Fermentation promoter for lactic acid bacteria

Abstract

The present invention provides a new technical means for promoting fermentation by lactic acid bacteria, and a new technical means for promoting succinic acid production by lactic acid bacteria in fermented milk. [Solution] Provided is a fermentation promoter for lactic acid bacteria, comprising at least one organic acid selected from malic acid and fumaric acid, for use in combination with a nucleic acid source, wherein the lactic acid bacteria is at least one species selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus kefiranofaciens subsp. kefirgranum, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus pentosus.

Description

Citation of Related Applications

[0001] This patent application claims the priority based on Japanese Patent Application No. 2019-239455 filed on December 27, 2019, and the entire disclosure content in such prior patent application is incorporated herein by reference.

Technical Field

[0002] The present invention relates to a novel fermentation promoter for lactic acid bacteria.

Background Art

[0003] In the industrial production of fermentation products and metabolites by microorganisms, shortening the fermentation time is important for reducing production costs and hygiene management. As a technology for promoting the fermentation of lactic acid bacteria and shortening the fermentation time, it has been reported that substances serving as raw materials for nucleic acids promote the fermentation of lactic acid bacteria (Patent Document 1). In addition, Patent Document 2 describes a microbial productivity improver containing an organic acid extract of Brassicaceae plant seeds as an active ingredient. However, from the viewpoint of efficiently producing fermented milk on an industrial scale, new technical means for simply promoting the metabolism and fermentation action of lactic acid bacteria are still needed.

[0004] On the other hand, with the recent increasing trend of health consciousness, it has been demanded to produce fermented milk with a good taste while reducing the usage amount of food additives. In particular, succinic acid is often used as a food additive because it is a umami substance and also has useful physiological functions such as an action of suppressing weight gain, an action of improving glucose tolerance, and an action of suppressing cancer cell proliferation. From the viewpoint of arousing consumers' demands, it is preferable that succinic acid is produced in fermented milk without using food additives.

[0005] In recent years, regarding the production mechanism of succinic acid, it has been reported that under anaerobic conditions, oxaloacetic acid may be produced from pyruvic acid or phosphoenolpyruvic acid via carbon dioxide fixation, and succinic acid may be produced through the reverse route of the TCA cycle (Non-Patent Document 1). However, there has been no report on the ability of lactic acid bacteria to utilize the reverse route of the TCA cycle.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

[0008] The present invention provides a new technical means for promoting the fermentation of lactic acid bacteria. Further, the present invention provides a new technical means for promoting the production of succinic acid by lactic acid bacteria in fermented milk.

[0009] The present inventors have now found that at least one organic acid selected from malic acid and fumaric acid can promote the fermentation of lactic acid bacteria. Furthermore, the present inventors have found that the above organic acid can promote the production of succinic acid by lactic acid bacteria in fermented milk. The present invention is based on such findings.

[0010] According to the present invention, the following inventions are included. [1] A fermentation promoter for lactic acid bacteria comprising at least one organic acid selected from malic acid and fumaric acid. [2]The fermentation promoter according to [1] for use in combination with a nucleic acid raw material. [3]The fermentation promoter according to [2], wherein the nucleic acid raw material is at least one selected from the group consisting of formic acid and a compound having a purine skeleton in which a hydrogen atom is bonded to the 2-position carbon atom. [4]The fermentation promoter according to any one of [1] to [3], wherein the lactic acid bacterium includes a bacterium belonging to the genus Lactobacillus. [5]The fermentation promoter according to [4], wherein the bacterium belonging to 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 bacterium starter comprising a lactic acid bacterium and at least one organic acid selected from malic acid and fumaric acid. [7]The lactic acid bacterium starter according to [6], further comprising a nucleic acid raw material. [8]A method for producing fermented milk, comprising subjecting a lactic acid bacterium to fermentation in the presence of at least one organic acid selected from malic acid and fumaric acid. [9]The method according to [8], comprising subjecting a lactic acid bacterium to fermentation in the coexistence of the organic acid and a nucleic acid raw material.

[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 in which a hydrogen atom is bonded to the 2-position carbon atom.

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

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

[12] The method according to

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

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

[12] , wherein the fermented milk contains a lactic acid bacterium and succinic acid.

[14] The fermented milk according to

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

[15] The fermented milk according to

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

[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.

[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.

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

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

[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 with a hydrogen atom bonded to the carbon atom at the 2-position.

[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.

[21] The method according to

[20] , comprising culturing lactic acid bacteria in the coexistence of the organic acid and the nucleic acid raw material.

[22] The method according to

[20] or

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

[23] The method according to

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

[24] The method according to

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

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

[26] The fermented milk according to

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

[27] The fermented milk according to

[25] or

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

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

[29] The production method according to

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

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

[31] The method according to

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

[0011] According to the present invention, the fermentation of lactic acid bacteria can be promoted. Further, according to the present invention, the production amount of succinic acid in fermented milk can be increased. The present invention can be advantageously used for promoting the fermentation and metabolism of lactic acid bacteria and producing fermentation metabolites such as extracellular polysaccharide (EPS) and peptides in a short time. In addition, the present invention can be advantageously used for increasing the production amount of succinic acid of lactic acid bacteria and reducing the addition amount of succinic acid as a food additive.

Brief Description of the Drawings

[0012]

Figure 1

[0013] Fermentation promoter One feature of the fermentation promoter of lactic acid bacteria of the present invention is that it comprises at least one organic acid selected from malic acid and fumaric acid.

[0014] In the fermentation promoter, the content of at least one organic acid selected from malic acid and fumaric acid is not particularly limited. For example, it is 0.1 to 100% by mass, preferably 50 to 100% by mass, and more preferably 80 to 100% by mass.

[0015] As the raw material of the organic acid in the fermentation promoter, commercially available products as food additives may be used, or synthetic products, preparations containing organic acids, etc. may be used.

[0016] The fermentation promoter may contain either one of malic acid and fumaric acid, or may contain both. The mass ratio of malic acid to fumaric acid (malic acid / fumaric acid) is not particularly limited, but for example, it is 0.1 to 10, preferably 0.2 to 5, more preferably 0.5 to 2, and even more preferably 0.6 to 1.5.

[0017] Malic acid The form of malic acid in the fermentation promoter is not particularly limited as long as it does not interfere with the effects of the present invention, and it may be contained in the agent in either the free acid or salt form. Examples of such salts include alkali metal salts such as potassium and sodium, or alkaline earth metal salts such as calcium and magnesium. The malic acid used in the culture may be either optical isomer, but preferably L-malic acid.

[0018] Malic acid or its salt can be added in an amount in the range of, for example, 0.001 to 75 mM, preferably 0.01 to 50 mM, more preferably 0.1 to 10 mM, even more preferably 0.5 to 10 mM, based on the total amount of the medium or raw milk. Therefore, it is preferable that malic acid is contained in the fermentation promoter in the above amount. Here, the total amount of the medium or raw milk refers to the total amount of all components other than the bacteria used for culturing, for example, the total amount of the medium or raw milk, malic acid and / or fumaric acid, and the nucleic acid raw material. The content of malic acid in the culture system of the present invention is measured by a high performance liquid chromatography (HPLC) method. Such measurement can be carried out by using a commercially available HPLC apparatus (for example, manufactured by Shimadzu Corporation) and a column (for example, ICSep ICE-ORH-801 (manufactured by TRANSGENOMIC)). More specifically, the above measurement can be carried out under the following conditions. Analyzer: LC20 system manufactured by Shimadzu Corporation, Column: ICSep ICE-ORH-801, 6.5 mm I.D. × 300 mm, used by connecting two columns in series, 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.

