Method for producing a 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 technology]

[0002] Aromatic lactates are metabolized from aromatic amino acids by various microorganisms. The aromatic lactates 3-phenyllactate, 4-hydroxyphenyllactate, and indole-3-lactate are ligands for the aryl hydrocarbon receptor (AhR) and bind to AhR. This activates AhR.

[0003] Activated AhR is known to have various functions. For example, activated AhR is known to have the effect of suppressing the excessive proliferation of intestinal stem cells (ISCs), repairing tissue damage, and suppressing the formation of colon cancer (Non-Patent Document 1). In addition, activated AhR has the effect of promoting goblet cell differentiation and mucin production. It is also known to have the effect of improving the intestinal barrier function (Non-Patent Document 2).

[0004] In addition, activated AhR regulates intestinal motility and maintains homeostasis in the intestinal tract. It is known that it has applications (Non-Patent Document 3).

[0005] In addition, it is known that AhR ligands are decreased in patients with inflammatory bowel disease (IBD) (Non-Patent Document 4), so activated AhR may have an anti-inflammatory effect. It is thought that. Indoleacrylic acid (IA) also inhibits the proliferation and proliferation of peripheral blood mononuclear cells (PBMCs) activated by lipopolysaccharide (LPS). The activity of the antibody has been reported to suppress the secretion of IL-6 and IL-1β by PBMCs (Non-Patent Document 2). Activated AhR is thought to have anti-inflammatory effects.

[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, activated AhR senses the bacterial signal molecule (quorum-sensing (QS) molecule) of Pseudomonas aeruginosa, and is therefore considered to be effective in preventing and improving infectious diseases (Non-Patent Document 6).

[0007] Indole-3-lactic acid is also found in fungi such as Penicillium bacteria, Escherichia coli, Bacillus cereus, etc. It was also reported that AhR activated by indole-3-lactic acid derived from Lactobacillus reuteri is involved in the induction of antibacterial activity against various bacteria. + It is known to have the effect of reprogramming T helper cells into immunoregulatory T cells (non- Patent document 2). In addition, indole-3-lactic acid has the effect of enhancing the effects of nerve growth factor (NGF). Therefore, it is believed to have an effect of promoting neurodevelopment (Non-Patent Document 7).

[0008] Indole-3-lactic acid, an aromatic lactic acid, is a lactic acid that is highly soluble in L-cysteine-containing MRS medium. It is known that it is produced by Bifidobacterium spp. in humans, and that human infant-type Bifidobacterium spp. produce it in large amounts. Examples of such human infant-type Bifidobacterium spp. include Bifidobacterium longum subsp. longum BB536 (NITE BP-02621), Bifidobacterium longum longum ATCC 15707, Bifidobacterium longum subsp. Infantis, Bifidobacterium longum subsp. 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 cell experiments have suggested that it may be useful in preventing necrotizing enterocolitis (NEC) (Non-Patent Document 9). It is also known that administration of culture supernatant of Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623) to a subject changes the expression of genes related to anti-inflammation (Patent Document 1).

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

[0011] It has also been reported that the addition of a reducing agent to the medium increases the amount of amino acid production by Corynebacterium glutamicum (Non-Patent Document 10).It has also been speculated that the addition of a reducing agent to the medium promotes the production of fermentation products by Bifidobacterium bacteria, lowering the pH (Non-Patent Document 11). [Prior art documents] [Chartered documents]

[0012]

Patent Document 1

Patent document 2

Non-licensed literature

[0013] [Non-licensed document 1] Immunity, 49(2):353-362 (2018) [Non-licensed document 2] Nat. Com., 9(1):3294 (2018) [Non-licensed document 3] Nature, 578(7794):284-289 (2020)

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0014] An objective of the present invention is to provide a technique for producing aromatic lactic acid in higher yields than ever before. [Means for solving the problem]

[0015] The present inventors discovered that when cells of a certain Bifidobacterium bacterium are cultured in a medium containing a certain protein hydrolysate and / or a reducing agent, aromatic lactic acid can be produced in higher yields than in the past, and thus completed the present invention.

[0016] That is, the present invention provides a method for producing a composition containing aromatic lactic acid, Bifidobacterium cells are cultured in a medium containing one or more selected from the group consisting of casein protein hydrolysate, whey protein hydrolysate, and a reducing agent. Process, and recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step; The present invention provides a method of manufacturing a semiconductor device comprising the steps of:

[0017] The method further comprises the steps of: In a preferred embodiment, the casein protein hydrolysate is a highly degraded casein protein hydrolysate.

[0018] The method also includes the step of: In a preferred embodiment, the whey protein hydrolysate is a highly degraded whey protein hydrolysate.

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

[0020] In a preferred embodiment of the production method, 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.

[0021] In a preferred embodiment of the production method, the recovering step includes a spray drying process or a freeze drying process.

[0022] The production method further comprises the step of: In a preferred embodiment, the strain is Lactobacillus longum subsp. infantis M-63 (NITE BP-02623).

[0023] The present invention also provides a method for increasing the amount of aromatic lactic acid produced by cells of a Bifidobacterium bacterium, comprising: The method may include culturing the bacterial cells in a medium containing one or more selected from the group consisting of casein protein hydrolysate, whey protein hydrolysate, and a reducing agent.

[0024] The present invention can also provide a processed bacterial cell product containing indole-3-lactic acid, the processed bacterial cell product being a Bifidobacterium bacterium, in which the amount of indole-3-lactic acid is 48 μg or more per 1 g in terms of solid content.

[0025] The present invention can also provide a processed product of bacterial cells of the genus Bifidobacterium containing 3-phenyllactic acid, wherein the amount of 3-phenyllactic acid is 60 μg or more per 1 g in terms of solid content.

[0026] The present invention can also provide a processed product of bacterial cells of the genus Bifidobacterium, which contains indole-3-lactic acid and 3-phenyllactic acid, and the total amount of the indole-3-lactic acid and the 3-phenyllactic acid is 110 μg or more per 1 g of solid content.

