Lactic acid bacterium fermentation promoter

Abstract

To provide means for promoting the fermentation of lactic acid bacteria, and also to provide means for promoting the production of succinic acid by lactic acid bacteria in fermented milk.SOLUTION: The present invention provides a lactic acid bacterium fermentation promoter, containing at least one organic acid selected from malic acid and fumaric acid.SELECTED DRAWING: None

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to Japanese Patent Application No. 2019-239455, filed on December 27, 2019, the entire disclosure of which is incorporated herein by reference. [Technical field]

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

[0003] In industrial production of microbial fermentation products and metabolites, shortening the fermentation time is important for reducing production costs and hygiene management. As a technology for promoting lactic acid bacteria fermentation and shortening the fermentation time, it has been reported that a substance that serves as a raw material for nucleic acids promotes lactic acid bacteria fermentation (Patent Document 1). In addition, Patent Document 2 describes a microbial productivity enhancer that contains an organic acid extract from the seeds of a cruciferous plant as an active ingredient. However, from the viewpoint of efficient production of 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 increase in health consciousness, there is a demand for producing fermented milk with good taste by reducing the amount of food additives used. In particular, succinic acid is often used as a food additive because it is an umami substance and also has useful physiological functions such as suppressing weight gain, improving glucose tolerance, and suppressing cancer growth. From the viewpoint of stimulating consumer demand, it is preferable to produce succinic acid in fermented milk without using food additives.

[0005] In recent years, it has been reported that, regarding the mechanism of succinic acid production, under anaerobic conditions, oxaloacetate is generated from pyruvate or phosphoenolpyruvate via carbon dioxide fixation, and succinic acid is produced via the reverse route of the TCA cycle (Non-Patent Document 1). However, there have been no reports on the ability of lactic acid bacteria to utilize the reverse route of the TCA cycle. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Tatsuo Hoshino, Microbiol. Cult. Coll., 2011, 27(2), p. 83-88 [Patent documents]

[0007] [Patent Document 1] JP 2017-221157 A [Patent Document 2] Japanese Patent Application Publication No. 04-141083 Summary of the Invention

[0008] The present invention provides a new technical means for promoting fermentation by lactic acid bacteria. Also, 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 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 these 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 source. [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 compounds having a purine skeleton in which a hydrogen atom is bonded to the carbon atom at the second position. [4] The fermentation promoter according to any one of [1] to [3], wherein the lactic acid bacteria comprises bacteria of the genus Lactobacillus. [5] The fermentation promoter described in [4], wherein the Lactobacillus bacterium 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 malic acid and fumaric acid. [7] The lactic acid bacteria starter according to [6], further comprising a nucleic acid source. [8] A method for producing fermented milk, comprising lactic acid bacteria fermenting in the presence of at least one organic acid selected from malic acid and fumaric acid. [9] The method according to [8], which comprises fermenting the organic acid with a nucleic acid source in the presence of the organic acid and the nucleic acid source.

[10] The method according to [9], wherein the nucleic acid source 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 position 2.

[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 bacteria.

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

[12] , The fermented milk comprises lactic acid bacteria and succinic acid.

[14] The fermented milk described in

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

[15] The fermented milk according to

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

[16] 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 fermenting the lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.

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

[17] , further comprising a nucleic acid source.

[19] The method for promoting lactic acid bacterium fermentation described in

[18] , wherein the nucleic acid source is at least one selected from formic acid and compounds having a purine skeleton in which a hydrogen atom is bonded to the carbon atom at position 2.

[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] , which comprises culturing lactic acid bacteria in the presence of the organic acid and a nucleic acid source.

[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 contains 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 having a succinic acid content of 0.15 mM or more based on the total volume 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 described in

[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 fermenting with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.

[29] The method according to

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

[30] A method for promoting the production of lactic acid bacteria fermentation metabolites (hereinafter also referred to as the "production promotion method"), comprising lactic acid bacteria 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 an exopolysaccharide (EPS).