[0019] Fumaric acid The form of fumaric acid in the fermentation promoter is not particularly limited as long as the effects of the present invention are not impaired, and it may be contained in the agent in either the form of free acid or salt. Examples of such salts include alkali metal salts such as potassium and sodium, alkaline earth metal salts such as calcium, and ammonium salts.

[0020] Also, fumaric acid can be added in an amount such that the content of fumaric acid or its salt is, for example, in the range of 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, based on the total amount of the medium or raw 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 in the same manner as malic acid.

[0021] Nucleic acid raw material From the viewpoint of more effectively promoting the fermentation of lactic acid bacteria, the fermentation promoter of the present invention is preferably used in combination with a nucleic acid raw material. The nucleic acid raw material may be contained as a constituent component of the fermentation promoter or used separately. Such a nucleic acid raw material is not particularly limited as long as it does not interfere with the effects of the present invention, and suitable examples include formic acid and compounds having a purine skeleton in which a hydrogen atom is bonded to the 2-position carbon atom.

[0022] Formic acid is known as a raw material constituting the purine skeleton of nucleic acids. The form of formic acid is not particularly limited as long as it does not interfere with the effects of the present invention, and it may be contained in the agent in either the free acid or salt form. Such salts include alkali metal salts such as potassium and sodium, alkaline earth metal salts such as calcium, and ammonium salts. The content of formic acid in the culture system of the present invention can be measured in the same manner as malic acid.

[0023] The compound having a purine skeleton refers to the following structure (purine skeleton):

Chemical formula

[0024] 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 the 2-position of the purine skeleton (the carbon atom represented by 2 in the above formula (I)). Such compounds include 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.

[0025] A purine nucleoside is a substance in which a purine base and a sugar (such as ribose or deoxyribose) are bonded, and it may be a ribonucleoside or a deoxyribonucleoside. Examples of purine nucleosides containing adenine or hypoxanthine as constituent elements include adenosine and inosine (ribonucleosides), and deoxyadenosine and deoxyinosine (deoxyribonucleosides).

[0026] A purine nucleotide is a substance in which one or more phosphate groups are bonded to a purine nucleoside, and it may be a ribonucleotide or a deoxyribonucleotide. A purine nucleotide may be a nucleoside monophosphate (nucleoside monophosphoric acid), a nucleoside diphosphate, or a nucleoside triphosphate. Examples of purine nucleotides containing adenine or hypoxanthine as constituent elements include adenylate (adenosine monophosphate or adenosine monophosphoric acid; AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), inosinate (inosine monophosphate or inosine monophosphoric acid; IMP), inosine diphosphate (IDP), inosine triphosphate (ITP), deoxyinosine monophosphate (dIMP), deoxyinosine diphosphate (dIDP), and deoxyinosine triphosphate (dITP).

[0027] Compounds having a purine skeleton with a hydrogen atom bonded to the 2-position carbon atom also include derivatives of purine bases, purine nucleosides, or purine nucleotides. In the present invention, the term "derivative" refers to a compound in which the purine base moiety, sugar residue moiety, and / or phosphate group moiety of a purine base, purine nucleoside, or purine nucleotide is chemically modified or a substituent is introduced.

[0028] Compounds having a purine skeleton with a hydrogen atom bonded to the 2-position carbon atom may be salts, such as salts of adenine, hypoxanthine, or purine nucleosides or purine nucleotides containing adenine or hypoxanthine as a constituent. In the present invention, preferred salts are alkali metal salts (e.g., sodium salts, potassium salts), and examples include sodium adenylate and sodium inosinate, but are not limited thereto.

[0029] Compounds having a purine skeleton with a hydrogen atom bonded to the 2-position carbon atom are selected from the group consisting of, for example, adenine, hypoxanthine, adenosine, inosine, deoxyadenosine, deoxyinosine, adenylate, inosinate, and salts thereof, and preferably inosinate. The content of inosinate in the culture system of the present invention can be measured using a kit by fluorescence method, Inosine Assay Kit, CELL BIOLABS.

[0030] The fermentation promoter of the present invention may use at least one, preferably 1 to 4 kinds, for example 1 to 3 kinds or 1 to 2 kinds of the compounds having a purine skeleton with a hydrogen atom bonded to the 2-position carbon atom as described above in combination.

[0031] When using a nucleic acid raw material in combination with an organic acid, the mass ratio of the organic acid to the nucleic acid raw material (organic acid / nucleic acid raw 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.

[0032] In addition, the nucleic acid raw material can be added in an amount such that the content of the nucleic acid raw material is, for example, in the range of 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, based on the total amount of the culture medium or raw milk.

[0033] In addition, the agent of the present invention can be provided as an agent containing, together with the above components, an additive that is optionally acceptable in terms of food hygiene or pharmaceutics. Examples of additives that are acceptable in terms of food hygiene or pharmaceutics include aqueous media such as water, solvents, solubilizing agents, lubricants, emulsifiers, isotonic agents, stabilizers, preservatives, antiseptics, surfactants, regulators, chelating agents, pH adjusters, buffers, excipients, thickeners, colorants, fragrances, or flavorings.

[0034] The agent of the present invention may be in any form such as a liquid, powder, granule, gel, solid, encapsulated body, or the like.

[0035] The fermentation promoter can be produced by appropriately mixing at least one organic acid selected from malic acid and fumaric acid and other optional components including nucleic acid. The resulting mixture can be further processed according to known formulation techniques such as dissolution in a solvent, powdering, granulation, gelation, solidification, encapsulation, or the like.

[0036] The fermentation-promoting effect of the present invention can be confirmed by culturing and fermenting lactic acid bacteria in raw milk added with a fermentation promoter, examining an index indicating the progress of the fermentation state, and as a result, the fermentation progresses faster compared to the control (the group without adding the fermentation promoter of the present invention). The index indicating the progress of the fermentation state is not particularly limited, but for example, the decrease in the pH value of the fermented milk accompanying the increase in the amount of lactic acid produced by the fermentation of lactic acid bacteria can be used as an index. Such pH is, for example, pH 4.6. Here, pH 4.6 can be set as the pH at which fermentation is sufficiently carried out and the fermentation is completed in the production of normal fermented milk. When the time until reaching pH 4.6 is shortened compared to the control, it can be determined that the fermentation promoter has a fermentation-promoting effect on lactic acid bacteria. The pH can be measured using a commercially available pH meter.

[0037] Lactic acid bacteria In the present invention, the lactic acid bacteria used for fermentation are not particularly limited as long as they do not interfere with the effects of the present invention, and they may be of animal origin or plant origin.

[0038] Preferred lactic acid bacteria of the present invention include bacteria belonging to the genus Lactobacillus, Streptococcus, Lactococcus, Enterococcus, Leuconostoc, and combinations thereof, and lactic acid bacteria containing Lactobacillus bacteria are preferred. Examples of lactic acid bacteria containing Lactobacillus bacteria include Lactobacillus bacteria, combinations of Lactobacillus bacteria and Streptococcus bacteria, combinations of Lactobacillus bacteria and Lactococcus bacteria, and the like. These lactic acid bacteria can be obtained, for example, from depository institutions such as ATCC, or commercially available ones can be appropriately used.

[0039] Examples of Lactobacillus bacteria include, for example, 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 sakei, etc. Preferred Lactobacillus bacteria are Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus pentosus, etc.Here, preferred Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus pentosus include 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, and Lactobacillus pentosus JCM 1558T strain, respectively.

