Fermentation accelerator for lactic acid bacteria

Abstract

The present invention relates to a fermentation promoter for lactic acid bacteria, which contains at least one organic acid selected from the group consisting of malic acid and fumaric acid.

Description

[0001] This application is a divisional application of an application filed on December 24, 2020, with application number 202080090813.8, and invention name “Fermentation promoter for lactic acid bacteria”.

[0002] Cross-reference to related applications

[0003] This patent application claims priority based on Japanese Patent Application No. 2019-239455 filed on December 27, 2019, and all disclosed contents in the above-mentioned prior patent application are incorporated herein by reference as a part of this specification. Technical Field

[0004] The present invention relates to a novel lactic acid bacteria fermentation promoter. Background Art

[0005] When industrially producing fermented products and metabolites based on microorganisms, the shortening of fermentation time is important in reducing manufacturing costs and health management. As a technology for shortening the fermentation time by promoting the fermentation of lactic acid bacteria, it is reported that the material that becomes the raw material of nucleic acid promotes the fermentation of lactic acid bacteria (patent document 1). In addition, patent document 2 records: a microbial productivity improver using organic acid extracts of cruciferous plant seeds as an effective ingredient. However, from the viewpoint of effectively producing fermented milk on an industrial scale, it is still necessary to simply promote the metabolism of lactic acid bacteria and the new technical means of fermentation.

[0006] On the other hand, with the increase of health awareness in recent years, it is sought to reduce the amount of food additives used to produce delicious fermented milk. In particular, succinic acid is a umami substance and has useful physiological functions such as weight gain inhibition, glucose tolerance improvement, and cancer proliferation inhibition, so it is often used as a food additive. From the viewpoint of attracting consumer demand, it is also preferable to produce succinic acid in fermented milk without using food additives.

[0007] In recent years, regarding the production mechanism of succinic acid, it has been reported that oxaloacetate is produced from pyruvic acid or phosphoenolpyruvic acid under anaerobic conditions by carbonic acid fixation, and succinic acid is produced through the reverse pathway of the TCA cycle (Non-Patent Document 1). However, there is no report that lactic acid bacteria can utilize the reverse pathway of the TCA cycle.

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent document 1: Tatsuo Hoshino, Microbiol. Cult. Coll., 2011, 27(2), p.83-88

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent Application Publication No. 2017-221157

[0013] Patent Document 2: Japanese Patent Application Laid-Open No. 04-141083 Summary of the invention

[0014] The present invention provides a novel technical means for promoting fermentation of lactic acid bacteria. In addition, the present invention provides a novel technical means for promoting production of succinic acid by lactic acid bacteria in fermented milk.

[0015] The present inventors have found that at least one organic acid selected from malic acid and fumaric acid can promote fermentation by lactic acid bacteria. Furthermore, the present inventors have found that the organic acid can promote the production of succinic acid by lactic acid bacteria in fermented milk. The present invention is based on the above findings.

[0016] The present invention includes the following inventions.

[0017] [1] A lactic acid bacteria fermentation promoter comprising at least one organic acid selected from malic acid and fumaric acid.

[0018] [2] The fermentation promoter according to [1], which is used in combination with a nucleic acid raw material.

[0019] [3] The fermentation promoter according to [2], wherein the nucleic acid raw material is at least one selected from formic acid and a compound having a purine skeleton in which a hydrogen atom is bonded to the carbon atom at the 2-position.

[0020] [4] The fermentation promoter according to any one of [1] to [3], wherein the lactic acid bacteria include bacteria of the genus Lactobacillus.

[0021] [5] The fermentation promoter according to [4], wherein the Lactobacillus bacteria is at least one selected from the group consisting of Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius and Lactobacillus pentosus.

[0022] [6] A lactic acid bacteria starter comprising lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid.

[0023] [7] The lactic acid bacteria starter according to [6], further comprising a nucleic acid raw material.

[0024] [8] A method for producing fermented milk, comprising fermenting with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.

[0025] [9] The method according to [8], which comprises carrying out lactic acid bacteria fermentation in the presence of the organic acid and the nucleic acid raw material.

[0026]

[10] The method according to [9], wherein the nucleic acid raw material is at least one selected from formic acid and a compound having a purine skeleton having a hydrogen atom bonded to the carbon atom at the 2-position.

[0027]

[11] The method according to any one of [8] to

[10] , wherein the fermented milk contains succinic acid.

[0028]

[12] The method according to

[11] , wherein the succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

[0029]

[13] Fermented milk obtained by the method according to any one of [8] to

[12] ,

[0030] The fermented milk contains lactic acid bacteria and succinic acid.

[0031]

[14] The fermented milk according to

[13] , wherein the succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

[0032]

[15] The fermented milk according to

[14] , wherein the content of the succinic acid is 0.15 mM or more relative to the total amount of the fermented milk.

[0033]

[16] The fermented milk according to any one of

[13] to

[15] , further comprising at least one organic acid selected from malic acid and fumaric acid.

[0034]

[17] A method for promoting lactic acid bacteria fermentation, comprising performing lactic acid bacteria fermentation in the presence of at least one organic acid selected from malic acid and fumaric acid.

[0035]

[18] The method for promoting lactic acid bacteria fermentation according to

[17] , further comprising a nucleic acid raw material.

[0036]

[19] The method for promoting lactic acid bacteria fermentation according to

[18] , wherein the nucleic acid raw material is at least one selected from formic acid and a compound having a purine skeleton having a hydrogen atom bonded to the carbon atom at the 2-position.

[0037]

[20] A method for producing a lactic acid bacteria starter, comprising culturing lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.

[0038]

[21] The method according to

[20] , which comprises culturing lactic acid bacteria in the co-presence of the organic acid and the nucleic acid raw material.

[0039]

[22] The method according to

[20] or

[21] , wherein the lactic acid bacteria starter contains lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid.

[0040]

[23] The method according to

[22] , wherein the lactic acid bacteria starter further contains succinic acid.

[0041]

[24] The method according to

[23] , wherein the succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

[0042]

[25] Fermented milk, wherein the content of succinic acid is 0.15 mM or more based on the total amount of the fermented milk.

[0043]

[26] The fermented milk according to

[25] , further comprising at least one organic acid selected from malic acid and fumaric acid.

[0044]

[27] The fermented milk according to

[25] or

[26] , wherein the succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

[0045]

[28] A method for producing lactic acid bacteria fermentation metabolites, comprising performing lactic acid bacteria fermentation in the presence of at least one organic acid selected from malic acid and fumaric acid.

[0046]

[29] The production method according to

[28] , wherein the lactic acid bacteria fermentation metabolite is extracellular polysaccharide (EPS).

[0047]

[30] A method for promoting the production of lactic acid bacteria fermentation metabolites (hereinafter also referred to as "production promotion method"), comprising performing lactic acid bacteria fermentation in the presence of at least one organic acid selected from malic acid and fumaric acid.

[0048]

[31] The method according to

[30] , wherein the lactic acid bacteria fermentation metabolite is extracellular polysaccharide (EPS).

[0049] According to the present invention, the fermentation of lactic acid bacteria can be promoted. In addition, according to the present invention, the production of succinic acid in fermented milk can be increased. The present invention can be advantageously used to promote the fermentation and metabolism of lactic acid bacteria, and to produce fermentation metabolites such as extracellular polysaccharides (EPS) and peptides in a short period of time. In addition, the present invention can be advantageously used to increase the production of succinic acid by lactic acid bacteria and reduce the amount of succinic acid added as a food additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a graph showing the change in EPS concentration with respect to the culture time in Test Example 9. DETAILED DESCRIPTION

[0051] Fermentation accelerator

[0052] One of the characteristics of the lactic acid bacteria fermentation promoter of the present invention is that it contains at least one organic acid selected from malic acid and fumaric acid.

[0053] The content of at least one organic acid selected from malic acid and fumaric acid in the fermentation accelerator is not particularly limited, and is, for example, 0.1 to 100% by mass, preferably 50 to 100% by mass, and more preferably 80 to 100% by mass.

[0054] As the raw material of the organic acid in the fermentation accelerator, a commercially available product as a food additive may be used, or a synthetic product, a preparation containing an organic acid, or the like may be used.

[0055] The fermentation promoter may contain either malic acid or fumaric acid, or both. The mass ratio of malic acid to fumaric acid (malic acid / fumaric acid) is not particularly limited, and is, for example, 0.1 to 10, preferably 0.2 to 5, more preferably 0.5 to 2, and even more preferably 0.6 to 1.5.

[0056] Malic Acid

[0057] The form of malic acid in the fermentation promoter is not particularly limited as long as it does not hinder the effect of the present invention, and it can be contained in the reagent in any form of free acid or salt. Examples of the above-mentioned salt include alkali metal salts such as potassium and sodium, and alkaline earth metal salts such as calcium and magnesium. The malic acid used in the culture is not limited to optical isomers, but is preferably L-malic acid.