[0011] According to the present invention, it is possible to promote fermentation of lactic acid bacteria. Also, according to the present invention, it is possible to increase the amount of succinic acid produced in fermented milk. The present invention can be advantageously used to promote fermentation and metabolism of lactic acid bacteria and produce fermentation metabolites such as extracellular polysaccharides (EPS) and peptides in a short period of time. Also, the present invention can be advantageously used to increase the amount of succinic acid produced by lactic acid bacteria and reduce the amount of succinic acid added as a food additive. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a chart showing the change in EPS concentration over time in Test Example 9. Description of the Invention

[0013] Fermentation accelerator One of the features 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.

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

[0015] As the raw material of the organic acid in the fermentation promoter, a commercially available product as a food additive, a synthetic product, a preparation containing an organic acid, etc. may be used.

[0016] The fermentation promoter may contain either one of the organic acids 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 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.

[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 the form of either a free acid or a salt. Examples of such salts 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 may be any optical isomer, but is preferably L-malic acid.

[0018] Malic acid or a salt thereof can be added in an amount ranging from 0.001 to 75 mM, preferably from 0.01 to 50 mM, more preferably from 0.1 to 10 mM, and even more preferably from 0.5 to 10 mM, relative to 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 in the culture, for example, the total amount of the medium or raw milk, malic acid and / or fumaric acid, and nucleic acid raw material. The content of malic acid in the culture system of the present invention is measured by high performance liquid chromatography (HPLC). Such a measurement can be performed using a commercially available HPLC device (e.g., Shimadzu Corporation) and column (e.g., ICSep ICE-ORH-801 (TRANSGENOMIC)). More specifically, the above measurement can be performed under the following conditions. Analytical equipment: Shimadzu LC20 system, column: ICSep ICE-ORH-801, 6.5 mm ID × 300 mm, two columns connected together, 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: electrical conductivity detector.

[0019] Fumaric acid The form of fumaric acid in the fermentation promoter is not particularly limited as long as it does not impair the effects of the present invention, and it may be contained in the agent in the form of either a free acid or a 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] Fumaric acid can be added in an amount such that the content of fumaric acid or a salt thereof 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, relative to the total amount of the medium or raw milk. Therefore, it is preferable that fumaric acid is contained in the fermentation promoter of the present invention in the above amount. The content of fumaric acid in the culture system of the present invention can be measured by the same method as that for malic acid.

[0021] Nucleic acid raw material The fermentation promoter of the present invention is preferably used in combination with a nucleic acid source from the viewpoint of more effectively promoting fermentation of lactic acid bacteria. The nucleic acid source may be included as a component of the fermentation promoter or may be used separately. Such a nucleic acid source 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 a compound having a purine skeleton with a hydrogen atom bonded to the carbon atom at the 2-position.

[0022] Formic acid is known as a raw material for forming the purine backbone of nucleic acid. 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 the form of either a free acid or a salt. Examples of 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 by the same method as that for malic acid.

[0023] A compound having a purine skeleton has the following structure (purine skeleton): [ka] [The numbers in formula (I) represent the position numbers of carbon atoms or nitrogen atoms] The term "purine" refers to a substance having the basic structure of the purine skeleton. Compounds having a purine skeleton are generally referred to as purines. Typical examples of compounds having a purine skeleton include purine bases, purine nucleosides, purine nucleotides, and salts thereof.

[0024] The compound having a purine skeleton used as a nucleic acid source of the present invention has a purine skeleton in which a hydrogen atom is bonded to the carbon atom at position 2 of the purine skeleton (the carbon atom represented by 2 in the above formula (I)). Examples of such compounds include adenine and hypoxanthine (purine bases), purine nucleosides containing adenine and hypoxanthine as building blocks, purine nucleotides containing adenine or hypoxanthine as building blocks, and salts thereof.