[0040] In addition, examples of Lactobacillus delbrueckii include Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus delbrueckii subspecies bulgaricus; Lactobacillus bulgaricus), Lactobacillus delbrueckii subsp. lactis (Lactobacillus delbrueckii subspecies lactis), Lactobacillus delbrueckii subsp. delbrueckii (Lactobacillus delbrueckii subspecies delbrueckii), Lactobacillus delbrueckii subsp. indicus (Lactobacillus delbrueckii subspecies indicus), and the like. Preferred Lactobacillus delbrueckii includes Lactobacillus delbrueckii subsp. bulgaricus and the like, and more preferably Lactobacillus delbrueckii subsp. bulgaricus (strain 2038, strain OLL 1073R-1, strain P1902901, strain OLL1171, strain OLL1255, strain OLL1247, strain OLL205013), and the like. Examples of Lactobacillus kefiranofaciens include Lactobacillus kefiranofaciens subsp. kefirgranum (Lactobacillus kefiranofaciens subspecies kefirgranum), and the like. Preferably, it is Lactobacillus kefiranofaciens subsp. kefirgranum JCM 8572T and the like.

[0041] In addition, Lactobacillus acidophilus JCM 1132T, Lactobacillus gasseri JCM 1131T, Lactobacillus rhamnosus JCM 1136T, Lactobacillus reuteri JCM 1112T, Lactobacillus salivarius JCM 1231T, Lactobacillus pentosus JCM 1558T, and Lactobacillus kefiranofaciens subsp. kefiranofaciens JCM 8572T can be obtained from the RIKEN BioResource Center (RIKEN BRC), Microbial Materials Development Laboratory (Japan Collection of Microorganisms) (RIKEN BRC-JCM, Japan) under accession numbers JCM 1132T, JCM 1131T, JCM 1136T, JCM 1112T, JCM 1231T, JCM 1558T, and JCM 8572T, respectively.

[0042] Lactobacillus delbrueckii subsp. bulgaricus 2038 can be isolated from "Meiji Bulgaria Yogurt" (registered trademark) using a commercially available selective medium for Lactobacillus spp. and is stored by Meiji Co., Ltd. (1-29-1, Shichikoku, Hachioji-shi, Tokyo 192-0919, Japan, Meiji Innovation Center).

[0043] Lactobacillus delbrueckii subsp. bulgaricus OLL 1073R-1 was deposited at the Patent Biological Depositary, National Institute of Advanced Industrial Science and Technology (1-1-1 Higashi 1-chome, Tsukuba, Ibaraki 305-8566, Japan) on February 22, 1999, and then transferred to international deposit and assigned the accession number FERM BP-10741. In addition, as described in Budapest Notification No. 282 (http: / / www.wipo.int / treaties / en / notifications / budapest / treaty_budapest_282.html), since the National Institute of Technology and Evaluation (IPOD, NITE) succeeded to the patent microorganism deposit business from the Patent Biological Depositary, National Institute of Advanced Industrial Science and Technology (IPOD, AIST), it is currently deposited at the Patent Biological Depositary, National Institute of Technology and Evaluation (IPOD, NITE) under the accession number FERM BP-10741.

[0044] Lactobacillus delbrueckii subsp. bulgaricus P1902901 is stored by Meiji Co., Ltd. (Meiji Innovation Center, 1-29-1 Shichikoku, Hachioji, Tokyo 192-0919, Japan).

[0045] Lactobacillus delbrueckii subsp. bulgaricus OLL1171 was internationally deposited as NITE BP-01569 on March 13, 2013, at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, an international deposit authority under the Budapest Treaty.

[0046] Lactobacillus delbrueckii subsp. bulgaricus OLL1255 was internationally deposited as NITE BP-76 on February 10, 2005, at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, an international deposit authority under the Budapest Treaty.

[0047] Lactobacillus delbrueckii subsp. bulgaricus OLL1247 was internationally deposited as NITE BP-01814 with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NITE), an international depositary authority under the Budapest Treaty, on March 6, 2014.

[0048] Lactobacillus delbrueckii subsp. bulgaricus OLL205013 was internationally deposited as NITE BP-02411 with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NITE), an international depositary authority under the Budapest Treaty, on February 3, 2017.

[0049] Examples of Streptococcus bacteria include Streptococcus thermophilus.

[0050] Examples of Lactococcus bacteria include Lactococcus lactis, Lactococcus plantarum, Lactococcus raffinolactis, and the like.

[0051] A preferred combination of Lactobacillus bacteria and Streptococcus bacteria is Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0052] The mixing ratio of lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid and nucleic acid raw materials may be appropriately set according to the type and properties of lactic acid bacteria, medium, raw milk, temperature conditions, and other fermentation conditions.

[0053] 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, more preferably 0.1 to 500.

[0054] The mass ratio of lactic acid bacteria to the above 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.

[0055] Lactic acid bacteria can be added in an amount of, for example, 0.001% by mass to 5% by mass, preferably 0.01% by mass to 2.5% by mass, more preferably 0.01% by mass to 2% by mass, and even more preferably 0.1% by mass to 1% by mass, based on the total amount of the medium or raw milk.

[0056] Lactic acid bacteria starter The organic acid selected from malic acid and fumaric acid as described above can be used together with lactic acid bacteria as a lactic acid bacteria starter. Therefore, according to a preferred embodiment of the present invention, there is provided a lactic acid bacteria starter comprising lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid.

[0057] The lactic acid bacteria starter includes those prepared by culturing lactic acid bacteria in a medium (for example, an activation medium) and undergoing intermediate fermentation. The lactic acid bacteria starter preferably includes lactic acid bacteria and the medium in which it is cultured as components. Therefore, the lactic acid bacteria starter may further contain succinic acid. Here, the succinic acid is preferably an endogenous organic acid produced by the lactic acid bacteria. In addition to those directly inoculated into the raw milk that is the source of fermented milk, the lactic acid bacteria starter includes those of the subsequent generations in which this lactic acid bacteria starter is inoculated into another medium to further grow (scale up) the lactic acid bacteria.

[0058] The lactic acid bacteria starter is basically used to ferment raw milk to obtain fermented milk. In addition to directly inoculating the raw milk with the lactic acid bacteria starter obtained by the present invention, the use of the lactic acid bacteria starter also includes culturing the lactic acid bacteria starter obtained by the present invention one or more times in a medium and then inoculating the raw milk with the lactic acid bacteria starter of the next generation and subsequent generations after the culturing.

[0059] The lactic acid bacteria starter may be used in combination with a nucleic acid raw material. Therefore, according to another aspect of the present invention, there is provided a lactic acid bacteria starter comprising lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid for use in combination with a nucleic acid raw material. It is preferable to use the above lactic acid bacteria starter in combination with a nucleic acid raw material. In the above combination, the nucleic acid raw material may be added to the medium or raw milk as a separate entity together with the lactic acid bacteria starter, or the nucleic acid raw material may be mixed as a constituent component with the lactic acid bacteria starter and used integrally. By using the lactic acid bacteria starter and the nucleic acid raw material in combination, the fermentation of lactic acid bacteria can be promoted more effectively. Therefore, according to one aspect, the above lactic acid bacteria starter may contain a nucleic acid raw material. Each aspect of malic acid, fumaric acid, nucleic acid raw material, and lactic acid bacteria in the lactic acid bacteria starter can be the same as the description of the fermentation promoter of the present invention.