[0058] Malic acid or its salt can be added in an amount of, for example, 0.001 to 75 mM, preferably 0.01 to 50 mM, more preferably 0.1 to 10 mM, and further preferably 0.5 to 10 mM relative to the total amount of the culture medium or raw milk. Therefore, malic acid is preferably contained in the fermentation promoter in such an amount as to be the above amount. Here, the total amount of the culture medium or raw milk refers to the total amount of all components other than the bacteria used in the culture, for example, the total amount of the culture medium or raw milk, malic acid and / or fumaric acid, and nucleic acid raw materials. The content of malic acid in the culture system of the present invention can be measured by high performance liquid chromatography (HPLC). Such a measurement can be carried out using a commercially available HPLC apparatus (e.g., manufactured by Shimadzu Corporation) and a column (e.g., ICSep ICE-ORH-801 (TRANSGENOMIC)). More specifically, the above measurement can be carried out under the following conditions. Analytical apparatus: LC20 system manufactured by Shimadzu Corporation, column: ICSep ICE-ORH-801, 6.5 mm ID×300 mm, two columns connected and used, mobile phase: 7.5 mM p-toluenesulfonic acid, reaction solution: 7.5 mM p-toluenesulfonic acid·150 μM EDTA (2NA)·30 mM Bis Tris, flow rate: 0.5 ml / min, injection volume: 10 μl, oven temperature: 55°C, detection: conductivity detector.

[0059] Fumaric Acid

[0060] The form of fumaric acid in the fermentation promoter is not particularly limited as long as it does not hinder the effect of the present invention, and can be contained in the reagent in any form of free acid or salt. Examples of the above-mentioned salt include alkali metal salts such as potassium and sodium, alkaline earth metal salts such as calcium, and ammonium salts.

[0061] In addition, the content of fumaric acid or its salt can be added in an amount of, for example, 0.001 to 10 mM, preferably 0.01 to 7.5 mM, more preferably 0.1 to 5 mM, and even more preferably 0.1 to 2.5 mM relative to the total amount of the culture medium or raw material milk. Therefore, fumaric acid is preferably contained in the fermentation promoter of the present invention in such an amount. The content of fumaric acid in the culture system of the present invention can be measured by the same method as malic acid.

[0062] Nucleic acid raw materials

[0063] For the fermentation promoter of the present invention, from the viewpoint of more effectively promoting the fermentation of lactic acid bacteria, it is preferably used in combination with a nucleic acid raw material. The nucleic acid raw material can be included as a constituent component of the fermentation promoter, or it can be used alone. As the above-mentioned nucleic acid raw material, there is no particular limitation as long as it does not hinder the effect of the present invention. As suitable examples, formic acid and a compound having a purine skeleton with a hydrogen atom bonded to the carbon atom at the 2nd position can be cited.

[0064] Formic acid is known as a raw material for the purine backbone of nucleic acid. The form of formic acid is not particularly limited as long as it does not hinder the effect of the present invention, and can be contained in the reagent in any form of free acid or salt. As the above-mentioned salt, alkali metal salts such as potassium and sodium, alkaline earth metal salts such as calcium, ammonium salts, etc. can be listed. The content of formic acid in the culture system of the present invention can be measured by the same method as malic acid.

[0065] The compound having a purine skeleton means a compound having the following structure (purine skeleton):

[0066]

[0067] [The numbers in formula (I) represent the position numbers of carbon atoms or nitrogen atoms]

[0068] As a substance with a basic skeleton. Compounds having a purine skeleton are generally also collectively referred to as purines. As compounds having a purine skeleton, typical examples include purine bases, purine nucleosides, purine nucleotides, or salts thereof.

[0069] The compound having a purine skeleton used as the nucleic acid raw material of the present invention has a purine skeleton in which a hydrogen atom is bonded to the carbon atom at position 2 (the carbon atom represented by 2 in the above formula (I)). Such a compound includes adenine and hypoxanthine (purine bases), purine nucleosides containing adenine and hypoxanthine as constituent elements, purine nucleotides containing adenine or hypoxanthine as constituent elements, and salts thereof.

[0070] Purine nucleosides are substances formed by the combination of purine bases and sugars (ribose, deoxyribose, etc.), and may be ribonucleosides or deoxyribonucleosides. Purine nucleosides containing adenine or hypoxanthine as constituent elements include, for example, adenosine and inosine (ribonucleosides), and deoxyadenosine and deoxyinosine (deoxyribonucleosides).

[0071] Purine nucleotides are substances formed by the combination of one or more phosphate groups with purine nucleosides, and may be ribonucleotides or deoxyribonucleotides. Purine nucleotides may be nucleoside monophosphates (nucleoside monophosphates), nucleoside diphosphates, or nucleoside triphosphates. Purine nucleotides comprising adenine or hypoxanthine as constituent elements include, for example, adenosine (adenosine monophosphate or adenosine monophosphate; AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), inosinic acid (inosine monophosphate or inosine monophosphate; IMP), inosine diphosphate (IDP), inosine triphosphate (ITP), deoxyinosine monophosphate (dIMP), deoxyinosine diphosphate (dIDP), and deoxyinosine triphosphate (dITP).

[0072] Compounds having a purine skeleton with a hydrogen atom bonded to the carbon atom at position 2 also include derivatives of purine bases, purine nucleosides, or purine nucleotides. In the present invention, "derivatives" refer to compounds in which the purine base part, sugar residue part, and / or phosphate part of a purine base, purine nucleoside, or purine nucleotide is chemically modified, or a substituent is introduced.

[0073] The compound having a purine skeleton with a hydrogen atom bonded to the carbon atom at position 2 may be a salt, such as adenine, hypoxanthine, or a salt of a purine nucleoside or purine nucleotide containing adenine or hypoxanthine as a constituent element. In the present invention, preferred salts include alkali metal salts (e.g., sodium salts, potassium salts), such as sodium adenylate and sodium inosinate, but are not limited to these.

[0074] The compound having a purine skeleton with a hydrogen atom bonded to the carbon atom at position 2 is selected from the group consisting of adenine, hypoxanthine, adenosine, inosine, deoxyadenosine, deoxyinosine, adenylic acid, inosinic acid, and their salts, preferably inosinic acid. It should be noted that the content of inosinic acid in the culture system of the present invention can be measured using a kit based on a fluorescence method, Inosine Assay Kit, CELL BIOLABS.

[0075] The fermentation promoter of the present invention may be used in combination with at least one, preferably 1 to 4, for example 1 to 3 or 1 to 2 of the above-mentioned compounds having a purine skeleton with a hydrogen atom bonded to the carbon atom at the 2-position.

[0076] When the nucleic acid material and the organic acid are used in combination, the mass ratio of the organic acid to the nucleic acid material (organic acid / nucleic acid material) is not particularly limited, and is, for example, 0.005 to 500, preferably 0.05 to 200, and more preferably 0.1 to 100.

[0077] The nucleic acid raw material can be added in an amount of, for example, 0.001 to 75 mM, preferably 0.01 to 50 mM, more preferably 0.1 to 10 mM, and even more preferably 0.5 to 2 mM relative to the total amount of the culture medium or raw material milk.

[0078] In addition, the agent of the present invention can be provided in the form of a reagent in which the above-mentioned components are mixed with additives acceptable to food hygiene or medicine as desired. As additives acceptable to food hygiene or medicine, aqueous media such as water, solvents, dissolution aids, lubricants, emulsifiers, isotonic agents, stabilizers, preservatives, preservatives, surfactants, regulators, chelating agents, pH regulators, buffers, excipients, thickeners, colorants, aromatics or spices, etc. can be listed.

[0079] The agent of the present invention may be in any form such as liquid, powder, granule, gel, solid, or encapsulated body.

[0080] The fermentation promoter can be produced by appropriately mixing at least one organic acid selected from malic acid and fumaric acid and other arbitrary components represented by nucleic acid. The obtained mixture can be further processed by dissolution in a solvent, powderization, granulation, gelation, solidification, encapsulation, etc. according to known formulation techniques.

[0081] Regarding the fermentation promoting effect of the present invention, lactic acid bacteria are cultured in raw milk to which a fermentation promoter is added to ferment it, and an index showing the progress of the fermentation state is investigated. As a result, it can be confirmed that the fermentation is progressing faster than the control (a group to which the fermentation promoter of the present invention is not added). As an index showing the progress of the fermentation state, there is no particular limitation, and for example, a decrease in the pH value of the fermented milk accompanied by an increase in the amount of lactic acid generated by the fermentation of lactic acid bacteria can be used as an index. As the above-mentioned pH, for example, pH 4.6. Here, for pH 4.6, it can be set to a pH at which fermentation is fully carried out and fermentation is terminated in the manufacture of conventional fermented milk. When the time required to reach pH 4.6 is shortened compared to the control, it can be judged that the fermentation promoter has a fermentation promoting effect on lactic acid bacteria. The pH can be measured using a commercially available pH meter.