[0027] The present invention also relates to a method for producing a bacterial cell of the genus Bifidobacterium, comprising the steps of: culturing cells of a bacterium of the genus Bifidobacterium in a medium containing one or more selected from the group consisting of a casein protein hydrolysate, a whey protein hydrolysate, and a reducing agent; 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; The present invention can provide a method for producing a composition for preventing or ameliorating inflammation, comprising:

[0028] In a preferred embodiment of the above-mentioned production method, the inflammation is necrotizing inflammation.

[0029] In a preferred embodiment of the above-mentioned production method, the necrotizing inflammation is necrotizing enterocolitis.

[0030] In a preferred embodiment of the above-mentioned production method, the composition is a food or drink composition.

[0031] In a preferred embodiment of the above-mentioned production method, the composition is a pharmaceutical composition. Effect of the Invention

[0032] According to the present invention, aromatic lactic acid can be produced in a higher yield than in the past. For example, when the bacteria of the genus Bifidobacterium are administered to a newborn baby who has a small amount of the bacteria in the intestine, individual differences occur in the colonization and metabolism of the bacteria in the intestine. However, if a composition rich in aromatic lactic acid can be produced according to the present invention, the newborn baby can be provided with a sufficient amount of aromatic lactic acid. [Brief description of the drawings]

[0033] [Figure 1] FIG. 2 is a diagram showing the results of Example 1 according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a diagram showing the results of Example 1 according to one embodiment of the present invention. [Diagram 3] FIG. 2 is a diagram showing the results of Example 1 according to one embodiment of the present invention. [Figure 4] FIG. 13 is a diagram showing the results of Example 2 according to an embodiment of the present invention. [Diagram 5] FIG. 13 is a diagram showing the results of Example 3 according to an embodiment of the present invention. [Figure 6] FIG. 13 is a diagram showing the results of Example 3 according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 expressed by weight (mass) unless otherwise specified.

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

[0036] The aromatic lactic acid of the present invention may be any one produced from aromatic amino acids by the bacterial cells of the Bifidobacterium genus. The aromatic lactic acid of the present invention may be one or more. Examples include 3-phenyllactic acid, 4-hydroxyphenyllactic acid, and indole-3-lactic acid. 3-phenyllactic acid is converted from tyrosine, 4-hydroxyphenyllactic acid is converted from phenylalanine, and indole-3-lactic acid is converted from tryptophan.

[0037] The casein protein hydrolysate of the present invention is any hydrolysate that can increase the amount of aromatic lactic acid produced from aromatic amino acids by culturing cells of Bifidobacterium. There are no particular limitations.

[0038] The average molecular weight of the casein protein hydrolysate in the present invention is determined based on the concept of number average molecular weight described below. The number average molecular weight, as described in, for example, the literature ("Basics of Polymer Science," edited by the Society of Polymer Science, pp. 116-119, Tokyo Kagaku Dojin Co., Ltd., 1978), indicates the average value of the molecular weight of a polymer compound based on different indices as follows: In other words, since polymeric compounds such as protein hydrolysates are heterogeneous substances and have a distribution of molecular weights, the molecular weight of protein hydrolysates must be expressed as an average molecular weight in order to handle them physicochemically, and the number average molecular weight (hereinafter sometimes abbreviated as Mn) is the average for the number of molecules.

[0039] In this specification, the average molecular weight of casein protein hydrolysate is measured and calculated by the following method: using high performance liquid chromatography, the hydrolysate was separated by elution with a polyhydroxyethyl aspartamide column (Poly LC, diameter 4.6 × 200 mm) in 20 mM sodium chloride, 50 mM glycerol, 1 ... The elution was performed with acid at a rate of 0.4 mL / min (see "High-Speed ​​Analysis of Proteins and Peptides" edited by Nobuo Ui et al.). (Liquid Chromatography", Chemical Special Issue No. 102, p. 241, Kagaku Dojin Co., Ltd., 1984). Detection is performed using a UV detector (Shimadzu Corporation), and the number average molecular weight is calculated by analyzing the data using a GPC analysis system (Shimadzu Corporation). As a standard for calculating the molecular weight, a protein and / or peptide with a known molecular weight may be appropriately used.

[0040] Protein hydrolysates generally contain free amino acids during production. In this specification, the free amino acid ratio is the ratio of the amount of free amino acids to the total amount of casein protein hydrolysate, and can be measured or calculated by the following method. For amino acids other than tryptophan, cysteine, and methionine, the sample is hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours, tryptophan is subjected to alkaline hydrolysis with barium hydroxide at 110°C for 22 hours, and cysteine ​​and methionine are treated with performic acid and then hydrolyzed with 6N hydrochloric acid at 110°C for 18 hours. Each is analyzed using an automatic amino acid analyzer (Hitachi, Model 835) and the masses of the amino acids are measured. The composition of each amino acid in the sample is measured by the above method, and the total mass of the amino acids in the sample is calculated by adding them up. The sample is then deproteinized with sulfosalicylic acid, and the mass of each remaining free amino acid is measured by the above method, and the total mass of the free amino acids in the sample is calculated by adding them up. From these values, the free amino acid content in the sample is calculated by the following formula. Free amino acid rate (mass%) = (mass of total free amino acids / mass of total amino acids) x 100

[0041] The casein protein used as the starting material for obtaining the casein protein hydrolysate may be, for example, various commercially available caseins, or caseins isolated from human, bovine, equine, ovine, caprine, or other milk by a standard method (e.g., isoelectric precipitation method), or caseins produced by genetic recombination technology, etc. Caseins are classified into α-casein, β-casein, and κ-casein, and any of these can be used in the present invention.