[0060] The viable count of lactic acid bacteria in the lactic acid bacteria starter is not particularly limited. For example, it is 1.0×10 4 ~1.0×10 13 cfu / g, preferably 1.0×10 5 ~1.0×10 12 cfu / g, and more preferably 1.0×10 6 ~1.0×10 11 cfu / g.

[0061] 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 and fumaric acid in the organic acid in the lactic acid bacteria starter can be the same as the mass ratio in the above fermentation promoter.

[0062] The lactic acid bacteria starter can be produced from lactic acid bacteria and optional components including the above-mentioned organic acids, nucleic acid raw materials, and medium components. Hereinafter, the details of a preferred production method of the lactic acid bacteria starter will be described. A preferred production method of the lactic acid bacteria starter includes a medium preparation step, a medium sterilization step, a lactic acid bacteria inoculation step, a culture step (medium fermentation step), and an organic acid addition step.

[0063] The medium preparation step is a step of preparing a medium (for example, an activation medium) for inoculating lactic acid bacteria. The medium is not particularly limited as long as it does not interfere with the effects of the present invention, and examples include media containing milk components, such as skim milk, skim concentrated milk, skim milk powder (reconstituted skim milk), and proteolysates of these skim milk components, whey, whey concentrate, whey powder (reconstituted whey), and proteolysates of these whey components, raw milk, sterilized milk (whole milk), whole concentrated milk, whole milk powder (reconstituted whole milk), and media containing proteolysates of these whole milk components, etc. Media containing skim milk, skim milk powder, and proteolysates of these skim milk components, whey, whey concentrate, whey powder, and proteolysates of these whey components are more preferred, and media containing skim milk, skim milk powder, whey, whey powder, etc. are even more preferred. Also, the above-mentioned medium may be the same as the raw milk described later. The above-mentioned medium preferably further contains yeast extract. The above-mentioned medium can be prepared by known methods such as mixing, dissolving, dispersing, and suspending the above-mentioned respective components.

[0064] The medium sterilization step is a step of sterilizing the medium prepared in the medium preparation step, for example, by heating. In the sterilization step, the heating temperature and heating time may be adjusted for heat treatment to a degree that can sterilize the contaminants in the medium. In the present invention, it is preferable to heat the medium to 80 °C or higher, 90 °C or higher, 95 °C or higher, or 100 °C or higher. Known methods can be used for heat sterilization. For example, in heat sterilization, heat treatment may be performed using a plate heat exchanger, a tube heat exchanger, a steam injection heating device, a steam infusion heating device, an electric heating device, an autoclave device, etc., or heat treatment may be performed using a jacketed tank. Note that the sterilization of the medium is not limited to heating, and it can also be performed by known methods such as ultraviolet irradiation.

[0065] The lactic acid bacteria addition (inoculation) step is a step of adding (inoculating) lactic acid bacteria to the sterilized medium. As the lactic acid bacteria to be added to the medium, frozen bacteria (for example, frozen concentrated bacteria, frozen pellets, freeze-dried powders, etc.) can be used. In the lactic acid bacteria addition step, it is preferable to add lactic acid bacteria to the medium at 0.05% by mass or more, more preferably at 0.05 - 10% by mass, and even more preferably at 0.1 - 5% by mass.

[0066] The culturing step is a step of culturing lactic acid bacteria in a medium to grow the lactic acid bacteria and obtain a lactic acid starter. The culturing time of the lactic acid bacteria is not particularly limited, and examples include 3 - 36 hours, preferably 5 - 30 hours, and more preferably 10 - 24 hours. When obtaining a lactic acid starter by culturing multiple times, the above culturing time refers to the culturing time for one time.

[0067] Also, in the culturing step, the temperature of the culture medium is preferably maintained in a fermentation temperature range of 30°C or higher. In particular, the temperature of the culture medium is preferably maintained at 30 to 50°C, more preferably maintained at 35 to 50°C. Also, in the culturing step, it is preferable to leave the culture medium standing without stirring. Here, "leaving standing" means not stirring the culture medium. For example, even if the container containing the culture medium is moved, if the inside of the culture medium is not stirred, it falls under "leaving standing". In this way, by leaving the culture medium standing during the culturing step, the growth of lactic acid bacteria can be promoted, and the time until the end of culturing can be shortened.

[0068] The step of adding an organic acid or the like is a step of adding at least one organic acid selected from malic acid and fumaric acid and, if desired, a nucleic acid raw material. The step of adding an organic acid or the like may be carried out at any time before, during, or after the culturing step. From the viewpoint of shortening the culturing time, it is preferably carried out before or during the culturing step. The step of adding an organic acid or the like can be carried out, for example, after the culturing step by collecting a predetermined amount of lactic acid bacteria or a lactic acid bacteria-containing culture medium thereof and adding an organic acid and, if desired, a nucleic acid raw material to such lactic acid bacteria or the lactic acid bacteria-containing culture medium thereof. Also, the step of adding an organic acid or the like may be carried out, for example, by adding an organic acid and, if desired, a nucleic acid raw material to the culture medium before or during the culturing step. Here, the time before the culturing step is not particularly limited and may 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, or simultaneous with the addition of lactic acid bacteria. The organic acid to be added is preferably dissolved in water and adjusted to a pH of 6.0 to 7.0.

[0069] Fermented milk According to the present invention, fermented milk can be efficiently produced by using the above fermentation promoter or lactic acid bacteria starter. Here, "fermented milk" includes "fermented milk", "lactic acid bacteria beverage in dairy products", and "lactic acid bacteria beverage" defined in the ordinance (Ordinance on Milk and Others) regarding the ingredient standards of milk and dairy products, and also includes yogurt and the like. For example, fermented milk refers to milk such as raw milk, cow's milk, special cow's milk, raw goat milk, pasteurized goat milk, raw ewe milk, adjusted milk, low-fat milk, non-fat milk, and processed milk, or milk containing not less than the equivalent amount of non-fat milk solids, which is fermented with lactic acid bacteria or yeast to make it paste-like or liquid-like, or those frozen, and includes hard yogurt, soft yogurt (paste-like fermented milk), or drinkable yogurt (liquid fermented milk).

[0070] Generally, hard yogurt such as plain yogurt is produced by filling a container with raw materials and then fermenting them (post-fermentation) (also referred to as "set yogurt"). On the other hand, soft yogurt and drinkable yogurt are produced by subjecting the fermented milk (pre-fermentation) to atomization treatment or homogenization treatment and then filling it into a container (also referred to as "stirred yogurt").

[0071] According to the present invention, by fermenting lactic acid bacteria in the presence of malic acid, fumaric acid, and further, if desired, a nucleic acid raw material, succinic acid can be contained in a high content in fermented milk without using a food additive. Therefore, according to a preferred embodiment of the present invention, there is provided a fermented milk comprising lactic acid bacteria and succinic acid, wherein the succinic acid is an endogenous organic acid produced by the lactic acid bacteria.