[0082] Lactic acid bacteria

[0083] In the present invention, the lactic acid bacteria used for fermentation are not particularly limited as long as they do not hinder the effects of the present invention, and may be animal-derived lactic acid bacteria or plant-derived lactic acid bacteria.

[0084] As preferred milk-acid bacteria of the present invention, can list lactobacillus (Lactobacillus) genus bacterium, streptococcus (Streptococcus) genus bacterium, lactococcus (Lactococcus) genus bacterium, enterococcus (Enterococcus) genus bacterium, leuconostoc (Leuconostoc) genus bacterium and their combination, preferably comprise the milk-acid bacteria of lactobacillus bacterium.As the milk-acid bacteria that comprises lactobacillus bacterium, for example can list the combination of lactobacillus bacterium, lactobacillus bacterium and streptococcus bacterium, the combination of lactobacillus bacterium and lactococcus bacterium etc.These milk-acid bacteria for example can obtain from storage institutions such as ATCC etc., also can suitably use commercially available product.

[0085] Examples of the bacteria of the genus Lactobacillus include Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus pentosus, Lactobacillus kefiranofaciens, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus amylovorous, Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus salivarius. plantarum and Lactobacillus sakei etc.Preferred Lactobacillus bacteria are Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus pentosus etc.Herein, as preferred Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius and Lactobacillus pentosus, Lactobacillus acidophilus JCM 1132T strain, Lactobacillus gasseri JCM 1131T strain, Lactobacillus rhamnosus JCM 1136T strain, Lactobacillus reuteri JCM1112T strain, Lactobacillus salivarius JCM 1231T strain and Lactobacillus pentosus JCM 1558T strain etc. are listed respectively.

[0086] In addition, examples of Lactobacillus delbrueckii include Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus delbrueckii bulgaricus), Lactobacillus delbrueckii subsp. lactis (Lactobacillus delbrueckii lactic acid subspecies), Lactobacillus delbrueckii subsp. delbrueckii (Lactobacillus delbrueckii subspecies), Lactobacillus delbrueckii subsp. indicus (Lactobacillus delbrueckii indicus), etc. Preferred Lactobacillus delbrueckii is Lactobacillus delbrueckii subsp. bulgaricus, and more preferably Lactobacillus delbrueckii subsp. bulgaricus (2038 strain, OLL1073R-1 strain, P1902901 strain, OLL1171 strain, OLL1255 strain, OLL1247 strain, OLL205013 strain), etc. Examples of Lactobacillus kefiranofaciens include Lactobacillus kefiranofaciens subsp. kefirgranum, and Lactobacillus kefiranofaciens subsp. kefirgranum is preferred, and Lactobacillus kefiranofaciens subsp. kefirgranum JCM 8572T is preferred.

[0087] In addition, Lactobacillus acidophilus JCM 1132T strain, Lactobacillus gasseri JCM 1131T strain, Lactobacillus rhamnosus JCM 1136T strain, Lactobacillus reuteri JCM 1112T strain, Lactobacillus salivarius JCM 1231T strain, Lactobacillus pentosus JCM 1558T strain, and Lactobacillus kumiss-like subsp. kumiss-like JCM 8572T strain can be obtained from the Japan Collection of Microorganisms (RIKEN BRC-JCM, Japan) of the RIKEN BRC under the accession numbers JCM 1132T, JCM 1131T, JCM 1136T, JCM 1112T, JCM 1231T, JCM 1558T, and JCM 8572T, respectively.

[0088] Lactobacillus delbrueckii subsp. bulgaricus 2038 strain can be isolated from "Meiji Bulgarian Yogurt" (registered trademark) using a commercially available selective medium for Lactobacillus, and is stored at Meiji Co., Ltd. (Meiji Innovation Center, 1-29-1 Shichikoku, Hachioji, Tokyo, Japan, 192-0919).

[0089] Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain was deposited on February 22, 1999 at the Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, International Patent Organism Depository (IPOD, NITE) (1-1-1-chuo-6, Higashi-1-chome, Tsukuba-shi, Ibaraki, Japan), and then transferred to the international depository for storage and assigned the deposit number FERM BP-10741. It should be noted that, as described in Budapest Notification No. 282 (http: / / www.wipo.int / treaties / en / notifications / budapest / treaty_budapest_282.html), the Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (IPOD, AIST) has taken over the patent microorganism deposit business, so the National Institute of Advanced Industrial Science and Technology International Patent Organism Depository (IPOD, NITE) is now deposited under the deposit number FERM BP-10741 was deposited.

[0090] Lactobacillus delbrueckii subsp. bulgaricus strain P1902901 was stored by Meiji Co., Ltd. (Meiji Innovation Center, 1-29-1 Shichikoku, Hachioji, Tokyo, Japan, 〒192-0919).

[0091] The Lactobacillus delbrueckii subsp. bulgaricus OLL1171 strain was internationally deposited on March 13, 2013 under the reference number NITE BP-01569 at the National Institute for Technology Evaluation's Patent Microbiological Depository, an international depository institution under the Budapest Treaty.

[0092] Lactobacillus delbrueckii subsp. bulgaricus OLL1255 strain was internationally deposited on February 10, 2005 under the designation NITE BP-76 at the National Institute for Technology Evaluation Patent Microbiological Depository, an international depository institution under the Budapest Treaty.

[0093] The Lactobacillus delbrueckii subsp. bulgaricus OLL1247 strain was internationally deposited on March 6, 2014 under the reference number NITE BP-01814 at the National Institute for Technology Evaluation's Patent Microbiological Depository, an international depository institution under the Budapest Treaty.

[0094] The Lactobacillus delbrueckii subsp. bulgaricus OLL205013 strain was internationally deposited on February 3, 2017 under the reference number NITE BP-02411 at the National Institute for Technology Evaluation Patent Microbiological Depository, an international depository institution under the Budapest Treaty.

[0095] Examples of bacteria belonging to the genus Streptococcus include Streptococcus thermophilus and the like.

[0096] Examples of the bacteria belonging to the genus Lactococcus include Lactococcus lactis, Lactococcus plantarum, and Lactococcus raffinolactis.

[0097] Preferred examples of the combination of Lactobacillus bacteria and Streptococcus bacteria include Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus.

[0098] The mixing ratio of lactic acid bacteria, at least one organic acid selected from malic acid and fumaric acid, and nucleic acid raw material can be appropriately set according to the types and properties of lactic acid bacteria, culture medium, raw milk, temperature conditions and other fermentation conditions.

[0099] The mass ratio of lactic acid bacteria to at least one organic acid selected from malic acid and fumaric acid (lactic acid bacteria / organic acid) is, for example, 0.001 to 500,000, preferably 0.01 to 5,000, and more preferably 0.1 to 500.

[0100] The mass ratio of lactic acid bacteria to the nucleic acid raw material (lactic acid bacteria / nucleic acid raw material) is, for example, 0.1 to 11,000, preferably 1 to 11,000, more preferably 10 to 1,100, and even more preferably 50 to 550.

[0101] Lactic acid bacteria can be added in an amount of, for example, 0.001 to 5% by mass, preferably 0.01 to 2.5% by mass, more preferably 0.01 to 2% by mass, and still more preferably 0.1 to 1% by mass, based on the total amount of the culture medium or raw material milk.

[0102] Lactic acid bacteria fermentation agent

[0103] The organic acid selected from malic acid and fumaric acid as described above can be used together with lactic acid bacteria and used as a lactic acid bacteria starter. Therefore, according to a preferred embodiment of the present invention, a lactic acid bacteria starter can be provided, which comprises lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid.

[0104] Lactic acid bacteria starter includes: those prepared by culturing lactic acid bacteria with a culture medium (for example, an activated culture medium) and undergoing intermediate fermentation. The lactic acid bacteria starter preferably includes lactic acid bacteria and a culture medium for culturing the lactic acid bacteria as constituent elements. Therefore, the lactic acid bacteria starter may further include succinic acid. Here, the succinic acid is preferably an endogenous organic acid produced by the lactic acid bacteria. In addition, the lactic acid bacteria starter includes those directly inoculated into the raw milk that is the source of fermented milk, and also includes the next generation and subsequent lactic acid bacteria starters in which the lactic acid bacteria starter is inoculated into other culture media and the lactic acid bacteria are further multiplied (expanded).

[0105] Lactic acid bacteria starter is basically used to ferment raw milk to obtain fermented milk. It should be noted that the use of lactic acid bacteria starter includes, in addition to directly inoculating the lactic acid bacteria starter obtained by the present invention into raw milk, further culturing the lactic acid bacteria starter obtained by the present invention in a culture medium once or more, and inoculating the next generation and subsequent lactic acid bacteria starter after the culturing into raw milk.