[0042] A general method for obtaining casein protein hydrolysate will be described below, but the method is not particularly limited thereto. The raw casein protein is dispersed and dissolved in water or warm water. There is no particular restriction on the concentration of the dissolving solution, but it is usually desirable to set the concentration in the range of about 5 to 15% in terms of protein 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 deterioration due to contamination by various bacteria. Next, an alkaline agent or an acid agent is added to the solution containing the casein protein, and the solution is sterilized. It is preferable to adjust the pH to or near the optimum pH of the hydrolase that uses H. The alkaline or acidic agent may be any alkaline or acidic agent that is acceptable for food or pharmaceutical use. Specifically, examples of alkaline agents include sodium hydroxide, potassium hydroxide, potassium carbonate, etc., and examples of acidic agents 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 is preferably an enzyme derived from an animal or a microorganism. The enzyme is preferably an endopeptidase. As the endopeptidase, various enzymes such as pancreatin, pepsin, trypsin, and elastase can be used. The term "derived from" means that the organism originally possesses the above-mentioned enzyme, 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 two or more types of enzymes are used, the enzyme reactions may be carried out simultaneously or separately. The solution to which the enzyme has been added is maintained at an appropriate temperature depending on the type of enzyme, for example, 30 to 60° C., preferably 45 to 55° C., to initiate hydrolysis of casein protein. The hydrolysis reaction time is determined by monitoring the decomposition rate of the enzyme reaction and continuing the reaction until a desired decomposition rate is reached.

[0044] The hydrolysis reaction is stopped by inactivating the enzyme in the hydrolysis solution, and can be carried out by a conventional heat inactivation treatment. The heating temperature and holding time of the heat inactivation treatment can be appropriately set to conditions that allow sufficient inactivation, taking into account the thermal stability of the enzyme used, and can be carried out, for example, at a temperature range of 80 to 130°C for a holding time of 30 minutes to 2 seconds. The pH of the obtained reaction solution can be adjusted to a range of 5.5 to 7 with an acid such as citric acid, if necessary.

[0045] The obtained solution containing the casein protein hydrolysate can be used as it is, or, if necessary, the solution can be concentrated by a known method to give a concentrate, which can then be dried by a known method to give a powder.

[0046] The casein protein hydrolysate of the present invention may be, for example, a highly hydrolyzed casein protein hydrolysate, a moderately hydrolyzed casein protein hydrolysate, or a low-hydrolyzed casein protein hydrolysate, which are classified according to the degree of hydrolysis. Either one or more of the above may be used. In addition, one or more selected from the group consisting of a highly hydrolyzed casein protein hydrolysate, a moderately hydrolyzed casein protein hydrolysate, and a low-hydrolyzed casein protein hydrolysate may be used.

[0047] The casein protein hydrolysate of the present invention is preferably one having a higher degree of hydrolysis, since the amount of aromatic lactic acid produced from aromatic amino acids by culturing the cells of Bifidobacterium bacteria is increased. That is, a moderately hydrolyzed casein protein hydrolysate is more preferable than a low-hydrolyzed casein protein hydrolysate, and a highly hydrolyzed casein protein hydrolysate is more preferable than a moderately hydrolyzed casein protein hydrolysate.

[0048] In this specification, the term "highly hydrolyzed casein protein hydrolysate" refers to a casein protein hydrolysate in which the proportion of peptides with 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). Specific examples include CU5000, C3500Ca, and MCH-30 (all manufactured by Morinaga Milk Industry Co., Ltd.). In addition to these, there are also other highly hydrolyzed casein protein hydrolysates. Mention may be made of the casein protein hydrolysate X.

[0049] The extensively hydrolyzed casein protein hydrolysate X can be prepared, for example, as follows. 10 kg of edible casein (Alacid 720, Fonterra, edible casein content: 88%) is dispersed in deionized water at a concentration of 10%, and dissolved by adjusting the pH to 7.0 with 10% sodium hydroxide solution. The resulting solution is heat sterilized at 80°C for 10 minutes, held at 55°C, adjusted to pH 9.0 with 10% sodium hydroxide solution, and hydrolyzed for 14 hours by adding 40 g of enzyme derived from Bacillus licheniformis (Amano Enzyme Co., Ltd.) and 400 g of Aspergillus oryzae (Shin Nippon Chemical Industry Co., Ltd.). The enzyme is then inactivated by heat treatment at 90°C for 10 minutes. The precipitate is then removed using diatomaceous earth silica 300SA (Chuo Silica Co., Ltd.), concentrated, and spray-dried to obtain a powdered highly decomposed casein protein hydrolysate X.

[0050] In this specification, moderately hydrolyzed casein protein hydrolysate refers to a casein protein hydrolysate 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] As used herein, the term "low-hydrolysis casein protein hydrolysate" refers to a casein protein hydrolysate 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 hydrolysate of the present invention is not particularly limited as long as it can increase the amount of aromatic lactic acid produced from aromatic amino acids by culturing cells of Bifidobacterium genus bacteria.

[0053] The average molecular weight of the whey protein hydrolysate in the present invention is calculated based on the concept of number average molecular weight, and the free amino acid rate is the ratio of the amount of free amino acids to the total amount of whey protein hydrolysate, and the measurement and calculation thereof are based on the above-mentioned explanations of the average molecular weight and the free amino acid rate of casein protein hydrolysate, respectively.

[0054] The whey protein used as the starting material for obtaining the whey protein hydrolysate can be, for example, one or a mixture of two or more selected from commercially available whey or whey separated by a known method from milk, skim milk, etc. (e.g., cheese whey, acid whey, membrane-separated whey, whey powder, desalted whey powder, etc.), or separated and purified whey protein concentrate (WPC), whey protein isolate (WPI), etc. The mixture can be mixed in any ratio. Furthermore, the whey protein may be derived from general milk or from plants such as soybeans (so-called "soybean whey" etc.), but is more preferably derived from milk, and even more preferably from cow's milk.

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

[0056] The aqueous whey protein solution is then subjected to a hydrolysis treatment, for example, an enzyme treatment, an acid treatment, an alkali treatment, a heat treatment, etc., and two or more of these treatments may be appropriately combined. The protease of the present disclosure may be derived from, for example, plants, animals, microorganisms, etc., and one or more of these may be used in combination. As the protease, an endoprotease is preferable. Examples of the endoprotease include serine proteases, metalloproteases, cysteine ​​proteases, and aspartic acid proteases, and one or more of these may be selected for use. It is preferred to use a protease. Proteases are further classified into alkaline proteases, neutral proteases and acidic proteases, of which neutral proteases are preferred.