[0072] 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, even more preferably 1 mM or more, still more preferably 3 mM or more, based on the total amount of the fermented milk. The preferable lower limit of the succinic acid content in the fermented milk of the present invention is 0.15 mM, preferably 1 mM, more preferably 3 mM, and the preferable upper limit is 50 mM, more preferably 20 mM, even more preferably 15 mM. According to a preferable embodiment of the present invention, when Lactobacillus delbrueckii subsp. bulgaricus is used as the lactic acid bacterium, the succinic acid content in the fermented milk is, for example, 1 mM or more, preferably 1.5 mM or more, more preferably 3 mM or more, based on the total amount of the fermented milk. Further, when Lactobacillus rhamnosus is used as the lactic acid bacterium, the succinic acid content in the fermented milk is, for example, 0.5 mM or more, preferably 1 mM or more, based on the total amount of the fermented milk. Further, when Lactobacillus salivarius is used as the lactic acid bacterium, the succinic acid content in the fermented milk is, for example, 0.15 mM or more, preferably 0.2 mM or more, based on the total amount of the fermented milk. The content of succinic acid in the fermented milk of the present invention is measured by a high performance liquid chromatography (HPLC) method. Such measurement can be easily performed by using a commercially available HPLC apparatus (for example, manufactured by Shimadzu Corporation) and a column (for example, ICSep ICE-ORH-801 (manufactured by TRANSGENOMIC)). The above measurement can be performed, for example, under the following conditions. Analyzer: LC20 system manufactured by Shimadzu Corporation, Column: ICSep ICE-ORH-801, 6.5 mm I.D. × 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.

[0073] Since the fermented milk is preferably produced in the presence of organic acids such as malic acid and fumaric acid, and further nucleic acid raw materials if desired, it may contain malic acid, fumaric acid, and nucleic acid raw materials.

[0074] The content of malic acid in fermented milk is, for example, 0.1 to 50 mM, preferably 0.1 to 45 mM, and more preferably 0.5 to 45 mM.

[0075] 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.

[0076] The content of nucleic acid raw material in fermented milk is, for example, 0.0001 to 5% by mass, preferably 0.0001 to 1.5% by mass.

[0077] The fermented milk of the present invention contains succinic acid at least twice as much as the fermented milk without adding at least one organic acid selected from malic acid and fumaric acid during production, preferably 2.5 times or more, more preferably 5 times or more. The upper limit is 30 times, preferably 20 times.

[0078] According to the present invention, fermented milk can be produced in the presence of an organic acid such as malic acid or fumaric acid and a nucleic acid raw material using the above fermentation promoter or lactic acid bacteria starter. Therefore, there is provided a method for producing fermented milk, which comprises fermenting raw milk with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid. Further, according to a preferred embodiment, the method for producing fermented milk is to perform lactic acid bacteria fermentation in the coexistence of the above organic acid and nucleic acid raw material. 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.

[0079] The raw milk preparation step is a step of preparing raw milk to which a lactic acid bacteria starter is inoculated. "Raw milk" is the raw material for fermented milk such as yogurt, and is also called yogurt mix, fermented milk mix, etc. In the present invention, known raw milk can be appropriately used. Raw milk includes both before sterilization and after sterilization.

[0080] Specific raw materials for the raw milk may include raw milk, pasteurized milk, skim milk, whole milk powder, skim milk powder, buttermilk, butter, cream, whey protein concentrate (WPC), whey protein isolate (WPI), α (alpha)-lactalbumin (La), β (beta)-lactoglobulin (Lg), etc. Gelatin that has been pre-warmed may be appropriately added. The raw milk is known and can be prepared according to known methods.

[0081] Preferred raw milk includes raw milk, skim milk, skim milk powder, and cream, but is not limited thereto. More preferred raw milk can 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 prevent the effects of the present invention, and is preferably 6 to 11% by mass, more preferably 7 to 10% by mass. Also, the content of fat in the raw milk is not particularly limited as long as it does not prevent the effects of the present invention, and preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass, and even more preferably 0.1 to 3% by mass can be exemplified, but is not limited thereto.

[0082] The raw milk sterilization step is a step of sterilizing the raw milk prepared in the raw milk preparation step, for example, by heating. The raw milk is preferably sterilized. Such sterilization includes, for example, sterilization by heating. As sterilization, the heating temperature and heating time can be adjusted for heat treatment to a degree that can sterilize the miscellaneous bacteria in the raw milk. For example, it is preferable to subject the raw milk to holding sterilization at 80°C or higher, preferably 90°C or higher. Known methods can be used for the heat treatment.

[0083] The lactic acid bacteria starter inoculation step is a step of inoculating (adding) a lactic acid bacteria starter to the above raw milk. As the above lactic acid bacteria starter, a lactic acid bacteria starter obtained through the method for producing the above lactic acid bacteria starter, or a lactic acid bacteria starter prepared and frozen by a normal method, or a lactic acid bacteria starter dried after freezing can be used.

[0084] Here, at least one organic acid selected from malic acid and fumaric acid and the nucleic acid raw material as an optional component are preferably added to the raw milk as components in the lactic acid bacteria starter. However, separately from the lactic acid bacteria starter, they may be contained in the raw milk as an additive or an endogenous component. The addition amounts of the lactic acid bacteria, organic acid, and nucleic acid raw material components in the raw milk can be the same as those described in the above fermentation accelerator.

[0085] The fermentation step is a step of fermenting the raw milk with a lactic acid bacteria starter. In the fermentation step, for example, the raw milk inoculated with the lactic acid bacteria starter is fermented while maintaining it in the fermentation temperature range to obtain fermented milk. In the present invention, known methods can be used in the fermentation step. Fermentation conditions such as the fermentation temperature can be appropriately adjusted in consideration of the type of raw milk and lactic acid bacteria (lactic acid bacteria starter), the type and flavor of the fermented milk to be prepared, etc. As a specific example, the fermentation temperature is about 30 to 50°C. At a temperature within this range, lactic acid bacteria generally tend to be active, so fermentation can proceed effectively. The fermentation temperature at this time is preferably about 30 to 45°C, more preferably about 35 to 43°C.

[0086] 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 fermentation time can be adjusted using the pH in the fermented milk reaching 4.6 as an index. The fermentation time is not limited, but examples include 2 hours to 36 hours, 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.

[0087] In addition, known devices and production conditions can be used for producing the fermented milk. For example, as a device for producing the fermented milk, for post-fermentation products, a fermentation chamber for performing fermentation after filling can be used, and for pre-fermentation products, a fermentation tank for performing fermentation and a line filter, a homogenizer, etc. for crushing the fermented milk card can be used. As production conditions, a deoxygenation device, etc. can be appropriately adopted.

[0088] As described above, succinic acid contained in fermented milk is a umami substance and an organic acid having useful physiological functions such as an action of suppressing weight gain, an action of improving glucose tolerance, and an action of suppressing cancer cell growth. Therefore, according to one aspect, the fermented milk of the present invention is provided as a composition for improving umami taste, suppressing weight gain, improving glucose tolerance, or suppressing cancer cell growth.

[0089] Other embodiments According to the present invention, as described above, at least one organic acid selected from malic acid and fumaric acid can be combined with a nucleic acid raw material as desired to promote the fermentation of lactic acid bacteria. Therefore, according to another aspect of the present invention, there is provided a method for promoting the fermentation of lactic acid bacteria, which comprises fermenting a medium (for example, a medium containing milk components) or raw milk with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid. Further, according to another preferred aspect, the method for promoting the fermentation of lactic acid bacteria comprises fermenting lactic acid bacteria in the coexistence of the organic acid and a nucleic acid raw material.

[0090] According to the present invention, as described above, it is possible to promote the fermentation and metabolism of lactic acid bacteria and produce fermentation metabolites in a short time. Therefore, according to still another aspect of the present invention, there is provided a method for producing or promoting the production of lactic acid bacteria fermentation metabolites, which comprises fermenting a medium (for example, a medium containing milk components) or raw milk with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid. Further, according to another preferred aspect, the method comprises fermenting lactic acid bacteria in the coexistence of the organic acid and a nucleic acid raw material. Preferred examples of the fermentation metabolites include succinic acid, extracellular polysaccharide (EPS), peptides, etc., and more preferably extracellular polysaccharide (EPS).