[0106] Lactic acid bacteria fermentation agent can be used in combination with nucleic acid raw materials. Therefore, according to other aspects of the present invention, a lactic acid bacteria fermentation agent can be provided, which is used in combination with nucleic acid raw materials and contains lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid. The above-mentioned lactic acid bacteria fermentation agent is preferably used in combination with nucleic acid raw materials. In the above-mentioned combination, the nucleic acid raw materials can be added to the culture medium or raw milk together with the above-mentioned lactic acid bacteria fermentation agent as independent individuals, or the nucleic acid raw materials can be mixed in the above-mentioned lactic acid bacteria fermentation agent as a constituent component and used as a whole. By combining the lactic acid bacteria fermentation agent and the nucleic acid raw materials, the fermentation of lactic acid bacteria can be more effectively promoted. Therefore, according to one aspect, the above-mentioned lactic acid bacteria fermentation agent can also contain nucleic acid raw materials. Each aspect of malic acid, fumaric acid, nucleic acid raw materials, and lactic acid bacteria in the lactic acid bacteria fermentation agent can be the same as the description of the fermentation promoter of the present invention.

[0107] The number of viable lactic acid bacteria in the lactic acid bacteria starter is not particularly limited, but is, for example, 1.0×10 4 ~1.0×10 13 cfu / g, preferably 1.0×10 5 ~1.0×10 12 cfu / g, more preferably 1.0×10 6 ~1.0×10 11 cfu / g.

[0108] The mass ratio of lactic acid bacteria to organic acid, the mass ratio of lactic acid bacteria to nucleic acid raw material, the mass ratio of organic acid to nucleic acid raw material, and the mass ratio of malic acid to fumaric acid in organic acid in the lactic acid bacteria starter may be the same as those in the fermentation promoter.

[0109] The lactic acid bacteria starter can be produced from lactic acid bacteria and any components represented by the above-mentioned organic acid, nucleic acid raw material, and medium components. The details of a suitable method for producing the lactic acid bacteria starter are described below.

[0110] A preferred method for producing a lactic acid bacteria starter comprises a culture medium preparation step, a culture medium sterilization step, a lactic acid bacteria inoculation step, a culture step (culture medium fermentation step), and an organic acid or the like addition step.

[0111] The culture medium preparation process is a process for preparing a culture medium (for example, an activated culture medium) for inoculating lactic acid bacteria. The culture medium is not particularly limited as long as it does not hinder the effect of the present invention. A culture medium comprising milk components can be listed, for example, preferably comprising skim milk, skim condensed milk, skim milk powder (reduced skim milk), and protein hydrolyzates of these skim milk components, whey, whey concentrate, whey powder (reduced whey), and protein hydrolyzates of these whey components, raw milk, sterilized milk (whole milk), whole fat condensed milk, whole milk powder (reduced whole milk), and protein hydrolyzates of these whole milk components, etc., more preferably comprising skim milk, skim milk powder, and protein hydrolyzates of these skim milk components, whey, whey concentrate, whey powder, and protein hydrolyzates of these whey components, further preferably comprising skim milk, skim milk powder, whey, whey powder, etc. The culture medium. In addition, the above-mentioned culture medium can be the same as the raw milk described later. The above-mentioned culture medium preferably further comprises yeast extract. The above-mentioned culture medium can be prepared by a known method such as mixing, dissolving, dispersing, or suspending the above-mentioned components.

[0112] The medium sterilization process is a process for sterilizing the medium prepared in the medium preparation process by, for example, heating. In the sterilization process, the heating temperature and the heating time are adjusted to perform heat treatment to the extent that the miscellaneous bacteria in the medium can be killed. In the present invention, it is preferred that the medium is heated to above 80°C, above 90°C, above 95°C, or above 100°C. Heat sterilization can be performed using known methods. For example, in heat sterilization, heat treatment can be performed using a plate heat exchanger, a tubular heat exchanger, a steam jet heating device, a steam injection heating device, an electric heating device, an autoclave device, etc., or heat treatment can be performed using a tank with a jacket. It should be noted that the sterilization of the culture medium is not limited to heating, and can also be performed by known methods such as ultraviolet irradiation.

[0113] The lactic acid bacteria addition (inoculation) step is a step of adding (inoculating) lactic acid bacteria to the sterilized culture medium. As the lactic acid bacteria added to the culture medium, frozen bacteria (e.g., frozen concentrated bacteria, frozen pellets, freeze-dried powder, etc.) can be used. In the lactic acid bacteria addition step, lactic acid bacteria are preferably added to the culture medium at 0.05 mass % or more, more preferably at 0.05 to 10 mass %, and further preferably at 0.1 to 5 mass %.

[0114] The culturing step is a step of culturing lactic acid bacteria in a culture medium to proliferate the lactic acid bacteria and obtain a lactic acid starter. The culturing time of lactic acid bacteria is not particularly limited, and for example, 3 to 36 hours, preferably 5 to 30 hours, and more preferably 10 to 24 hours can be cited. It should be noted that, in the case of obtaining a lactic acid bacteria starter by culturing multiple times, the above-mentioned culturing time refers to the culturing time for one time.

[0115] In addition, during the culturing process, the temperature of the culture medium is preferably maintained within a fermentation temperature range of 30°C or above. In particular, the temperature of the culture medium is preferably maintained at 30 to 50°C, more preferably at 35 to 50°C. In addition, during the culturing process, it is preferred that the culture medium be allowed to stand in advance without stirring. Here, "standing" means that the culture medium is not stirred. For example, the container containing the culture medium may be moved, and as long as the culture medium is not stirred, it is considered "standing". In this way, by allowing the culture medium to stand between culturing processes, the proliferation of lactic acid bacteria can be promoted, and the time required to complete the culturing can be shortened.

[0116] The organic acid and the like addition step is a step of adding at least one organic acid selected from malic acid and fumaric acid and adding a nucleic acid raw material as desired. The organic acid and the like addition step can be implemented at any time before the culture step, during the culture step, or after the culture step, but from the viewpoint of shortening the culture time, it is preferably implemented before the culture step or during the culture step. The organic acid and the like addition step can be implemented, for example, by collecting a predetermined amount of lactic acid bacteria or a culture medium containing the lactic acid bacteria after the culture step, adding an organic acid to the lactic acid bacteria or a culture medium containing the lactic acid bacteria and adding a nucleic acid raw material as desired. In addition, the organic acid and the like addition step can be implemented, for example, by adding an organic acid to the culture medium before or during the culture step, and adding a nucleic acid raw material as desired. Here, before the culture step, there is no particular limitation, and it can be any time before the culture medium sterilization step, after the culture medium sterilization step, before the lactic acid bacteria addition step, after the lactic acid bacteria addition step, and at the same time as the addition of lactic acid bacteria. It is preferred that the added organic acid is dissolved in water to adjust the pH to 6.0 to 7.0.

[0117] Fermented milk

[0118] According to the present invention, fermented milk can be effectively produced using the above-mentioned fermentation promoter or lactic acid bacteria fermentation agent. Here, "fermented milk" refers to: "fermented milk" specified in the ministerial order on the composition standards of milk and dairy products (milk, etc.), including "dairy lactic acid bacteria beverages" and "lactic acid bacteria beverages", and also includes yogurt, etc. For example, fermented milk refers to: milk such as raw milk, cow's milk, special milk, raw goat's milk, sterilized goat's milk, raw sheep's milk, formula milk, low-fat milk, fat-free milk and processed milk, or milk containing equal to or more non-fat milk solids, fermented with lactic acid bacteria or yeast, made into a paste or liquid or frozen, including hard yogurt, soft yogurt (paste-like fermented milk) or drinkable yogurt (liquid fermented milk).

[0119] Generally, hard yogurt such as plain yogurt can be produced by filling raw materials into a container and then fermenting (post-fermentation) (also called "set yogurt"). On the other hand, soft yogurt and drinkable yogurt can be produced by micronizing and homogenizing fermented milk (pre-fermentation) and then filling it into a container (also called "stirred yogurt").