[0025] Purine nucleosides are substances in which a purine base is bound to a sugar (such as ribose or deoxyribose), and may be either ribonucleosides or deoxyribonucleosides. Purine nucleosides that contain adenine or hypoxanthine as a component include, for example, adenosine and inosine (ribonucleosides), and deoxyadenosine and deoxyinosine (deoxyribonucleosides).

[0026] A purine nucleotide is a substance in which one or more phosphate groups are bound to a purine nucleoside, and may be a ribonucleotide or a deoxyribonucleotide. A purine nucleotide may be a nucleoside monophosphate, a nucleoside diphosphate, or a nucleoside triphosphate. Purine nucleotides containing adenine or hypoxanthine as building blocks include, for example, adenylic acid (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).

[0027] Compounds having a purine skeleton in which a hydrogen atom is bonded to the carbon atom at position 2 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 portion, sugar residue portion, and / or phosphate group portion of a purine base, purine nucleoside, or purine nucleotide has been chemically modified or a substituent has been introduced.

[0028] A compound having a purine skeleton with a hydrogen atom bonded to the carbon atom at position 2 may be a salt, for example, a salt of adenine, hypoxanthine, or a purine nucleoside or purine nucleotide containing adenine or hypoxanthine as a component. In the present invention, preferred salts are alkali metal salts (e.g., sodium salts, potassium salts), including, but not limited to, sodium adenylate and sodium inosinate.

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

[0030] 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 in which a hydrogen atom is bonded to the carbon atom at position 2.

[0031] When the nucleic acid source is used in combination with an organic acid, the mass ratio of the organic acid to the nucleic acid source (organic acid / nucleic acid source) 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] Furthermore, the nucleic acid source can be added in an amount such that the content of the nucleic acid source relative to the total amount of the medium or raw milk 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.

[0033] The agent of the present invention can be provided as an agent containing the above-mentioned components and, if desired, additives that are food hygienically or pharma- ceutically acceptable. Examples of food hygienically or pharma- ceutically acceptable additives include aqueous media such as water, solvents, solubilizers, lubricants, emulsifiers, isotonicity agents, stabilizers, preservatives, antiseptics, surfactants, regulators, chelating agents, pH regulators, buffers, excipients, thickeners, colorants, fragrances, or flavors.

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

[0035] The fermentation promoter can be produced by appropriately mixing at least one organic acid selected from malic acid and fumaric acid with other optional components including nucleic acids. The resulting mixture can be further treated according to known formulation techniques, such as dissolving in a solvent, powdering, granulating, gelling, solidifying, encapsulating, etc.

[0036] The fermentation promoting effect of the present invention can be confirmed by culturing and fermenting lactic acid bacteria in raw milk to which a fermentation promoter has been added, examining an indicator of the progress of the fermentation state, and finding that the fermentation progresses faster than that of a control (a group to which the fermentation promoter of the present invention has not been added). The indicator of the progress of the fermentation state is not particularly limited, but for example, a decrease in the pH value of fermented milk with an increase in the amount of lactic acid produced by fermentation of lactic acid bacteria can be used as an indicator. An example of such a pH is pH 4.6. Here, pH 4.6 can be set as the pH at which fermentation is sufficiently performed and fermentation is terminated in the production of normal fermented milk. If the time to reach 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 impair the effects of the present invention, and may be of animal or plant origin.

[0038] The preferred lactic acid bacteria of the present invention include Lactobacillus, Streptococcus, Lactococcus, Enterococcus, Leuconostoc, and combinations thereof, and preferably includes Lactobacillus-containing lactic acid bacteria.The Lactobacillus-containing lactic acid bacteria include Lactobacillus, Lactobacillus and Streptococcus, Lactobacillus and Lactococcus, and the like.These lactic acid bacteria can be obtained from, for example, ATCC or other storage facilities, or can be used as appropriate from commercially available products.

