromatography (HPLC), the whey protein hydrolysate has a ratio of peptides having a molecular weight of less than 500 of 30% or more and less than 50%. The method of claim 10.

12. The method according to claim 10 or 11, wherein the inflammation is necrotizing inflammation.

13. The method of claim 12, wherein the necrotizing inflammation is necrotizing enterocolitis.

14. The method according to any one of claims 10 to 13, wherein the composition is a food or drink composition.

15. The method according to any one of claims 10 to 13, wherein the composition is a pharmaceutical composition.

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