[0120] According to the present invention, by fermenting with lactic acid bacteria in the presence of malic acid, fumaric acid, and further in the presence of a desired nucleic acid raw material, succinic acid can be contained in a high content in fermented milk even without using food additives. Therefore, according to a preferred embodiment of the present invention, a fermented milk can be provided, which contains lactic acid bacteria and succinic acid, and the succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

[0121] The succinic acid content in the fermented milk is, for example, 0.15 mM or more, preferably 0.2 mM or more, more preferably 0.7 mM or more, further preferably 1 mM or more, and further preferably 3 mM or more relative to the total amount of the fermented milk. The preferred lower limit of the succinic acid content in the fermented milk of the present invention is 0.15 mM, preferably 1 mM, and more preferably 3 mM, and the preferred upper limit is 50 mM, more preferably 20 mM, and further preferably 15 mM. According to a preferred embodiment of the present invention, when Lactobacillus delbrueckii subspecies bulgaricus is used as lactic acid bacteria, the succinic acid content in the fermented milk is, for example, 1 mM or more, preferably 1.5 mM or more, and more preferably 3 mM or more relative to the total amount of the fermented milk. In addition, when Lactobacillus rhamnosus is used as lactic acid bacteria, the succinic acid content in the fermented milk is, for example, 0.5 mM or more, preferably 1 mM or more relative to the total amount of the fermented milk. In addition, when Lactobacillus salivarius is used as lactic acid bacteria, the succinic acid content in the fermented milk is, for example, 0.15 mM or more, preferably 0.2 mM or more relative to the total amount of the fermented milk. The content of succinic acid in the fermented milk of the present invention can be measured by high performance liquid chromatography (HPLC). Such a measurement can be easily performed by using a commercially available HPLC apparatus (e.g., manufactured by Shimadzu Corporation) and a column (e.g., ICSep ICE-ORH-801 (TRANSGENOMIC)). As the above-mentioned measurement, for example, it can be performed according to the following conditions. Analytical apparatus: LC20system manufactured by Shimadzu Corporation, column: ICSep ICE-ORH-801, 6.5mm ID×300mm, two connected and used, mobile phase: 7.5mM p-toluenesulfonic acid, reaction solution: 7.5mM p-toluenesulfonic acid·150μM EDTA (2NA)·30mM Bis Tris, flow rate: 0.5ml / min, injection volume: 10μl, oven temperature: 55°C, detection: conductivity detector.

[0122] Fermented milk can be suitably produced in the presence of an organic acid such as malic acid and fumaric acid and, if desired, a nucleic acid raw material, and therefore may contain malic acid, fumaric acid, and a nucleic acid raw material.

[0123] The content of malic acid in the fermented milk is, for example, 0.1 to 50 mM, preferably 0.1 to 45 mM, and more preferably 0.5 to 45 mM.

[0124] The content of fumaric acid in fermented milk is, for example, 0.1 to 10 mM, preferably 0.1 to 5 mM, and more preferably 0.5 to 1 mM.

[0125] The content of the nucleic acid raw material in the fermented milk is, for example, 0.0001 to 5% by mass, and preferably 0.0001 to 1.5% by mass.

[0126] The fermented milk of the present invention contains succinic acid at least twice, preferably at least 2.5 times, and more preferably at least 5 times, as compared to fermented milk to which at least one organic acid selected from malic acid and fumaric acid is not added during production. The upper limit is 30 times, preferably 20 times.

[0127] According to the present invention, fermented milk can be produced using the above-mentioned fermentation promoter or lactic acid bacteria fermentation agent in the presence of an organic acid such as malic acid, fumaric acid, and a nucleic acid raw material. Therefore, a method for producing fermented milk can be provided, which comprises: fermenting raw milk by lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid. In addition, according to a preferred embodiment, the method for producing fermented milk is to perform lactic acid bacteria fermentation in the coexistence of the above-mentioned organic acid and nucleic acid raw material.

[0128] More specifically, the method for producing fermented milk preferably includes a raw milk preparation step, a raw milk sterilization step, a lactic acid bacteria starter inoculation step, and a fermentation step.

[0129] The raw milk preparation process is a process for preparing raw milk inoculated with a lactic acid bacteria starter. "Raw milk" is the raw material of fermented milk such as yogurt, and is therefore also referred to as yogurt mixture, fermented milk mixture, etc. In the present invention, known raw milk can be appropriately used. Raw milk includes raw milk before sterilization and raw milk after sterilization.

[0130] Specific raw materials of raw milk may include raw milk, sterilized milk, skim milk, whole milk powder, skim milk powder, buttermilk, butter, cream, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (La), β-lactoglobulin (Lg), etc. Preheated gelatin, etc. may also be added as appropriate. Raw milk is well known and can be prepared according to a well-known method.

[0131] Preferred raw milks include raw milk, skim milk, skim milk powder, and cream, but are not limited to these. More preferred raw milks include skim milk and skim milk powder. The content of non-fat milk solids in the raw milk used in the present invention is not particularly limited as long as it does not hinder the effect of the present invention, and is preferably 6 to 11% by mass, and more preferably 7 to 10% by mass. In addition, the content of fat components in the raw milk is not particularly limited as long as it does not hinder the effect of the present invention, and can be exemplified as preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass, and further preferably 0.1 to 3% by mass, but is not limited to these.

[0132] The raw milk sterilization step is a step of sterilizing the raw milk prepared in the raw milk preparation step by, for example, heating. The raw milk is preferably sterilized. As the above-mentioned sterilization, for example, sterilization based on heating can be cited. As the sterilization, the heating temperature and the heating time are adjusted to perform heat treatment to the extent that the miscellaneous bacteria in the raw milk can be killed. For example, it is preferred to sterilize the raw milk at a high temperature of 80°C or more, preferably 90°C or more. A known method can be used for the heating treatment.

[0133] The lactic acid bacteria starter inoculation step is a step of inoculating (adding) the lactic acid bacteria starter into the raw milk. As the lactic acid bacteria starter, a lactic acid bacteria starter obtained by the manufacturing method of the lactic acid bacteria starter or a lactic acid bacteria starter prepared by a conventional method and frozen or dried after freezing can be used.

[0134] Here, at least one organic acid selected from malic acid and fumaric acid, and the nucleic acid raw material of any component are preferably added to the raw milk as a constituent component of the lactic acid bacteria starter, but may also be contained in the raw milk as an additive or as an endogenous component separately from the lactic acid bacteria starter. The amount of each component of the lactic acid bacteria, organic acid, and nucleic acid raw material added to the raw milk may be the same as the amount described in the above-mentioned fermentation promoter.

[0135] The fermentation process is a process of fermenting raw milk by a lactic acid bacteria starter. In the fermentation process, for example, fermented milk is obtained by fermenting raw milk inoculated with a lactic acid bacteria starter while maintaining it within a fermentation temperature range. In the present invention, the fermentation process can use a known method. Fermentation conditions such as the fermentation temperature can be appropriately adjusted in consideration of the types of raw milk, lactic acid bacteria (lactic acid bacteria starter), the type of fermented milk to be prepared, the flavor, etc. As a specific example, as a fermentation temperature, about 30 to 50°C can be listed. If it is a temperature within this range, lactic acid bacteria generally work easily, so fermentation can be carried out effectively. As the fermentation temperature at this time, it is preferably about 30 to 45°C, and more preferably about 35 to 43°C.

[0136] The fermentation time can be appropriately adjusted according to the lactic acid bacteria (lactic acid bacteria starter) used, the fermentation temperature, etc. For example, the pH in the fermented milk can be 4.6 as an indicator to adjust the fermentation time. As the above-mentioned fermentation time, there is no limitation, for example, 2 hours to 36 hours can be listed, preferably 2.5 hours to 24 hours, and more preferably 4 hours to 24 hours. The pH can be measured using a commercially available pH meter.

[0137] It should be noted that the apparatus and manufacturing conditions for producing fermented milk can be known. For example, as an apparatus for producing fermented milk, a fermentation chamber for fermentation after filling can be used for post-fermented products, and a fermentation tank for fermentation and a line filter and a homogenizer for breaking up fermented milk curd can be used for pre-fermented products. As manufacturing conditions, a deoxygenation device can be appropriately used.

[0138] As described above, succinic acid contained in fermented milk is an umami substance and an organic acid having useful physiological functions such as weight gain inhibition, glucose tolerance improvement, and cancer growth inhibition. Therefore, according to one embodiment, the fermented milk of the present invention can be provided in the form of a composition for improving umami taste, for inhibiting weight gain, for improving glucose tolerance, or for inhibiting cancer growth.

[0139] Other methods

[0140] As described above, according to the present invention, by combining at least one organic acid selected from malic acid and fumaric acid with a desired nucleic acid raw material, the fermentation of lactic acid bacteria can be promoted. Therefore, according to another embodiment of the present invention, a method for promoting the fermentation of lactic acid bacteria can be provided, which includes fermenting a culture medium (e.g., a culture medium containing milk components) or raw milk by lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid. In addition, according to another preferred embodiment, the method for promoting the fermentation of lactic acid bacteria includes fermenting lactic acid bacteria in the presence of the organic acid and the nucleic acid raw material.