[0039] Examples of the Lactobacillus bacteria include Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus pentosus, Lactobacillus kefiranofaciens, Lactobacillus helveticus, and Lactobacillus johnsonii. johnsonii, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus amylovorous, Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus sakei, etc. Preferred Lactobacillus bacteria include Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, and 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] Examples of Lactobacillus delbrueckii include Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. delbrueckii, and Lactobacillus delbrueckii subsp. indicus. Preferred Lactobacillus delbrueckii is Lactobacillus delbrueckii subsp. bulgaricus, and more preferably Lactobacillus delbrueckii subsp. bulgaricus (2038 strain, OLL 1073R-1 strain, P1902901 strain, OLL1171 strain, OLL1255 strain, OLL1247 strain, OLL205013 strain), etc. Examples of Lactobacillus kefiranofaciens include Lactobacillus kefiranofaciens subsp. kefirgranum, and preferably Lactobacillus kefiranofaciens subsp. kefirgranum JCM 8572T, etc.

[0041] 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 kefiranofaciens subsp. kefirgranum JCM 8572T strain were obtained from the Japan Collection of Microorganisms (RIKEN BRC-JCM, Japan) under the accession numbers JCM 1132T, JCM 1131T, JCM 1136T, JCM 1112T, JCM 1231T, and JCM 8572T, respectively. Available under JCM 1558T, JCM 8572T.

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

[0043] Lactobacillus delbrueckii subsp. bulgaricus OLL 1073R-1 strain was deposited on February 22, 1999 at the National Institute of Advanced Industrial Science and Technology (6-1-1 Central, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), and was subsequently transferred to international depository status and assigned the accession number FERM BP-10741. As stated in Budapest Notification No. 282 (http: / / www.wipo.int / treaties / en / notifications / budapest / treaty_budapest_282.html), the National Institute of Technology and Evaluation (IPOD, NITE) has taken over the patent microorganism deposit business from the National Institute of Advanced Industrial Science and Technology (IPOD, AIST). Therefore, the patent is currently deposited at the National Institute of Technology and Evaluation (IPOD, NITE) under the deposit number FERM BP-10741.

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

[0045] Lactobacillus delbrueckii subsp. bulgaricus OLL1171 strain has been internationally deposited with the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary, an international depository authority under the provisions of the Budavest Treaty, as NITE BP-01569 on March 13, 2013.

[0046] Lactobacillus delbrueckii subsp. bulgaricus OLL1255 strain has been internationally deposited as NITE BP-76 on February 10, 2005 at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation, which is the international depository authority under the provisions of the Budavest Treaty.

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

[0048] The Lactobacillus delbrueckii subsp. bulgaricus OLL205013 strain has been internationally deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary, an international depository authority under the provisions of the Budavest Treaty, as NITE BP-02411 on February 3, 2017.

[0049] An example of the genus Streptococcus is Streptococcus thermophilus.

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

[0051] The combination of bacteria of the genus Lactobacillus and bacteria of the genus Streptococcus preferably includes Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

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

[0053] The mass ratio of the lactic acid bacteria to at least one organic acid selected from malic acid and fumaric acid (lactic acid bacteria / organic acid) is, for example, 0.001-500,000, preferably 0.01-5,000, and more preferably 0.1-500.

[0054] The mass ratio of the lactic acid bacteria to the nucleic acid source (lactic acid bacteria / nucleic acid source) is, for example, 0.1-11,000, preferably 1-11,000, more preferably 10-1,100, and even more preferably 50-550.

[0055] The 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 even more preferably 0.1% to 1% by mass, relative to 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 as a lactic acid bacteria starter together with lactic acid bacteria. 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] Lactic acid bacteria starters include those prepared by culturing lactic acid bacteria in a medium (e.g., activation medium) and undergoing intermediate fermentation. Lactic acid bacteria starters preferably contain lactic acid bacteria and the medium in which they are cultured as components. Thus, 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. Lactic acid bacteria starters also include those directly inoculated into raw milk, which is the source of fermented milk, as well as those of the next generation or later inoculated into another medium to further grow (scale up) the lactic acid bacteria.