[0057] The proteolytic enzyme may be a commercially available product. Examples of the proteolytic enzyme include Bioprase (manufactured by Nagase Biochemical Industries, Ltd.), Proleather (manufactured by Amano Enzyme Co., Ltd.), Protease S (manufactured by Amano Enzyme Co., Ltd.), PTN6.0S (manufactured by Novozymes Co., Ltd.), Savinase (manufactured by Novozymes Co., Ltd.), GODO BAP (manufactured by Godo Shusei ... Tease N (Amano Enzyme Co., Ltd.), GODO BNP (Godoh Shusei Co., Ltd.), Newt Examples of enzymes that can be used include enzyme ... Among these, one or more neutral proteases selected from subtilisin (eg, bioprase), trypsin (eg, PTN6.0S), and bachillolysin (eg, protease N) are preferred, and more preferably, these three types are used in combination.

[0058] The amount of endoprotease used relative to the whey protein is not particularly limited, and may be appropriately adjusted depending on the substrate concentration, enzyme titer, reaction temperature, reaction time, and the like. By appropriately adjusting the hydrolysis conditions using the protease, a whey protein hydrolysate having a specific average molecular weight range can be obtained. Before hydrolysis with the protease, the pH of the raw milk protein solution can be adjusted to the optimum pH for the enzyme to be used using food-safe salts such as potassium carbonate and sodium hydroxide. The reaction temperature of the protease is desirably within the optimum temperature range for the enzyme used. The reaction retention time of the proteolytic enzyme may be appropriately adjusted so as to achieve the specific non-protein nitrogen ratio. Hydrolysis with the protease may be terminated by inactivating the enzyme by heating, for example, for 1 to 3 seconds when inactivating at 100°C or higher (preferably 110 to 130°C), and for 3 to 40 minutes when inactivating at 60°C or higher but lower than 100°C.

[0059] In the production of the whey protein hydrolysate according to the present invention, when a solution with an unadjusted calcium concentration is hydrolyzed, the resulting hydrolyzed liquid may be desalted as described above to adjust the calcium concentration. The enzyme is then inactivated by heating in a conventional manner. The reaction heating temperature and reaction retention time can be appropriately set to conditions that allow sufficient inactivation, taking into consideration the thermal stability of the enzyme used. After inactivation by heating, the enzyme is cooled in a conventional manner and can be used as it is, or can be concentrated to obtain a concentrated liquid as necessary, and can also be dried to obtain a powder product.

[0060] Furthermore, when the aqueous whey protein solution is hydrolyzed by acid treatment or alkali treatment, the pH of the aqueous whey protein solution may be adjusted before treatment. In the case of treatment by pH adjustment, the pH of the aqueous whey protein solution is preferably pH 5 or less or pH 9 or more, more preferably pH 4 or less or pH 10 or more. The aqueous solution thus pH-treated can be left or stirred at room temperature for several minutes or more, preferably 5 minutes to 1 hour, to obtain an acid-treated or alkali-treated hydrolysate. Here, "room temperature" refers to about 4 to 40°C, preferably 10 to 30°C. The aqueous whey protein solution may be hydrolyzed by heat treatment. The pH of the aqueous whey protein solution may be unadjusted, or may be pH adjusted (specifically, acidic (pH 5 or less), neutral (pH 6 to 8), or alkaline (pH 8 or more)). The heat treatment may be performed at about 4 to 100° C. under the same conditions as those of the acid-alkali treatment.

[0061] The obtained whey protein hydrolysate can exert its effect even if used in an unpurified state, but may be further subjected to appropriate separation and purification by known methods. For example, the obtained whey protein hydrolysate can be subjected to molecular weight fractionation to obtain a whey protein hydrolysate that satisfies a specific range of average molecular weight and amino acid release rate. For molecular weight fractionation, for example, methods such as ultrafiltration and gel filtration can be used, which can increase 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 desired gel filtration agent for size exclusion chromatography may be used. Furthermore, in order to desalt or remove impurities, or to increase purity, known separation and purification methods (for example, ion exchange resins, etc.) may be used.

[0062] The whey protein hydrolysate of the present invention may be, for example, a highly degraded whey protein hydrolysate, a moderately degraded whey protein hydrolysate, or a low-degraded whey protein hydrolysate, according to the degree of hydrolysis. Either one or more of them may be used. In addition, one or more selected from the group consisting of a highly degraded whey protein hydrolysate, a moderately degraded whey protein hydrolysate, and a low-degraded whey protein hydrolysate may be used.

[0063] The whey protein hydrolysate of the present invention is preferably one having a higher degree of hydrolysis, since the amount of aromatic lactic acid produced from aromatic amino acids by culturing the cells of Bifidobacterium bacteria is higher. That is, the whey protein hydrolysate having a higher degree of hydrolysis is more preferable than the whey protein hydrolysate having a lower degree of hydrolysis, and the whey protein hydrolysate having a higher degree of hydrolysis is more preferable than the whey protein hydrolysate having a lower degree of hydrolysis.

[0064] In this specification, the term "highly degraded whey protein hydrolysate" refers to a whey protein hydrolysate in which the proportion of peptides with 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). Specific examples include WU2900 (manufactured by Morinaga Milk Industry Co., Ltd.).

[0065] In this specification, the term "moderately hydrolyzed whey protein hydrolysate" refers to a whey protein hydrolysate 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). (Manufactured by Dairy Products Co., Ltd.)

[0066] As used herein, a low-degradation whey protein hydrolysate refers to a whey protein hydrolysate 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).