[0091] Further, according to still another aspect of the present invention, there is provided at least one organic acid selected from malic acid and fumaric acid for promoting the fermentation of lactic acid bacteria. Further, according to another preferred aspect, the organic acid ferments lactic acid bacteria in the coexistence of a nucleic acid raw material.

[0092] Further, according to still another aspect of the present invention, there is provided the 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. Further, according to a preferred still another aspect, the above organic acid is used in combination with a nucleic acid raw material.

[0093] Further, according to still another aspect of the present invention, there is provided the use of at least one organic acid selected from malic acid and fumaric acid in the production of a lactic acid bacteria starter. Further, according to a preferred still another aspect, the above organic acid is used in combination with a nucleic acid raw material.

[0094] Further, according to still another aspect of the present invention, there is provided the use of at least one organic acid selected from malic acid and fumaric acid in the production of fermented milk, which comprises fermenting lactic acid bacteria in the presence of the organic acid. Further, according to a preferred still another aspect, the above organic acid is used in combination with a nucleic acid raw material. According to a more preferred still another aspect, the above fermented milk contains succinic acid, and the succinic acid may be an endogenous organic acid produced by lactic acid bacteria.

[0095] Each of the above aspects of the method for promoting fermentation of lactic acid bacteria, the organic acid, and the use can be carried out according to the description regarding the production methods of the fermentation promoter, the lactic acid bacteria starter, and the fermented milk of the present invention.

Examples

[0096] Hereinafter, the present invention will be described more specifically by way of examples, but the technical scope of the present invention is not limited to these examples. Unless otherwise specified, all ratios used in the present invention are by mass. Also, unless otherwise specified, the units and measurement methods described in this specification are based on JIS standards.

[0097] Test Example 1: Examination of the fermentation promoting effect of malic acid or fumaric acid on Lactobacillus delbrueckii subsp. bulgaricus (hereinafter also referred to as "L. bulgaricus") First, frozen bacteria of the following Lactobacillus delbrueckii subsp. bulgaricus strains were prepared. (1) Lactobacillus delbrueckii subsp. bulgaricus 2038 (hereinafter also referred to as "2038") (2) Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (hereinafter also referred to as "OLL 1073R-1") (3) Lactobacillus delbrueckii subsp. bulgaricus P1902901 (hereinafter also referred to as "P1902901") (4) Lactobacillus delbrueckii subsp. bulgaricus OLL1171 (hereinafter also referred to as "OLL1171") (5) Lactobacillus delbrueckii subsp. bulgaricus OLL1255 (hereinafter also referred to as "OLL1255") (6) Lactobacillus delbrueckii subsp. bulgaricus OLL1247 (hereinafter also referred to as "OLL1247") (7) Lactobacillus delbrueckii subsp. bulgaricus OLL205013 (hereinafter also referred to as "OLL205013")

[0098] The above strain was activated in an activation medium before use. As the activation medium, a 10% reduced skim milk medium containing 0.1% by mass of yeast extract, which was sterilized at 121°C for 7 minutes, was used. Here, the 10% reduced skim milk medium is a 10% aqueous solution of skim milk powder (1% fat, 34% protein, 54% lactose, 8% ash, 96% non-fat milk solids) (manufactured by Meiji Co., Ltd.) (in the above medium, the lactose content is 5.4% by mass and the non-fat milk solids content is 9.6% by mass). 0.1% by mass of the frozen bacteria was added to the above activation medium (relative to the amount of the activation medium), and static culture was 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 the activation medium) was added to another activation medium, and static culture was carried out at 37°C for 16 hours to obtain a lactic acid bacteria starter.

[0099] Thereafter, fermentation was carried out using raw milk as a fermentation medium. As the fermentation medium, a 10% reduced skim milk medium to which formic acid was added to a final concentration of 1 mM and which was heated to 95°C for pasteurization was used. In addition, an aqueous solution of fumaric acid or malic acid adjusted to a pH of about 6.5 with NaOH before sterilization was added to the above fermentation medium to a final concentration of 1 mM. The pH of the above fermentation medium was about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated into the above fermentation medium at 0.5% by mass (relative to the amount of the fermentation medium), and static culture was carried out at 40°C to carry out fermentation. The fermentation time was defined as the time required to reach pH 4.6. The pH was measured using a commercially available pH meter. The obtained results are shown in Table 1. From Table 1, it can be seen that the fermentation of all 7 strains of L. bulgaricus was promoted by the addition of malic acid or fumaric acid. The fermentation promoting effects of malic acid and fumaric acid on L. bulgaricus were almost the same. The fermentation shortening time varied depending on the strain and was from 40 minutes to 12 hours and 15 minutes.

[0100]

Table 1

[0101] Test Example 2-1: Examination of the concentrations of malic acid and fumaric acid showing a fermentation promoting effect on L. bulgaricus strain 2038 Fermentation was carried out using a fermentation medium (raw milk). As the fermentation medium, a 10% reduced skim milk medium supplemented with formic acid to a final concentration of 1 mM and sterilized by heating to 95°C was used. In addition, an aqueous solution of fumaric acid or malic acid adjusted to a pH of about 6.5 with NaOH before sterilization was added to the above fermentation medium to the final concentrations shown in Tables 3 and 4. The above fermentation medium was inoculated with a lactic acid bacteria starter of L. bulgaricus strain 2038 at 0.5% (relative to the amount of the fermentation medium) and statically cultured at 40°C to carry out fermentation. The fermentation time was defined as the time required to reach pH 4.6.

[0102] The measurement of the organic acid concentration after the fermentation was carried out as follows. 0.4 g of the obtained fermented product (fermented milk) was diluted 2-fold with pure water, 20 μL of Carrez reagent I (53.5% (w / v) zinc sulfate) was added and vortexed, and 20 μl of Carrez reagent II (17.2% (w / v) potassium ferrocyanide) was added and vortexed. Centrifugation was performed at 4°C, 20620 g for 10 minutes, and the supernatant filtered through a 0.22 μm filter was analyzed under the conditions of Table 2.

[0103]

Table 2

[0104] The results obtained are shown in Tables 3 to 5. From Tables 3 and 4, it was confirmed that L. bulgaricus strain 2038 had a fermentation promoting effect in 0.01 - 2.5 mM of fumaric acid and 0.01 - 50 mM of malic acid. Also, both malic acid and fumaric acid showed a fermentation promoting effect even at a low concentration of 0.01 mM. As a result of measuring the organic acid concentration after fermentation, the concentrations of the added malic acid and fumaric acid decreased, and the concentration of succinic acid increased (Table 5). That is, it was suggested that the added malic acid and fumaric acid were metabolized in the direction of malic acid → fumaric acid → succinic acid by the reductive TCA cycle.

[0105]

Table 3

[0106]

Table 4

[0107]

Table 5

[0108] Test Example 2-2: Succinic acid production amount when malic acid and fumaric acid are added in Lactobacillus delbrueckii subsp. bulgaricus The strains shown in Tables 6 and 7 were activated in the activation medium and then used. As the activation medium, a 10% reduced skim milk medium containing 0.1% by mass of yeast extract, which had been sterilized 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 content 1% by mass, protein 34% by mass, lactose 54% by mass, ash 8% by mass, non-fat milk solids 96%) (manufactured by Meiji Co., Ltd.) (in the above medium, lactose content 5.4% by mass, non-fat milk solids 9.6% by mass). 0.1% by mass of the frozen bacteria (relative to the amount of the activation medium) was added to the above activation medium, and static culture was 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 the activation medium) was added to another activation medium, and static culture was carried out at 37°C for 16 hours to obtain a lactic acid bacteria starter.