[0141] As described above, according to the present invention, the fermentation and metabolism of lactic acid bacteria can be promoted, and fermentation metabolites can be produced in a short time. Therefore, according to a further other embodiment of the present invention, a method for producing lactic acid bacteria fermentation metabolites or a method for promoting production can be provided, which includes fermenting lactic acid bacteria such as a culture medium (for example, a culture medium containing milk components) or raw milk in the presence of at least one organic acid selected from malic acid and fumaric acid. In addition, according to another preferred embodiment, the above method includes fermenting lactic acid bacteria in the presence of the above organic acid and nucleic acid raw material. As the above fermentation metabolites, preferably succinic acid, extracellular polysaccharides (EPS), peptides, etc., more preferably extracellular polysaccharides (EPS).

[0142] In addition, according to a further embodiment of the present invention, at least one organic acid selected from malic acid and fumaric acid for promoting fermentation of lactic acid bacteria can be provided. In addition, according to a further preferred embodiment, the organic acid is used to ferment lactic acid bacteria in the presence of a nucleic acid raw material.

[0143] In addition, according to another embodiment of the present invention, use of at least one organic acid selected from malic acid and fumaric acid in the production of a fermentation promoter for lactic acid bacteria can be provided. In addition, according to another preferred embodiment, the organic acid is used in combination with a nucleic acid raw material.

[0144] According to another preferred embodiment of the present invention, at least one organic acid selected from malic acid and fumaric acid can be used in the production of a lactic acid bacteria starter. According to another preferred embodiment, the organic acid is used in combination with a nucleic acid raw material.

[0145] In addition, according to a further embodiment of the present invention, there can be provided a use of at least one organic acid selected from malic acid and fumaric acid in the manufacture of fermented milk, which comprises fermenting with lactic acid bacteria in the presence of the organic acid. In addition, according to a further preferred embodiment, the organic acid is used in combination with a nucleic acid raw material. According to a further preferred embodiment, the fermented milk contains succinic acid, which may be an endogenous organic acid produced by lactic acid bacteria.

[0146] The above-mentioned method for promoting fermentation of lactic acid bacteria, organic acid, and each form of use can be implemented according to the description of the fermentation promoter, lactic acid bacteria starter, and fermented milk production method of the present invention.

[0147] Example

[0148] Hereinafter, the present invention will be described in more detail by way of examples, but protection scope of the present invention is not limited to these examples. It should be noted that, unless otherwise specified, all ratios used in the present invention are based on mass. In addition, unless otherwise specified, the units and measuring methods described in this specification are based on JIS standards.

[0149] Test Example 1: Effect of malic acid or fumaric acid on Lactobacillus delbrueckii subspecies bulgaricus (hereinafter also referred to as Study on the fermentation promoting effect of "L. bulgaricus")

[0150] First, frozen bacteria of the following Lactobacillus delbrueckii subsp. bulgaricus strain were prepared.

[0151] (1) Lactobacillus germanica subspecies bulgaricus 2038 (hereinafter referred to as "2038")

[0152] (2) Lactobacillus germanica subspecies bulgaricus OLL1073R-1 (hereinafter also referred to as "OLL 1073R-1")

[0153] (3) Lactobacillus germanica subspecies bulgaricus P1902901 (hereinafter also referred to as "P1902901")

[0154] (4) Lactobacillus germanica subspecies bulgaricus OLL1171 (hereinafter also referred to as "OLL1171")

[0155] (5) Lactobacillus germanica subspecies bulgaricus OLL1255 (hereinafter also referred to as "OLL1255")

[0156] (6) Lactobacillus germanica subspecies bulgaricus OLL1247 (hereinafter also referred to as "OLL1247")

[0157] (7) Lactobacillus germanica subspecies bulgaricus OLL205013 (hereinafter also referred to as "OLL205013")

[0158] In the activation medium, the above-mentioned strain is activated and used. As the activation medium, a substance obtained by sterilizing a 10% by mass reduced skim milk medium containing 0.1% by mass yeast extract at 121°C for 7 minutes is used. Here, the 10% by mass reduced skim milk medium is a 10% by mass aqueous solution of skim milk powder (fat component 1% by mass, protein 34% by mass, lactose 54% by mass, ash 8% by mass, non-fat milk solids 96% by mass) (made by Meiji Co., Ltd.) (in the above-mentioned medium, lactose content is 5.4% by mass, non-fat milk solids 9.6% by mass). 0.1% by mass of frozen bacteria (relative to the amount of activation medium) is added to the above-mentioned activation medium, and static culture is carried out at 37°C for 16 hours to obtain an activation solution. 0.1% by mass of the obtained activation solution (relative to the amount of activation medium) is added to another activation medium, and static culture is carried out at 37°C for 16 hours to obtain a lactic acid bacteria starter.

[0159] Then, fermentation was performed using a fermentation medium as raw milk. As a fermentation medium, a 10% reduced skim milk medium to which formic acid was added in a manner to a final concentration of 1 mM was sterilized at 95°C. In addition, an aqueous fumaric acid solution or an aqueous malic acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added in the above-mentioned fermentation medium in a manner to a final concentration of 1 mM. The pH of the above-mentioned fermentation medium is about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated in the above-mentioned fermentation medium at 0.5% by mass (relative to the amount of the fermentation medium), and was statically cultured at 40°C for fermentation. The fermentation time is the time required to reach pH 4.6. The pH was measured using a commercially available pH meter. The results obtained are shown in Table 1. As can be seen from Table 1, the fermentation of all 7 strains of Lactobacillus bulgaricus was promoted by adding malic acid or fumaric acid. Malic acid and fumaric acid have almost the same fermentation promoting effect on L. bulgaricus. The fermentation shortening time varies depending on the strain, ranging from 40 minutes to 12 hours and 15 minutes.

[0160] [Table 1]

[0161] Table 1 Effects of malic acid and fumaric acid on the fermentation promotion of Lactobacillus delbrueckii subsp. bulgaricus

[0162]

[0163] Test Example 2-1: Malic acid and fumaric acid showing fermentation promoting effect on Lactobacillus bulgaricus 2038 strain Concentration in Research

[0164] Fermentation was carried out using a fermentation medium (raw milk). As the fermentation medium, a 10% reduced skim milk medium to which formic acid was added in a manner to a final concentration of 1 mM was sterilized at 95°C. In addition, an aqueous fumaric acid solution or an aqueous malic acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added to the above-mentioned fermentation medium in a manner to a final concentration as described in Tables 3 and 4. 0.5% of a lactic acid bacteria starter (relative to the amount of the fermentation medium) of Lactobacillus bulgaricus 2038 was inoculated into the above-mentioned fermentation medium, and fermentation was carried out by static culture at 40°C. The fermentation time is the time required to reach pH 4.6.

[0165] The concentration of organic acids after fermentation was determined as follows. 0.4 g of the obtained fermented product (fermented milk) was diluted 2-fold with pure water, 20 μL of Carey's reagent I (53.5% (w / v) zinc sulfate) was added and vortexed, and 20 μL of Carey's reagent II (17.2% (w / v) potassium cyanide) was added and vortexed. Centrifugation was performed at 4°C, 20620 g, and 10 minutes, and the supernatant was filtered with a 0.22 μm filter under the conditions of Table 2 and analyzed.

[0166] [Table 2]

[0167] Table 2 Organic acid analysis conditions

[0168]

[0169] The obtained results are shown in Tables 3 to 5. According to Tables 3 and 4, Lactobacillus bulgaricus 2038 strain confirmed the fermentation promoting effect in 0.01 to 2.5 mM fumaric acid and 0.01 to 50 mM malic acid. In addition, malic acid and fumaric acid showed the fermentation promoting effect even at such a low concentration of 0.01 mM. The organic acid concentration after fermentation was measured, and the concentration of added malic acid and fumaric acid decreased, while the concentration of succinic acid increased (Table 5). That is, it is suggested that the added malic acid and fumaric acid are metabolized in the direction of malic acid → fumaric acid → succinic acid through the reduced TCA pathway.

[0170] [Table 3]

[0171] Table 3 Fermentation promotion effect of fumaric acid on Lactobacillus delbrueckii subspecies bulgaricus 2038 strain

[0172]

[0173] [Table 4]

[0174] Table 4 Fermentation promotion effect of malic acid on Lactobacillus delbrueckii subspecies 2038

[0175]

[0176] [Table 5]

[0177] Table 5 Concentrations of malic acid, fumaric acid and succinic acid after fermentation of Lactobacillus delbrueckii subspecies bulgaricus 2038 strain

[0178]

[0179] Test Example 2-2: Production of succinic acid in Lactobacillus delbrueckii subspecies bulgaricus when malic acid and fumaric acid were added quantity

[0180] In the activation medium, the strains shown in Tables 6 and 7 were activated and used. As the activation medium, a substance obtained by sterilizing a 10% reduced skim milk medium containing 0.1% yeast extract at 121°C for 7 minutes was used. Here, the 10% reduced skim milk medium is a 10% aqueous solution of skim milk powder (fat component 1% by mass, protein 34% by mass, lactose 54% by mass, ash 8% by mass, non-fat milk solids 96% by mass) (made by Meiji Co., Ltd.) (in the above-mentioned medium, lactose content 5.4% by mass, non-fat milk solids 9.6% by mass). 0.1% by mass of frozen bacteria (relative to the amount of activation medium) was added to the above-mentioned activation medium, and the activation solution was obtained by static culture at 37°C for 16 hours. 0.1% by mass of the obtained activation solution (relative to the amount of activation medium) was added to another activation medium, and the lactic acid bacteria starter was obtained by static culture at 37°C for 16 hours.