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

[0068] The Bifidobacterium bacterium of the present invention is not particularly limited as long as it can produce aromatic lactic acid from aromatic amino acids. For example, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, Bifidobacterium lactic acid bacterium, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, Bifidobacterium longum subsp. longum ... Bacterium bifidum, Bifidobacterium adenovirus 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. Bifidobacterium longum subsp. longum is sometimes simply written as Bifidobacterium longum. Bifidobacterium longum subsp. infantis is sometimes simply written 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 breve ATCC15700, Bifidobacterium Bifidobacterium breve FERM BP-11175, Bifidobacterium breve M-16V (NITE BP-02622), Bifidobacterium bifidum ATCC29521, Bifidobacterium adolescentis Bifidobacterium adolescentis ATCC15703, Bifidobacterium angulatum ATCC27535 ), Bifidobacterium dentium DSM20436, Bifidobacterium psudocatenulatum ATCC27919, Bifidobacterium animalis subsp. lactis DSM10140 DSM10140), Bifidobacterium animalis subsp. animalis AT CC25527 (Bifidobacterium animalis subsp. animalis ATCC25527), Bifidobacterium pseudolongum subsp. globosum JCM5820 (Bifidobacterium pseudolongum subsp. pseudolongm ATCC25526), ​​Bifidobacterium pseudolongum subsp. pseudolongm ATCC25526, Bifidobacterium thermophilus ATCC25527, Bifidobacterium pseudolongum subsp. globosum JCM5820 (Bifidobacterium pseudolongum subsp. pseudolongm ATCC25526), Bifidobacterium thermophilum ATCC25525 and the like can be used. The Bifidobacterium bacteria can be one or more species. Also, one or more strains can be used.

[0070] Bacteria assigned ATCC numbers were obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America. ) can be obtained from Bacteria that have been assigned DSM numbers can be obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany. Bacteria that have been assigned JCM numbers can be obtained from the Japan Collection of Microorganisms (RIKEN BioResource Center, Microbial Materials Development Division, Postal Code: 305-0074, Address: 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture).

[0071] Bifidobacterium longum subsp. longum BB536 (NITE BP-02621) was deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on January 26, 2018. This was an international deposit made under the Budapest Treaty under the accession number NITE BP-02621. Bifidobacterium longum subsp. infantis M-63 (NITE BP-02623) was deposited at the National Institute of Technology and Evaluation (NITE BP-02623) Patent Microorganisms Deposit Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818) on January 26, 2018. ) under the accession number NITE BP-02623, and was made as an international deposit under the Budapest Treaty. Bifidobacterium breve FERM BP-11175 was deposited on August 25, 2009 at the National Institute of Advanced Industrial Science and Technology (currently the National Institute of Technology and Evaluation Patent Organism Depositary, Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under the accession number FERM BP-11175 as an internationally recognized international transfer agent under the Budapest Treaty. A deposit has been made. Bifidobacterium breve M-16V (NITE BP-02622) was deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on January 26, 2018. The international deposit was made under the Budapest Treaty with the accession number.

[0072] In addition, Bifidobacterium longum subsp. longum BB536 (NITE BP-02621) of the present invention is not limited to the strain deposited or registered with a specified institution under the name of the bacterium (hereinafter, for convenience of explanation, also referred to as the "deposited strain"), but also includes substantially equivalent strains thereto (also referred to as the "derived strain" or "derived strain"). In other words, "Bifidobacterium longum subsp. longum BB536 (NITE BP-02621)" is not limited to the strain deposited with the above depository institution under the accession number NITE BP-02621, but also includes substantially equivalent strains thereto. The term "strain substantially equivalent to the deposited strain" refers to a strain that belongs to the same species as the deposited strain, that can obtain the effects of the present invention, and that has an identity of preferably 98% or more, more preferably 99% or more, and even more preferably 100% in the base sequence of its 16SrRNA gene to the base sequence of the 16SrRNA gene of the deposited strain, and that preferably has the same mycological properties as the deposited strain. A strain substantially equivalent to the above-mentioned deposited strain may be, for example, a derived strain having the deposited strain as a parent strain. Examples of derived strains include strains bred from the deposited strain and strains that have naturally arisen from the deposited strain. Breeding methods include modification by genetic engineering techniques and modification by mutation treatment. Examples of mutation treatment include irradiation with X-rays, irradiation with ultraviolet rays, and modification by N-methyl-N'-nitrophenyl ether. Examples of such mutations include treatment with mutagens such as tro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate. Strains that have arisen naturally from the deposited strain include strains that have arisen naturally during use of the deposited strain. Such strains include mutant strains that have arisen naturally by culturing (e.g., subculturing) the deposited strain. Derivative strains may be constructed by a single modification, or by two or more modifications. This also applies to the above-mentioned deposited strains to which accession numbers have been assigned.

[0073] In the present invention, the cells of the Bifidobacterium bacteria to be cultured in the culturing step may be cultured in advance as follows. The culture method is not particularly limited as long as the Bifidobacterium bacteria can grow. For example, a method commonly used for culturing Bifidobacterium bacteria can be used as is or with appropriate modifications. The culture temperature may be, for example, 25 to 50°C, and preferably 35 to 42°C. The culture can be preferably performed under anaerobic conditions, for example, while aerating anaerobic gas such as carbon dioxide gas. The culture can also be performed under microaerobic conditions such as liquid stationary culture. The culture can be performed, for example, until the Bifidobacterium bacteria grow to a desired extent.

[0074] The medium used for the culture is not particularly limited as long as the Bifidobacterium bacteria can grow. For example, a medium normally used for the culture of Bifidobacterium bacteria can be used as it is or after appropriate modification. That is, as the carbon source, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and blackstrap molasses can be used depending on the assimilation. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. In addition, as the inorganic salt, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, and the like can be used. In addition, organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract can be used. In addition, media commonly used for culturing Bifidobacterium breve include, specifically, Reinforced Clostridial medium, de Man, Rogosa, and Sharpe medium (MRS medium), modified MRS medium (mMRS medium), TOS propionate medium (TOSP medium), and TOSP Mup medium (TOSP Mup). Examples of media include TOS propionate mupirocin medium.