[0058] Lactic acid bacteria starters are basically used to ferment raw milk to obtain fermented milk. The use of the lactic acid bacteria starter includes not only direct inoculation of the raw milk with the lactic acid bacteria starter obtained by the present invention, but also inoculation of the raw milk with the lactic acid bacteria starter of the next generation or later after culturing the lactic acid bacteria starter obtained by the present invention in a medium one or more times.

[0059] The lactic acid bacteria starter may be used in combination with a nucleic acid source. Therefore, according to another aspect of the present invention, a lactic acid bacteria starter is provided, which comprises lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid, for use in combination with a nucleic acid source. The lactic acid bacteria starter is preferably used in combination with a nucleic acid source, and in the combination, the nucleic acid source may be added separately to a medium or raw milk together with the lactic acid bacteria starter, or the lactic acid bacteria starter may be mixed with the nucleic acid source as a component and used together. By using the lactic acid bacteria starter in combination with the nucleic acid source, the fermentation of the lactic acid bacteria can be promoted more effectively. Therefore, according to one aspect, the lactic acid bacteria starter may contain a nucleic acid source. The aspects of malic acid, fumaric acid, nucleic acid source, and lactic acid bacteria in the lactic acid bacteria starter can all be the same as those described for the fermentation promoter of the present invention.

[0060] The viable cell count of the lactic acid bacteria in the lactic acid bacteria starter is not particularly limited, but may be, 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.

[0061] The mass ratio of lactic acid bacteria to organic acid in the lactic acid bacteria starter, 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 can be the same as the mass ratios in the above-mentioned fermentation promoter.

[0062] The lactic acid bacteria starter can be produced from lactic acid bacteria and any other components, including the above organic acids, nucleic acid raw materials, and medium components. A preferred method for producing the lactic acid bacteria starter is described below in detail. A preferred method for producing a 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 a step of adding an organic acid or the like.

[0063] The medium preparation step is a step of preparing a medium (e.g., activation medium) inoculated with lactic acid bacteria. The medium is not particularly limited as long as it does not interfere with the effects of the present invention, but examples thereof include a medium containing a milk component, such as skim milk, skim concentrated milk, skim milk powder (reconstituted skim milk), and protein hydrolysates of these skim milk components, whey, whey concentrate, whey powder (reconstituted whey), and protein hydrolysates of these whey components, raw milk, pasteurized milk (whole fat milk), whole fat concentrated milk, whole milk powder (reconstituted whole fat milk), and protein hydrolysates of these whole fat milk components, etc., and more preferably a medium containing skim milk, skim milk powder, and protein hydrolysates of these skim milk components, whey, whey concentrate, whey powder, and protein hydrolysates of these whey components, and even more preferably a medium containing skim milk, skim milk powder, whey, whey powder, etc. In addition, the above medium may be the same as the raw milk described below. The above medium preferably further contains yeast extract. The medium can be prepared by a known method such as mixing, dissolving, dispersing, or suspending each of the above 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 to a degree that can sterilize the bacteria in the medium, and the medium may be heated. In the present invention, the medium is preferably heated to 80°C or higher, 90°C or higher, 95°C or higher, or 100°C or higher. A known method may be used for heat sterilization. For example, in heat sterilization, the heat treatment may be performed using a plate-type heat exchanger, a tube-type heat exchanger, a steam injection type heating device, a steam infusion type heating device, an electric heating device, an autoclave device, or the like, and the heat treatment may be performed using a jacketed tank. The sterilization of the medium is not limited to heating, and may also be performed by a known method 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. Frozen bacteria (e.g., frozen concentrated bacteria, frozen pellets, freeze-dried powder, etc.) can be used as the lactic acid bacteria to be added to the medium. In the lactic acid bacteria addition step, the lactic acid bacteria are preferably added to the medium at 0.05% by mass or more, more preferably at 0.05 to 10% by mass, and even more preferably at 0.1 to 5% by mass.