[0109] Subsequently, fermentation was carried out using the fermented medium as raw milk. As the fermented medium, a 10% by mass reduced skim milk medium to which formic acid was added to a final concentration of 1 mM and sterilized by heating to 95°C was used. In addition, an aqueous solution of fumaric acid or malic acid adjusted to a pH of about 6.5 with NaOH before sterilization was added to the above fermented medium to a final concentration of 1 mM. The pH of the above fermented medium was about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated into the above fermented medium (2.5% by mass based on the amount of the fermented medium), and static culture was carried out at 40°C to conduct fermentation. The fermentation time was defined as the time required to reach pH 4.6. The pH was measured using a commercially available pH meter. The obtained results are shown in Tables 6 and 7. From Table 6, in any of the L. delbrueckii subsp. bulgaricus strains, the concentration of succinic acid increased due to the addition of malic acid and fumaric acid. Further, from Table 7, similar to Test Example 1, the fermentation of all 7 strains of L. bulgaricus was promoted by the addition of malic acid or fumaric acid.

[0110]

Table 6

[0111]

Table 7

[0112] Test Example 3: Examination of the fermentation promoting effect of fumaric acid on lactic acid bacteria species other than Lactobacillus delbrueckii subsp. bulgaricus First, reference strains of the following bacterial species other than Lactobacillus delbrueckii subsp. bulgaricus were prepared. (8) Lactobacillus acidophilus JCM 1132T (9) Lactobacillus gasseri JCM 1131T (10) Lactobacillus rhamnosus JCM 1136T (11) Lactobacillus reuteri JCM 1112T (12) Lactobacillus salivarius JCM 1231T (13)Lactobacillus pentosus JCM 1558T

[0113] The above strain was activated in an activation medium before use. As the activation medium, a 10% reduced skim milk medium containing 0.1% yeast extract, which was sterilized at 121°C for 7 minutes, was used. Frozen bacteria were added to the above activation medium at 1% (relative to the amount of the activation medium), and 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) was added to another activation medium, and statically cultured at 37°C for 24 hours to obtain a lactic acid bacteria starter.

[0114] Thereafter, 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 to a final concentration of 1 mM and sterilized by reaching 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 fermentation medium to a final concentration of 1 mM. The lactic acid bacteria starter of each strain activated above was inoculated into the above fermentation medium at 1% (relative to the amount of the fermentation medium), and statically cultured at 37°C to carry out fermentation. The fermentation time was the time required to reach pH 4.6. The results obtained are shown in Table 8. The addition of fumaric acid promoted fermentation in L. acidophilus, L. gasseri, L. rhamnosus, L. reuteri, L. salivarius, and L. pentosus.

[0115]

Table 8

[0116] Test Example 4: Examination of the fermentation promoting effect of malic acid on lactic acid bacteria species other than Lactobacillus delbrueckii subsp. bulgaricus First, reference strains of the following bacterial species were prepared. (12)Lactobacillus salivarius JCM 1231T (13)Lactobacillus pentosus JCM 1558T (14)Lactobacillus kefiranofaciens subsp. kefirgranum JCM 8572T

[0117] The experiment was conducted in the same manner as in Test Example 3, except that malic acid was added instead of fumaric acid. The results obtained are shown in Table 9. The addition of malic acid promoted fermentation in L. salivarius, L. pentosus, and Lactobacillus kefiranofaciens subsp. kefirgranum.

[0118]

Table 9

[0119] Test Example 5: Measurement of the concentrations of malic acid, fumaric acid and succinic acid in L. rhamnosus and L. salivarius The measurement of the organic acid concentration after the fermentation of L. rhamnosus and L. salivarius in Test Example 3 or 4 was carried out in the same manner as in Test Example 2-1. The results obtained are shown in Table 10. The addition of malic acid or fumaric acid promoted the production of succinic acid in L. rhamnosus and L. salivarius.

[0120]

Table 10

[0121] Test Example 6: Examination of the fermentation promoting effect of fumaric acid on Lactobacillus delbrueckii subsp. bulgaricus when inosinic acid is added Frozen bacteria of the following Lactobacillus delbrueckii subsp. bulgaricus strains were prepared. (1) Lactobacillus delbrueckii subsp. bulgaricus 2038 (2) Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (3) Lactobacillus delbrueckii subsp. bulgaricus P1902901 (4)Lactobacillus delbrueckii subsp. bulgaricus OLL1171 (5)Lactobacillus delbrueckii subsp. bulgaricus OLL1255 (6)Lactobacillus delbrueckii subsp. bulgaricus OLL1247 (7)Lactobacillus delbrueckii subsp. bulgaricus OLL205013

[0122] The above strains were activated in the activation medium before use. As the activation medium, a 10% reduced skim milk medium containing 0.1% yeast extract, which was sterilized at 121 °C for 7 minutes, was used. Frozen bacteria were added to the above activation medium at 1% (relative to the amount of the activation medium), and 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) was added to another activation medium, and statically cultured at 37 °C for 24 hours to obtain a lactic acid bacteria starter.

[0123] Thereafter, fermentation was carried out using a fermentation medium (raw milk). As the fermentation medium, a 10% reduced skim milk medium to which inosinic acid was added to a final concentration of 1 mM and which was heated to 95 °C for pasteurization 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 fermentation medium to a final concentration of 1 mM. The lactic acid bacteria starter of each strain activated above was inoculated into the above fermentation medium at 1% (relative to the amount of the fermentation medium), and statically cultured at 37 °C to carry out fermentation. The fermentation time was the time required to reach pH 4.6. The pH was measured using a pH meter. The obtained results are shown in Table 11. From Table 11, it was found that the addition of fumaric acid promoted the fermentation of all 7 strains of L. bulgaricus.

[0124]

Table 11

[0125] Test Example 7: Examination of the fermentation promoting effect of malic acid on Lactobacillus delbrueckii subsp. bulgaricus when inosinic acid is added Frozen bacteria of the following Lactobacillus delbrueckii subsp. bulgaricus strains were prepared. (1) Lactobacillus delbrueckii subsp. bulgaricus 2038 (2) Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (3) Lactobacillus delbrueckii subsp. bulgaricus P1902901 (4) Lactobacillus delbrueckii subsp. bulgaricus OLL1171 (5) Lactobacillus delbrueckii subsp. bulgaricus OLL1247

[0126] The procedure was the same as in Test Example 6, except that malic acid was added instead of fumaric acid. The results obtained are shown in Table 12. Fermentation was promoted in the L. bulgaricus 2038 strain, OLL1073R-1 strain, P1902901 strain, OLL1171 strain, and OLL1247 strain by the addition of malic acid.

[0127]

Table 12

[0128] Test Example 8: Influence of malic acid or fumaric acid on succinic acid production of Lactobacillus delbrueckii subsp. bulgaricus when inosinic acid is added The strains shown in Tables 13 and 14 were activated in an activation medium and then used. As the activation medium, a 10% reduced skim milk medium containing 0.1% by mass of yeast extract, which was sterilized 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 content 1%, protein content 34%, lactose content 54%, ash content 8%, non-fat milk solids content 96%) (manufactured by Meiji Co., Ltd.) (in the above medium, lactose content 5.4% by mass, non-fat milk solids content 9.6% by mass). 0.1% by mass of frozen bacteria (relative to the amount of the activation medium) was added to the above activation medium, and static culture was 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 the activation medium) was added to another activation medium, and static culture was carried out at 37°C for 16 hours to obtain a lactic acid bacteria starter.