[0075] The medium used in the culturing step is not particularly limited as long as the bacterial cell 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 that can be safely ingested by a subject. For example, 5% sweet whey powder (Sweet whey powder, Examples include milk media containing 5% skim milk (SWP) and milk media containing 5% skim milk (SM). Examples of the carbon source include, but are not limited to, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysates, and blackstrap molasses, depending on the assimilation ability. Examples of the nitrogen source include ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates. Examples of the inorganic salts include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract can also be used. It's fine.

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

[0077] The method for producing a 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 recovery step 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, freeze-drying, heating, centrifugation, microfiltration, ultrafiltration, etc. In addition, since aromatic lactic acid is contained in the culture supernatant of the culture obtained in the culturing step, the recovery step may include the above-mentioned treatment performed on the culture supernatant after the culturing step.

[0079] The recovery step may or may not include a step of removing the cells of the Bifidobacterium bacteria cultured in the culture step, but it is preferable that it does not include such a step. Therefore, the fraction containing aromatic lactic acid recovered in the recovery step may or may not include the cells of the Bifidobacterium bacteria cultured in the culture step, but it is preferable that it does include such a step.

[0080] When the recovery step does not include a step of removing the cells of the Bifidobacterium bacteria cultured in the culture step, the recovery step may or may not include a treatment that kills the cells, but it is preferable that the recovery step does not include the cells, and it is preferable that the recovery step is a treatment that increases the survival rate of the cells.Therefore, when the fraction containing aromatic lactic acid recovered in the recovery step includes the cells of the Bifidobacterium bacteria cultured in the culture step, the cells may be live cells, killed cells, or both, but live cells are preferred, and live cells are preferred.This is because when the cells are live cells or include live cells, the effect of probiotics can be expected.

[0081] The amount of aromatic lactic acid contained in the culture obtained in the culturing step can be measured according to a conventional method. For example, as in the examples described below, aromatic lactic acid can be extracted from the culture supernatant, 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 amount of aromatic lactic acid produced by Bifidobacterium bacteria, comprising: Cultivating the bacterial cells in a medium containing one or more selected from the group consisting of casein protein hydrolysate, whey protein hydrolysate, and a reducing agent. It is.

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

[0084] Bifidobacterium bacteria, aromatic lactic acid, casein protein hydrolysate, whey The above-mentioned contents are incorporated herein by reference with regard to the steps of culturing the protein hydrolysate, the reducing agent, and the cells of the Bifidobacterium bacteria.

[0085] The amount of aromatic lactic acid produced when the bacterial cells are cultured in a medium containing a casein protein hydrolysate is preferably at least 1.2 times, more preferably at least 1.5 times, even more preferably at least 1.7 times, and even more preferably at least 2 times, the amount of aromatic lactic acid produced when the bacterial cells are cultured in a medium not containing a casein protein hydrolysate. On the other hand, the upper limit is preferably as high as possible, for example, not more than 20 times.

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

[0087] The amount of aromatic lactic acid produced when the bacterial cells are cultured in a medium containing a reducing agent is preferably 1.2 times or more, more preferably 1.5 times or more, and even more preferably 1.8 times or more, compared to the amount of aromatic lactic acid produced when the bacterial cells are cultured in a medium not containing a reducing agent. On the other hand, the higher the upper limit, the better, but it is, for example, 20 times or less.

[0088] The amount of aromatic lactic acid produced when the bacterial cells are cultured in a medium containing a casein protein hydrolysate and a reducing agent is preferably at least 2 times, more preferably at least 2.5 times, even more preferably at least 3 times, and even more preferably at least 3.5 times, the higher the upper limit, the better, but is, for example, 30 times or less, compared to the amount produced when the bacterial cells are cultured in a medium not containing a casein protein hydrolysate and a reducing agent.

[0089] The amount of aromatic lactic acid produced when the bacterial cells are cultured in a medium containing a whey protein hydrolysate and a reducing agent is preferably at least 2 times, more preferably at least 2.5 times, even more preferably at least 3 times, and even more preferably at least 3.5 times, the higher the upper limit, the better, for example, 30 times or less, compared to the amount of aromatic lactic acid produced when the bacterial cells are cultured in a medium not containing a whey protein hydrolysate and a reducing agent.

[0090] Another aspect of the present invention is a processed product of bacterial cells containing indole-3-lactic acid, the processed product being a bacterial cell of the genus Bifidobacterium, in which the indole-3-lactic acid is present in an amount of 48 μg or more per gram in terms of solid content, preferably 50 μg or more, more preferably 60 μg or more, even more preferably 100 μg or more, and particularly preferably 150 μg or more per gram in terms of solid content.

[0091] Another aspect of the present invention is a processed product of bacterial cells containing 3-phenyllactic acid, the processed product being a bacterial cell of the genus Bifidobacterium, in which the 3-phenyllactic acid is 60 μg or more per 1 g in terms of solid content, preferably 140 μg or more, more preferably 150 μg or more, even more preferably 200 μg or more, and particularly preferably 250 μg or more per 1 g in terms of solid content.

[0092] Another aspect of the present invention is a processed product of bacterial cells containing indole-3-lactic acid and 3-phenyllactic acid, the total amount of the indole-3-lactic acid and the 3-phenyllactic acid being 110 μg or more per gram in terms of solid content. The total amount of the indole-3-lactic acid and the 3-phenyllactic acid is preferably 190 μg or more per gram in terms of solid content, more preferably 200 μg or more, even more preferably 300 μg or more, and particularly preferably 400 μg or more. stomach.

[0093] Another aspect of the present invention is A step of culturing Bifidobacterium bacteria in a medium containing one or more selected from the group consisting of casein protein hydrolysate, whey protein hydrolysate, and a reducing agent; 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; The present invention relates to a method for producing a composition for preventing or ameliorating inflammation, comprising the steps of:

[0094] The above-mentioned contents are incorporated herein by reference with regard to the step of culturing Bifidobacterium bacteria in a medium containing one or more selected from the group consisting of casein protein hydrolysate, whey protein hydrolysate, and a reducing agent, and the step of recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step.