[0066] The culture step is a step of culturing lactic acid bacteria in a medium, proliferating the lactic acid bacteria, and obtaining a lactic acid starter. The culture time of the lactic acid bacteria is not particularly limited, but may be, for example, 3 to 36 hours, preferably 5 to 30 hours, and more preferably 10 to 24 hours. When the lactic acid bacteria starter is obtained by culturing multiple times, the above culture time refers to the culture time for one culture.

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

[0068] The organic acid, etc. addition step is a step of adding at least one organic acid selected from malic acid and fumaric acid, and optionally a nucleic acid raw material. The organic acid, etc. addition step may be performed before, during, or after the culture step, but from the viewpoint of shortening the culture time, it is preferable to perform it before or during the culture step. The organic acid, etc. addition step can be performed, for example, after the culture step, by collecting a predetermined amount of lactic acid bacteria or a lactic acid bacteria-containing medium, and adding an organic acid and optionally a nucleic acid raw material to the lactic acid bacteria or a lactic acid bacteria-containing medium. The organic acid, etc. addition step may be performed, for example, by adding an organic acid and optionally a nucleic acid raw material to the medium before or during the culture step. Here, the time before the culture step is not particularly limited, and may be any of the following times: before the medium sterilization step, after the medium sterilization step, before the lactic acid bacteria addition step, after the lactic acid bacteria addition step, and simultaneously with the addition of the lactic acid bacteria. The organic acid to be added is preferably dissolved in water and the pH is adjusted to 6.0 to 7.0.

[0069] Fermented milk According to the present invention, fermented milk can be efficiently produced using the fermentation promoter or lactic acid bacteria starter. Here, "fermented milk" includes "fermented milk," "dairy lactic acid bacteria beverage," and "lactic acid bacteria beverage" defined in the Ministerial Ordinance on the Ingredient Standards of Milk and Dairy Products (Milk, etc. Ministerial Ordinance), and also includes yogurt, etc. For example, fermented milk refers to milk such as raw milk, cow's milk, special cow's milk, raw goat's milk, pasteurized goat's milk, raw sheep's milk, ingredient-adjusted milk, low-fat milk, non-fat milk, and processed milk, or milk containing non-fat milk solids equivalent to or greater than these, fermented with lactic acid bacteria or yeast to form a paste or liquid, or to freeze these, and includes hard yogurt, soft yogurt (paste-like fermented milk), and drink yogurt (liquid fermented milk).

[0070] Generally, hard yogurt such as plain yogurt is produced by filling a container with ingredients and then fermenting it (post-fermentation) (also called "set yogurt"), whereas soft yogurt and drinkable yogurt are produced by atomizing and homogenizing fermented milk (pre-fermentation) and then filling it into a container (also called "stirred yogurt").

[0071] According to the present invention, by carrying out lactic acid fermentation in the presence of malic acid, fumaric acid, and optionally a nucleic acid source, it is possible to obtain a high succinic acid content in fermented milk without using food additives. Thus, according to a preferred embodiment of the present invention, there is provided 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, and even more preferably 3 mM or more, based on 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 even more preferably 15 mM. According to a preferred 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, and more preferably 3 mM or more, based on the total amount of the fermented milk. 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. 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 succinic acid content in the fermented milk of the present invention is measured by high performance liquid chromatography (HPLC). Such a measurement can be easily performed using a commercially available HPLC device (e.g., Shimadzu Corporation) and column (e.g., ICSep ICE-ORH-801 (TRANSGENOMIC)). The above measurement can be performed, for example, under the following conditions. Analytical equipment: Shimadzu LC20 system, column: ICSep ICE-ORH-801, 6.5 mm ID × 300 mm, two columns connected together, 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: electrical conductivity detector.

[0073] Fermented milk is preferably produced in the presence of an organic acid such as malic acid or fumaric acid, and further, if desired, a nucleic acid raw material, and therefore may contain malic acid, fumaric acid, and a nucleic acid raw material.

[0074] 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.

[0075] The content of fumaric acid in the 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 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.