[0181] Then, fermentation was performed using a fermentation medium as raw milk. As a fermentation medium, a 10% reduced skim milk medium to which formic acid was added in a manner to a final concentration of 1 mM was sterilized at 95°C. In addition, an aqueous fumaric acid solution or an aqueous malic acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added in the above-mentioned fermentation medium in a manner to a final concentration of 1 mM. The pH of the above-mentioned fermentation medium is about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated in the above-mentioned fermentation medium at 2.5% by mass (relative to the amount of the fermentation medium), and static culture was performed at 40°C for fermentation. The fermentation time is the time required to reach pH 4.6. The pH was measured using a commercially available pH meter. The results obtained are shown in Tables 6 and 7. According to Table 6, for any Lactobacillus delbrueckii subspecies bulgaricus strain, the concentration of succinic acid increased by adding malic acid and fumaric acid. Furthermore, according to Table 7, similarly to Test Example 1, the fermentation of all 7 strains of Lactobacillus bulgaricus was promoted by adding malic acid or fumaric acid.

[0182] [Table 6]

[0183] Table 6 Organic acid concentration in the fermentation product at the end of fermentation (pH 4.6)

[0184]

[0185] [Table 7]

[0186] Table 7 Effect of malic acid and fumaric acid on the fermentation promotion of Lactobacillus delbrueckii subsp. bulgaricus

[0187]

[0188] Test Example 3: Fermentation promotion of fumaric acid with respect to lactic acid bacteria other than Lactobacillus delbrueckii subsp. bulgaricus Research on the effect

[0189] First, standard strains of the following bacterial species other than Lactobacillus delbrueckii subsp. bulgaricus were prepared.

[0190] (8) Lactobacillus acidophilus JCM 1132T

[0191] (9) Lactobacillus gasseri JCM 1131T

[0192] (10) Lactobacillus rhamnosus JCM 1136T

[0193] (11) Lactobacillus reuteri JCM 1112T

[0194] (12) Lactobacillus salivarius JCM 1231T

[0195] (13) Lactobacillus pentosus JCM 1558T

[0196] The above strains are activated in an activation medium before use. As an activation medium, a substance obtained by sterilizing a 10% reduced skim milk medium containing 0.1% yeast extract at 121°C for 7 minutes is used. 1% of frozen bacteria (relative to the amount of the activation medium) is added to the above activation medium, and the mixture is statically cultured at 37°C for 24 hours to obtain an activation solution. 1% of the obtained activation solution (relative to the amount of the activation medium) is added to another activation medium, and the mixture is statically cultured at 37°C for 24 hours to obtain a lactic acid bacteria starter.

[0197] Then, fermentation was carried out using a fermentation medium (raw milk). As a fermentation medium, a substance obtained by sterilizing a 10% reduced skim milk medium to which formic acid was added in a manner to a final concentration of 1 mM was used. In addition, an aqueous fumaric acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added in the above-mentioned fermentation medium in a manner to a final concentration of 1 mM. 1% of the lactic acid bacteria starter (relative to the amount of the fermentation medium) of each strain activated in the above-mentioned fermentation medium was inoculated, and fermentation was carried out by static culture at 37°C. The fermentation time is the time required to reach pH 4.6. The results obtained are shown in Table 8. Fermentation is promoted in Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus pentosus by adding fumaric acid.

[0198] [Table 8]

[0199] Table 8 Fermentation promotion effect of fumaric acid

[0200]

[0201] Test Example 4: Fermentation promoting effect of malic acid on lactic acid bacteria other than Lactobacillus delbrueckii subspecies bulgaricus Fruit Research

[0202] First, standard strains of the following bacterial species were prepared.

[0203] (12) Lactobacillus salivarius JCM 1231T

[0204] (13) Lactobacillus pentosus JCM 1558T

[0205] (14) Lactobacillus kumiss subsp. kumiss JCM 8572T

[0206] The same procedure as in Test Example 3 was carried out except that malic acid was added instead of fumaric acid. The obtained results are shown in Table 9. The addition of malic acid promoted fermentation in Lactobacillus salivarius, Lactobacillus pentosus, and Lactobacillus kumiss-like subsp. kumiss-like.

[0207] [Table 9]

[0208] Table 9 Fermentation promotion effect of malic acid

[0209]

[0210] Test Example 5: Determination of the concentrations of malic acid, fumaric acid and succinic acid in Lactobacillus rhamnosus and Lactobacillus salivarius

[0211] The organic acid concentrations after the fermentation of Lactobacillus rhamnosus and Lactobacillus salivarius in Test Example 3 or 4 were measured in the same manner as in Test Example 2-1. The obtained results are shown in Table 10. The addition of malic acid and fumaric acid promoted the production of succinic acid in Lactobacillus rhamnosus and Lactobacillus salivarius.

[0212] [Table 10]

[0213] Table 10 Concentrations of malic acid, fumaric acid, and succinic acid after fermentation of Lactobacillus rhamnosus and Lactobacillus salivarius

[0214]

[0215] Test Example 6: Effect of fumaric acid on the fermentation promotion of Lactobacillus delbrueckii subsp. bulgaricus when inosinic acid is added Research

[0216] Frozen bacteria of the following Lactobacillus delbrueckii subsp. bulgaricus strains were prepared.

[0217] (1) Lactobacillus germanica subspecies bulgaricus 2038

[0218] (2) Lactobacillus germanica subspecies bulgaricus OLL1073R-1

[0219] (3) Lactobacillus germanica subspecies bulgaricus P1902901

[0220] (4) Lactobacillus germanica subspecies bulgaricus OLL1171

[0221] (5) Lactobacillus germanica subspecies bulgaricus OLL1255

[0222] (6) Lactobacillus germanica subspecies bulgaricus OLL1247

[0223] (7) Lactobacillus germanica subspecies bulgaricus OLL205013

[0224] The above strains are activated in an activation medium before use. As an activation medium, a substance obtained by sterilizing a 10% reduced skim milk medium containing 0.1% yeast extract at 121°C for 7 minutes is used. 1% of frozen bacteria (relative to the amount of the activation medium) is added to the above activation medium, and the mixture is statically cultured at 37°C for 24 hours to obtain an activation solution. 1% of the obtained activation solution (relative to the amount of the activation medium) is added to another activation medium, and the mixture is statically cultured at 37°C for 24 hours to obtain a lactic acid bacteria starter.

[0225] Then, fermentation was carried out using a fermentation medium (raw milk). As a fermentation medium, a substance obtained by sterilizing a reduced skim milk medium to which 10% inosinic acid was added at a final concentration of 1 mM at 95°C was used. In addition, an aqueous fumaric acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added to the above-mentioned fermentation medium in a manner to a final concentration of 1 mM. 1% of the lactic acid bacteria starter (relative to the amount of the fermentation medium) of each strain activated in the above-mentioned fermentation medium was inoculated, and the fermentation was carried out by static culture at 37°C. The fermentation time is the time required to reach pH 4.6. The pH was measured using a pH meter. The results obtained are shown in Table 11. According to Table 11, the fermentation of all 7 strains of Lactobacillus bulgaricus was promoted by adding fumaric acid.

[0226] [Table 11]

[0227] Table 11 Effect of fumaric acid on the fermentation promotion of Lactobacillus delbrueckii subsp. bulgaricus when inosinic acid was added

[0228]

[0229] Test Example 7: Effect of malic acid on the fermentation promotion of Lactobacillus delbrueckii subsp. bulgaricus when inosinic acid is added Research

[0230] Frozen bacteria of the following Lactobacillus delbrueckii subsp. bulgaricus strains were prepared.

[0231] (1) Lactobacillus germanica subspecies bulgaricus 2038

[0232] (2) Lactobacillus germanica subspecies bulgaricus OLL1073R-1

[0233] (3) Lactobacillus germanica subspecies bulgaricus P1902901

[0234] (4) Lactobacillus germanica subspecies bulgaricus OLL1171

[0235] (5) Lactobacillus germanica subspecies bulgaricus OLL1247

[0236] The same procedure as in Test Example 6 was carried out except that malic acid was added instead of fumaric acid. The obtained results are shown in Table 12. The addition of malic acid promoted fermentation in L. bulgaricus 2038 strain, OLL1073R-1 strain, P1902901 strain, OLL1171 strain, and OLL1247 strain.