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

[0096] In preparing a formulation, the fraction containing the aromatic lactic acid recovered in the recovery step can be used as an active ingredient to prepare a formulation according to a conventional method.

[0097] The aromatic lactic acid-containing fraction recovered in the recovering step is formulated as an active ingredient to obtain a composition for preventing or ameliorating inflammation. The inflammation is preferably necrotizing inflammation, and the necrotizing inflammation is preferably necrotizing enterocolitis.

[0098] In addition, as described in the Background Art, aromatic lactic acid can activate AhR, The composition is useful not only for preventing or improving inflammation, but also for other actions of activated AhR. That is, the compositions can be used for the following purposes: suppression of excessive proliferation of intestinal stem cells, repair of tissue damage, suppression of colon cancer formation, promotion of goblet cell differentiation, promotion of mucin production, improvement of intestinal barrier function, regulation of intestinal motility, maintenance of homeostasis in the intestinal tract, anti-inflammatory, promotion of L-22 production, prevention and improvement of infectious diseases, antibacterial, intraepithelial CD4 + To reprogram T helper cells into immunoregulatory T cells, to enhance the effect of nerve growth factor (NGF), and to promote neurodevelopment. Examples of reasons for this include advancing

[0099] The fraction containing the aromatic lactic acid recovered in the recovery process can be incorporated as an active ingredient into a food or beverage composition, and as one embodiment of the composition, it can be processed into a food or beverage composition for preventing or improving inflammation.

[0100] Such food and drink compositions may be in any form, such as liquid, paste, gel-like solid, powder, etc., and may be food and drink, including, in addition to tablet confectionery, liquid food, etc., wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.; instant foods such as instant noodles, cup noodles, retort / prepared foods, pre-cooked canned foods, microwave foods, instant soups / stews, instant miso soup / cleansing soups, canned soups, freeze-dried foods, and other instant foods; canned agricultural products, canned fruit, jams / mackerels, etc. - Agricultural processed products such as malts, pickles, boiled beans, dried agricultural goods, cereals (processed grain products), etc.; processed marine products such as canned seafood, fish ham and sausage, seafood paste products, marine delicacies, tsukudani (fried fish paste), etc.; processed livestock products such as canned livestock paste, livestock ham and sausage, etc.; dairy products such as processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, prepared milk powder, cream, other dairy products, etc.; fats and oils such as butter, margarines, vegetable oils, etc.; soy sauce, miso, sauces, processed tomato seasonings, mirin, etc., Basic seasonings such as vinegar; complex seasonings and foods such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen foods such as raw frozen foods, semi-cooked frozen foods, and cooked frozen foods; sweets such as caramel, candy, gummies, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, dessert sweets, jellies, and other sweets; beverages such as carbonated drinks, natural fruit juice, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit particles, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, and other beverages; other commercially available foods such as baby food, furikake, and ochazuke nori; infant formula; enteral nutrition; special purpose foods, health functional foods (foods for specified health uses, foods with nutritional functions, and foods with functional claims); and nutritional supplements.

[0101] In addition, the fraction containing the aromatic lactic acid recovered in the recovery step can be incorporated as an active ingredient into a pharmaceutical composition, and as one embodiment of the composition, it can be processed into a pharmaceutical composition for preventing or ameliorating inflammation.

[0102] The dosage form of the pharmaceutical composition is not particularly limited, and specific examples include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, ointments, patches, eye drops, and nasal drops. In addition, when preparing the pharmaceutical composition, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavorings, diluents, surfactants, and injection solvents that are usually used as pharmaceutical carriers can be used.

[0103] As the pharmaceutical carrier, various organic or inorganic carriers can be used depending on the dosage form. Examples of the carrier in the case of a solid preparation include an excipient, a binder, a disintegrant, a lubricant, a stabilizer, and a flavoring agent.

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

[0105] Examples of binders include gelatin, polyvinylpyrrolidone, macrogol, and the like, in addition to the above-mentioned excipients.

[0106] Examples of disintegrants include the above-mentioned excipients, as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone.

[0107] Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and geranium stearate; 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 silicic anhydride and silicic acid hydrate; starch derivatives, and the like.

[0108] Examples of stabilizers include paraoxybenzoates such as methylparaben and propylparaben. Examples of the solvent include esters; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; and sorbic acid.

[0109] Examples of flavoring agents include sweeteners, acidulants, and fragrances. In the case of a liquid preparation for oral administration, examples of the carrier used include a solvent such as water, a flavoring agent, and the like. EXAMPLES

[0110] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0111] [Example 1] Evaluation of aromatic lactic acid production In this example, the bacteria Bifidobacterium longum subsp. longum BB536, Bifidobacterium longum subsp. infantis M-63, and Bifidobacterium lactis DSM10140 were cultured in a milk medium having the following composition. T The bacteria were cultured and the amounts of 3-phenyllactic acid (3-PLA), 4-hydroxyphenyllactic acid (4-OH PLA), and indole-3-lactic acid (ILA) in the culture supernatant were measured. The production of aromatic lactic acid was evaluated.

[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% sweet whey powder (SWP), highly hydrolyzed casein protein hydrolysate, and moderately hydrolyzed whey protein hydrolysate, respectively.

[0113] (2) Extraction of aromatic lactic acid from culture supernatant The supernatant was dispensed into 1.5 ml screw-capped tubes (50 μl each) 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 added to each tube at 250 μl with a glass pipette. After thorough mixing with a vortex, the mixture was placed in a shaking broth. The mixture was mixed at 1,200 rpm for 5 minutes in a cold incubator at 37°C. Centrifuge at 16,000 x g for 3 minutes at 4°C. After that, 200 μl of the supernatant was collected from each tube into a 1.5 ml Eppendorf tube. 178 μl of MilliQ water was added to each supernatant, mixed thoroughly by vortexing, and centrifuged at 16,000×g at 4°C for 3 minutes. 250 μl of the supernatant was collected in a 1.5 ml screw-cap tube, being careful not to suck up the pellet, and dried by processing in a centrifugal evaporator for more than 3 hours. It solidified.