[0077] The fermented milk of the present invention contains at least twice as much succinic acid, preferably at least 2.5 times, and more preferably at least 5 times, as compared with fermented milk to which at least one organic acid selected from malic acid and fumaric acid has not been added during production. The upper limit is 30 times, and preferably 20 times.

[0078] According to the present invention, fermented milk can be produced using the above fermentation promoter or lactic acid bacteria starter in the presence of an organic acid such as malic acid or fumaric acid, or a nucleic acid raw material. Thus, a method for producing fermented milk is provided, 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. In a preferred embodiment, the method for producing fermented milk involves fermenting with lactic acid bacteria in the coexistence of the organic acid and the nucleic acid raw material. More specifically, the method for producing fermented milk preferably includes a raw material milk preparation step, a raw material milk sterilization step, a lactic acid bacteria starter inoculation step, and a fermentation step.

[0079] The raw material milk preparation step is a step of preparing raw material milk to be inoculated with a lactic acid bacteria starter. The "raw material milk" is a raw material for fermented milk such as yogurt, and is also called yogurt mix or fermented milk mix. In the present invention, any known raw material milk can be used as appropriate. The raw material milk includes both pre- and post-sterilization milk.

[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-lactalbumin (La), β-lactoglobulin (Lg), etc. Pre-warmed gelatin, etc. may be added as appropriate. 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 includes 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 impede the effects of the present invention, and is preferably 6 to 11% by mass, more preferably 7 to 10% by mass. The content of fat in the raw milk is not particularly limited as long as it does not impede the effects of the present invention, and is 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, 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. Examples of such sterilization include sterilization by heating. Sterilization can be performed by adjusting the heating temperature and heating time to a degree that can sterilize the germs in the raw milk. For example, it is preferable to sterilize the raw milk by reaching a temperature of 80°C or higher, preferably 90°C or higher. A known method can be used for the heat treatment.

[0083] The lactic acid bacteria starter inoculation step is a step of inoculating (adding) the raw milk with a lactic acid bacteria starter. As the lactic acid bacteria starter, a lactic acid bacteria starter obtained through the above-mentioned method for producing a lactic acid bacteria starter, a lactic acid bacteria starter prepared by a conventional method and frozen, or a lactic acid bacteria starter that has been frozen and then dried can be used.

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

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

[0086] The fermentation time may be adjusted appropriately depending on the lactic acid bacteria (lactic acid bacteria starter) used, the fermentation temperature, etc. For example, the fermentation time can be adjusted using the pH of the fermented milk reaching 4.6 as an index. The fermentation time is not limited, but may be, for example, 2 to 36 hours, preferably 2.5 to 24 hours, and more preferably 4 to 24 hours. The pH can be measured using a commercially available pH meter.

[0087] The apparatus and production conditions for producing fermented milk may be publicly known. For example, the apparatus for producing fermented milk may be a fermentation chamber for filling a product for post-fermentation and a fermentation tank for pre-fermentation and a line filter or homogenizer for crushing fermented milk curds. As the production conditions, a deoxygenation device or the like may be appropriately adopted.

[0088] 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 suppression, glucose tolerance improvement, and cancer growth suppression. Therefore, in one embodiment, the fermented milk of the present invention is provided as a composition for improving umami, suppressing weight gain, improving glucose tolerance, or suppressing cancer growth.

[0089] Other Aspects According to the present invention, as described above, lactic acid bacteria fermentation can be promoted by optionally combining at least one organic acid selected from malic acid and fumaric acid with a nucleic acid raw material. Therefore, according to another aspect of the present invention, there is provided a method for promoting lactic acid bacteria fermentation, which comprises fermenting a medium (e.g., a medium containing a milk component) or raw milk with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid. In another preferred aspect, the method for promoting lactic acid bacteria fermentation comprises fermenting with lactic acid bacteria in the coexistence of the organic acid and the nucleic acid raw material.