[0237] [Table 12]

[0238] Table 12 Effect of malic acid on the fermentation promotion of Lactobacillus delbrueckii subsp. bulgaricus when inosinic acid was added

[0239]

[0240] Test Example 8: Effect of malic acid or fumaric acid on succinic acid production by Lactobacillus delbrueckii subsp. bulgaricus when inosinic acid was added The impact of life

[0241] In the activation medium, the strains shown in Tables 13 and 14 were activated and used. As the activation medium, a substance obtained by sterilizing a 10% by mass reduced skim milk medium containing 0.1% by mass yeast extract at 121°C for 7 minutes was used. Here, the 10% by mass reduced skim milk medium is a 10% by mass aqueous solution of skim milk powder (fat component 1% by mass, protein 34% by mass, lactose 54% by mass, ash 8% by mass, non-fat milk solids 96% by mass) (made by Meiji Co., Ltd.) (in the above-mentioned medium, lactose content 5.4% by mass, non-fat milk solids 9.6% by mass). 0.1% by mass of frozen bacteria (relative to the amount of activation medium) was added to the above-mentioned activation medium, and the activation solution was obtained by static culture at 37°C for 16 hours. 0.1% by mass of the obtained activation solution (relative to the amount of activation medium) was added to another activation medium, and the lactic acid bacteria starter was obtained by static culture at 37°C for 16 hours.

[0242] Then, fermentation was performed using a fermentation medium as raw milk. As a fermentation medium, a substance obtained by sterilizing a 10% reduced skim milk medium to which inosinic acid was added at a final concentration of 1 mM at 95°C was used. In addition, an aqueous fumaric acid solution or an aqueous malic acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added to the above-mentioned fermentation medium in a manner of a final concentration of 1 mM. The pH of the above-mentioned fermentation medium is about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated in the above-mentioned fermentation medium at 2.5% by mass (relative to the amount of the fermentation medium), and static culture was performed at 40°C for fermentation. The fermentation time is the time required to reach pH 4.6. The pH was measured using a commercially available pH meter. The results obtained are shown in Tables 13 and 14. According to Table 13, in a culture medium containing inosinic acid, in any Lactobacillus delbrueckii subspecies bulgaricus strain, the concentration of succinic acid increased by adding malic acid and fumaric acid. Therefore, not only in the medium with formic acid, but also in the medium with inosinic acid, by adding malic acid and fumaric acid, the succinic acid production of Lactobacillus delbrueckii subspecies bulgaricus increased. In addition, according to Table 14, as in Test Example 6, by adding malic acid or fumaric acid, the fermentation of all 7 strains of Lactobacillus bulgaricus was promoted.

[0243] [Table 13]

[0244] Table 13 Organic acid concentration in the fermentation product at the end of fermentation (pH 4.6)

[0245]

[0246] [Table 14]

[0247] Table 14 The fermentation promoting effect of malic acid and fumaric acid on Lactobacillus delbrueckii subsp. bulgaricus

[0248]

[0249] Experimental Example 9: Effect of malic acid or fumaric acid on the proliferation and metabolite production of Lactobacillus delbrueckii subsp. bulgaricus Impact

[0250] Prepare frozen bacteria of Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain.

[0251] The composition of the culture medium is based on Table 15 below. The raw material components other than anhydrous crystalline glucose were dissolved in Elix water, and after adjusting to pH 6.65 with sodium hydroxide, the volume was fixed to 70% of the weight of the medium input. Anhydrous crystalline glucose was dissolved in Elix water and the volume was fixed to 30% of the weight of the medium input. When adding malic acid or fumaric acid, it was dissolved in Elix water together with the raw material components other than anhydrous crystalline glucose to a final concentration of 4 mM and added.

[0252] The sugar solution and the raw material components other than sugar were sterilized by heat treatment in an autoclave at 110° C. for 1 minute, and then aseptically mixed before use.

[0253] [Table 15]

[0254] Table 155 Culture medium composition

[0255]

[0256] 1.5 kg of the above culture medium was adjusted to pH 5.4 using 6N potassium carbonate, and 0.30% by mass of frozen bacteria (relative to the amount of the culture medium) was inoculated. The upper surface was aerated with nitrogen at a flow rate of 0.5 L / min, and cultured at 37°C while stirring at 150 rpm.

[0257] The changes in EPS concentration with respect to culture time are shown in Figure 1 .

[0258] like Figure 1As shown, it was confirmed that when malic acid or fumaric acid was added, the EPS concentration increased compared with the control. It can be seen that this is because the addition of malic acid and fumaric acid can promote the production of EPS, which is a metabolite of Lactobacillus delbrueckii subsp. bulgaricus. In addition, when malic acid or fumaric acid was added, the EPS concentration increased in a short period of time compared with the control, thus confirming again that malic acid or fumaric acid promotes the fermentation of Lactobacillus delbrueckii subsp. bulgaricus.

[0259] The EPS concentration was measured under the following conditions.

[0260] Weigh 1.5 g of culture medium and add 10 g of ultrapure water (MilliQ). Add 100% trichloroacetic acid and mix thoroughly. Centrifuge (4°C, 13400 g, 10 minutes) and recover the supernatant. Add 5 ml of 10% trichloroacetic acid solution to the precipitate to suspend it, centrifuge again and recover the supernatant. Add twice the amount of cooled 99.5% ethanol as much as the supernatant, pour and mix, and let stand overnight at -20°C.

[0261] Centrifuge (4°C, 13400g, 20 minutes) and remove the supernatant, add 20 ml of cooled 66% ethanol and suspend the precipitate. Centrifuge (4°C, 13400g, 20 minutes) and remove the supernatant, air-dry to remove the ethanol, add ultrapure water and make the volume to 10 ml, and use the obtained product as the analysis sample.

[0262] The EPS concentration of the analyzed samples was determined using the phenol-sulfuric acid method.

[0263]

[0264]

[0265] To be filled in by the receiving office

[0266]

[0267] To be filled in by the International Bureau

[0268]

Claims

1. A lactic acid bacteria fermentation promoter comprising at least one organic acid selected from malic acid and fumaric acid.

2. The fermentation promoter according to claim 1, which is used in combination with a nucleic acid raw material.

3. The fermentation promoter according to claim 2, wherein The nucleic acid raw material is at least one selected from formic acid and a compound having a purine skeleton in which a hydrogen atom is bonded to the carbon atom at the 2-position.

4. The fermentation promoter according to any one of claims 1 to 3, wherein The lactic acid bacteria include bacteria of the genus Lactobacillus.

5. The fermentation promoter according to claim 4, wherein The bacteria of the genus Lactobacillus is at least one selected from the group consisting of Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus pentosus.

6. A lactic acid bacteria starter comprising lactic acid bacteria and at least one organic acid selected from the group consisting of malic acid and fumaric acid. 7 . The lactic acid bacteria starter according to claim 6 , further comprising a nucleic acid raw material.

8. A method for producing fermented milk, comprising fermenting with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid. 9 . The method according to claim 8 , comprising fermenting with lactic acid bacteria in the presence of the organic acid and the nucleic acid raw material.

10. The method according to claim 8 or 9, wherein: The fermented milk contains succinic acid.

11. The method according to claim 10, wherein: The succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

12. Fermented milk obtained by the method according to any one of claims 8 to 11, The fermented milk contains lactic acid bacteria and succinic acid.

13. The fermented milk according to claim 12, wherein The succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

14. The fermented milk according to claim 13, wherein The content of succinic acid is 0.15 mM or more based on the total amount of fermented milk. 15 . The fermented milk according to claim 12 , further comprising at least one organic acid selected from the group consisting of malic acid and fumaric acid.

16. A method for promoting lactic acid bacteria fermentation, comprising performing lactic acid bacteria fermentation in the presence of at least one organic acid selected from malic acid and fumaric acid. 17 . The method for promoting fermentation of lactic acid bacteria according to claim 16 , further comprising a nucleic acid raw material.

18. A method for producing a lactic acid bacteria starter, comprising culturing lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.

19. The method according to claim 18, comprising culturing lactic acid bacteria in the presence of the organic acid and the nucleic acid raw material.

20. The method according to claim 18 or 19, wherein: The lactic acid bacteria starter contains lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid.

21. The method according to claim 20, wherein: The lactic acid bacteria starter culture further comprises succinic acid.

22. The method according to claim 21, wherein: The succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

23. A fermented milk, wherein: The content of succinic acid is 0.15 mM or more based on the total amount of fermented milk. The fermented milk according to claim 23, further comprising at least one organic acid selected from the group consisting of malic acid and fumaric acid.

25. The fermented milk according to claim 23 or 24, wherein The succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

26. A method for producing lactic acid bacteria fermentation metabolites, comprising performing lactic acid bacteria fermentation in the presence of at least one organic acid selected from malic acid and fumaric acid.

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