[0114] (3) Derivatization of aromatic lactic acid Methoxyamine hydrochloride, adjusted to 20 mg / ml in pyridine as a solvent, was added in an amount of 80 μl to each dried sample. The tubes were sonicated for 10 minutes or more until no residue was visible in the tubes. After treatment at 30°C and 1200 rpm in a shaking block incubator for 90 minutes or more, 40 μl of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added. The tubes were then treated at 37°C and 1200 rpm in a shaking block incubator for 30 minutes or more, centrifuged at 16000×g for 3 minutes, and 70 μl of the supernatant was analyzed by GC-MS. was provided.

[0115] (4) Measurement of aromatic lactic acid content using GC-MS The sample (3) was subjected to GC-MS. GC-MS was performed using a GC-MS QP2010 Ultra and a Smart metabolite database (Shimadzu Corporation), and quantification was performed by the internal standard method. The amount of aromatic lactic acid was measured by GC-MS under the following analytical conditions.

[0116] [Table 1]

[0117] (5) Results B. longum BB536, B. infantis M-63, B. lactis DSM10140 T 3-PLA produced by each of the bacteria The amounts of 4-OH PLA and ILA produced were measured (Figures 1, 2, and 3). B. longum BB536 and B. infantis M-63 were cultured in the presence of B. lactis DSM10140. T The amount of aromatic lactic acid produced was higher than that of the other bacteria. It was found that there are many

[0118] [Example 2] Culture medium examination In this example, a medium for enabling Bifidobacterium bacteria to produce aromatic lactic acid at a high level was examined.

[0119] (1) Examination of medium components 27 kinds of protein hydrolysates with different decomposition degrees and origins were added to 5% SWP, and B. longum BB536 was cultured in each medium for 9 hours. The amount of aromatic lactic acid was measured using GC-MS in Example 1. The 27 types of protein hydrolysates used were casein protein hydrolysate, whey protein hydrolysate, peptides derived from rice protein (rice protein hydrolysate), or peptides derived from pea protein (pea protein hydrolysate), and were selected from those with low to high degrees of hydrolysis. A comparison of the amount of aromatic lactic acid produced when each protein hydrolysate was used revealed that the amount of 3-PLA and ILA produced was higher when highly hydrolyzed casein protein hydrolysate was used. The top five protein hydrolysates with the highest ILA and 3-PLA production are shown in Tables 2 and 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 of aromatic lactate In addition to the control medium containing 5% SWP and 2% highly degraded casein protein hydrolysate A, A control medium further containing 0.6% trisodium citrate, a control medium further containing 0.05% A medium containing L-cysteine ​​hydrochloride and a control medium containing 1.0% sodium ascorbate were prepared. B. longum BB536 was cultured in each medium for 9 hours, and the total amount of aromatic lactic acid (the 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, it was observed that the addition of a reducing agent to the medium tended to increase the total amount of aromatic lactic acid (Figure 4). From the above, it is considered that the addition of a reducing agent is effective in increasing the total amount of aromatic lactic acid.

[0123] [Example 3] Verification of aromatic lactic acid production in a medium containing skim milk In this example, the effect of using a medium containing skim milk on the production of aromatic lactic acid was examined. A medium containing casein protein hydrolysate A, and a control medium containing 2% highly hydrolyzed casein protein hydrolysate A. The control medium was supplemented with 2% moderately hydrolyzed whey protein hydrolyzate B, and the control medium was supplemented with 2% moderately hydrolyzed whey protein hydrolyzate B and 0.6% trisodium citrate (cit). B. longum BB536 was cultured in each medium for 9 hours and then cultured in the same manner as in Example 1. The amounts of 3-PLA and ILA in the medium were measured by GC-MS in the same manner. As a result, similar to the results of the SWP study (Examples 1 and 2), the protein hydrolysate It was suggested that the amount of 3-PLA and ILA increased by adding and / or a reducing agent, and that the production of 3-PLA and ILA was particularly increased when the highly hydrolyzed casein protein hydrolysate was combined with a reducing agent (Figures 5 and 6).

[0124] [Example 4] Recovery of fractions containing aromatic lactic acid In the production of PCM (probiotic conditioned medium), aromatic lactic acid is extracted from the culture supernatant. The fraction containing the 3-PLA is recovered by freeze-drying or spray-drying, which removes water and pulverizes it. The 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, the following is required for the milk component medium: (i) Addition of highly degraded protein hydrolysates (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 casein protein hydrolysate; and recovering a fraction containing aromatic lactic acid from the culture obtained in the culturing step; Including, The casein protein hydrolysate is a casein protein hydrolysate having a proportion 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). Manufacturing method.

2. The method of claim 1 , wherein the medium contains whey protein hydrolysate.

3. A manufacturing method described in claim 1 or 2, wherein the culture medium contains a reducing agent.

4. The method according to any one of claims 1 to 3, 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.

5. The method according to any one of claims 1 to 4, wherein the recovering step comprises a spray-drying process or a freeze-drying process.

6. The Bifidobacterium bacterium is Bifidobacterium longum subsp. longum BB536 (NITE BP-02621) and / or Bifidobacterium longum sa The method according to any one of claims 1 to 5, wherein the strain is B. infantis M-63 (NITE BP-02623).

7. A method for increasing the amount of aromatic lactic acid produced by Bifidobacterium bacteria, comprising: Cultivating the bacterial cells in a medium containing a casein protein hydrolysate, The casein protein hydrolysate is a casein 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). It is a protein hydrolysate, method.

8. Cultivating Bifidobacterium bacteria in a medium containing casein 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, The casein protein hydrolysate is a casein protein hydrolysate having a proportion 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). A method for producing a composition for preventing or ameliorating inflammation.

9. The method of claim 8, wherein the inflammation is necrotizing inflammation.

10. The method according to claim 9, wherein the necrotizing inflammation is necrotizing enterocolitis.

11. The method according to any one of claims 8 to 10, wherein the composition is a food or drink composition.

12. The method according to any one of claims 8 to 10, wherein the composition is a pharmaceutical composition.

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