[0129] Thereafter, fermentation was carried out using raw milk as a fermentation medium. As the fermentation medium, a 10% reduced skim milk medium to which inosinic acid was added to a final concentration of 1 mM and sterilized by reaching a temperature of 95°C was used. In addition, an aqueous solution of fumaric acid or malic acid adjusted to a pH of about 6.5 with NaOH before sterilization was added to the above fermentation medium to a final concentration of 1 mM. The pH of the above fermentation medium was about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated into the above fermentation medium at 2.5% by mass (relative to the amount of the fermentation medium), and static culture was carried out at 40°C to carry out fermentation. The fermentation time was the time required to reach pH 4.6. The pH was measured using a commercially available pH meter. The obtained results are shown in Tables 13 and 14. From Table 13, in the medium containing inosinic acid, in any L. delbrueckii subsp. bulgaricus strain, the addition of malic acid and fumaric acid increased the concentration of succinic acid. Therefore, it was shown that the addition of malic acid and fumaric acid increased the succinic acid production amount of L. delbrueckii subsp. bulgaricus not only in the formic acid-added medium but also in the inosinic acid-added medium. Also, from Table 14, similar to Test Example 6, the addition of malic acid or fumaric acid promoted the fermentation of all 7 strains of L. bulgaricus.

[0130]

Table 13

[0131] [Table 14]

[0132] Test Example 9: Influence of malic acid or fumaric acid on the growth and metabolite production of Lactobacillus delbrueckii subsp. bulgaricus Frozen bacteria of Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 were prepared.

[0133] The composition of the medium followed Table 15 below. Raw material components other than anhydrous crystalline glucose were dissolved in Elix water, adjusted to pH 6.65 using sodium hydroxide, and then made up to 70% by weight of the medium charge. Anhydrous crystalline glucose was dissolved in Elix water and made up to 30% by weight of the medium charge. When adding malic acid or fumaric acid, it was dissolved and added together with the raw material components other than anhydrous crystalline glucose in Elix water to a final concentration of 4 mM. Note that the sugar solution and raw material components other than sugar were each heat-sterilized by autoclaving at 110°C for 1 minute and then aseptically mixed for use.

[0134] [Table 15]

[0135] 1.5 kg of the above medium was adjusted to pH 5.4 using 6N potassium carbonate, inoculated with frozen bacteria at 0.30% by mass (relative to the medium volume), and cultured at 37°C with stirring at 150 rpm while passing nitrogen gas over the surface at a flow rate of 0.5 L / min. The change in EPS concentration over the culture time is shown in Fig. 1. As shown in Fig. 1, when malic acid or fumaric acid was added, it was confirmed that the EPS concentration increased compared to the control. From this, it was found that the production of EPS, a metabolite of L. delbrueckii subsp. bulgaricus, could be promoted by adding malic acid or fumaric acid. Also, when malic acid or fumaric acid was added, the EPS concentration increased in a shorter time compared to the control, so it was reconfirmed that malic acid or fumaric acid promoted the fermentation of L. delbrueckii subsp. bulgaricus.

[0136] The EPS concentration was measured under the following conditions. 1.5 g of the culture broth was weighed, and MilliQ water was added to make it 10 g. 100% trichloroacetic acid was added and mixed well, and after centrifugation (4 °C, 13400 g, 10 min), the supernatant was collected. 5 ml of 10% trichloroacetic acid solution was added to the precipitate and suspended, and then centrifuged again to collect the supernatant. 2 times the volume of cooled 99.5% ethanol was added to the supernatant, and after inverting and mixing, it was left standing at -20 °C overnight. Centrifugation (4 °C, 13400 g, 20 min) was performed to remove the supernatant, 20 ml of cooled 66% ethanol was added to suspend the precipitate. Centrifugation (4 °C, 13400 g, 20 min) was performed to remove the supernatant, and after air-drying to remove ethanol, MilliQ water was added and made up to 10 ml to obtain the analysis sample. The EPS concentration of the analysis sample was measured by the phenol-sulfuric acid method.

Claims

1. A method for producing fermented milk containing lactic acid bacteria and succinic acid, comprising fermenting with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid, wherein the lactic acid bacteria is at least one selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus 2038, Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1, Lactobacillus delbrueckii subsp. bulgaricus OLL1171, Lactobacillus delbrueckii subsp. bulgaricus OLL1255, Lactobacillus delbrueckii subsp. bulgaricus OLL1247, Lactobacillus delbrueckii subsp. bulgaricus OLL205013, Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactobacillus acidophilus JCM 1132T, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus pentosus.

2. The production method according to claim 1, wherein the lactic acid bacteria is at least one selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus 2038, Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1, Lactobacillus delbrueckii subsp. bulgaricus OLL1171, Lactobacillus delbrueckii subsp. bulgaricus OLL1255, Lactobacillus delbrueckii subsp. bulgaricus OLL1247, Lactobacillus delbrueckii subsp. bulgaricus OLL205013, Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactobacillus acidophilus JCM 1132T, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus pentosus.

3. The lactic acid bacterium is at least one selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus strain 2038, Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1, Lactobacillus delbrueckii subsp. bulgaricus strain OLL1171, Lactobacillus delbrueckii subsp. bulgaricus strain OLL1255, Lactobacillus delbrueckii subsp. bulgaricus strain OLL1247, Lactobacillus delbrueckii subsp. bulgaricus strain OLL205013, Lactobacillus acidophilus strain JCM 1132T, Lactobacillus gasseri strain JCM 1131T, Lactobacillus rhamnosus strain JCM 1136T, Lactobacillus salivarius strain JCM 1231T, Lactobacillus pentosus strain JCM 1558T, and Lactobacillus kefiranofaciens subsp. kefiranofaciens strain JCM 8572T. The manufacturing method according to claim 1.

4. The lactic acid bacterium is at least one selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus strain 2038, Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1, Lactobacillus delbrueckii subsp. bulgaricus strain OLL1171, Lactobacillus delbrueckii subsp. bulgaricus strain OLL1255, Lactobacillus delbrueckii subsp. bulgaricus strain OLL1247, Lactobacillus delbrueckii subsp. bulgaricus strain OLL205013, Lactobacillus gasseri strain JCM 1131T, Lactobacillus rhamnosus strain JCM 1136T, Lactobacillus salivarius strain JCM 1231T, Lactobacillus pentosus strain JCM 1558T, and Lactobacillus kefiranofaciens subsp. kefiranofaciens strain JCM 8572T. The manufacturing method according to any one of claims 1 to 3.

5. The lactic acid bacterium is at least one selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus 2038 strain, Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain, Lactobacillus delbrueckii subsp. bulgaricus OLL1171 strain, Lactobacillus delbrueckii subsp. bulgaricus OLL1255 strain, Lactobacillus delbrueckii subsp. bulgaricus OLL1247 strain, and Lactobacillus delbrueckii subsp. bulgaricus OLL205013 strain. The production method according to any one of claims 1 to 4.

6. The lactic acid bacterium is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain. The production method according to any one of claims 1 to 5.

7. The succinic acid is an endogenous organic acid produced by the lactic acid bacterium. The production method according to any one of claims 1 to 6.

8. The fermented milk further contains at least one organic acid selected from malic acid and fumaric acid. The production method according to any one of claims 1 to 7.

9. The fermented milk contains Streptococcus thermophilus. The production method according to any one of claims 1 to 8.

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