[0090] According to the present invention, as described above, fermentation and metabolism of lactic acid bacteria can be promoted, and fermentation metabolites can be produced in a short time. Therefore, according to yet 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 lactic acid bacteria fermenting a medium (e.g., a medium containing a milk component) or raw milk in the presence of at least one organic acid selected from malic acid and fumaric acid. In another preferred aspect, the method comprises lactic acid bacteria fermentation in the coexistence of the organic acid and a nucleic acid raw material. The fermentation metabolites are preferably succinic acid, extracellular polysaccharides (EPS), peptides, etc., and more preferably extracellular polysaccharides (EPS).

[0091] According to yet another aspect of the present invention, there is provided at least one organic acid selected from malic acid and fumaric acid for promoting fermentation by lactic acid bacteria. According to yet another preferred aspect, the organic acid is fermented by lactic acid bacteria in the presence of a nucleic acid source.

[0092] According to yet another aspect of the present invention, there is provided a 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. According to yet another preferred aspect, the organic acid is used in combination with a nucleic acid source.

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

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

[0095] The above-mentioned method for promoting lactic acid bacteria fermentation, the organic acid, and each aspect of use can be carried out in accordance with the descriptions regarding the fermentation promoter, the lactic acid bacteria starter, and the method for producing fermented milk of the present invention. EXAMPLES

[0096] The present invention will be described in more detail below with reference to 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 weight. Furthermore, unless otherwise specified, the units and measurement methods described in this specification are in accordance with 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 strain were prepared. (1) Lactobacillus delbrueckii subsp. bulgaricus 2038 (hereinafter 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 strains were activated in the activation medium before use. As the activation medium, a 10% by mass reduced skim milk medium containing 0.1% by mass yeast extract was used, which was sterilized at 121°C for 7 minutes. Here, the 10% by mass reduced skim milk medium is a 10% by mass aqueous solution of skim milk powder (fat 1% by mass, protein 34% by mass, lactose 54% by mass, ash 8% by mass, non-fat milk solids 96% by mass) (manufactured by Meiji Co., Ltd.) (lactose content 5.4% by mass, non-fat milk solids 9.6% by mass in the above medium). 0.1% by mass (relative to the amount of the activation medium) of frozen bacteria was added to the above activation medium, and statically cultured at 37°C for 16 hours to obtain an activation liquid. 0.1% by mass (relative to the amount of the activation medium) of the obtained activation liquid was added to another activation medium, and statically cultured at 37°C for 16 hours to obtain a lactic acid bacteria starter.

[0099] Thereafter, fermentation was carried out using a fermentation medium as the raw milk. As the fermentation medium, a 10% by mass reduced skim milk medium to which formic acid was added 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 fermentation medium to a final concentration of 1 mM. The pH of the fermentation medium was about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated into the fermentation medium at 0.5% by mass (relative to the amount of the fermentation medium), and the mixture was statically cultured 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 results are shown in Table 1. It can be seen from Table 1 that the addition of malic acid or fumaric acid promoted the fermentation of all seven strains of L. bulgaricus. The fermentation promoting effects of malic acid and fumaric acid on L. bulgaricus were almost the same. The shortened fermentation time varied depending on the strain, ranging 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 that show fermentation promoting effects on L. bulgaricus 2038 strain Fermentation was carried out using a fermentation medium (raw milk). The fermentation medium used was a 10% reduced skim milk medium to which formic acid was added to a final concentration of 1 mM, which was sterilized at 95°C. In addition, an aqueous fumaric acid solution or an aqueous malic acid solution, which had been adjusted to a pH of about 6.5 with NaOH before sterilization, was added to the fermentation medium to the final concentrations shown in Tables 3 and 4. A lactic acid bacteria starter of L. bulgaricus 2038 strain was inoculated into the fermentation medium at 0.5% (relative to the amount of the fermentation medium), and the mixture was statically cultured at 40°C to carry out fermentation. The fermentation time was defined as the time required to reach a pH of 4.6.

[0102] The organic acid concentration after fermentation was measured as follows: 0.4 g of the obtained fermented product (fermented milk) was diluted 2-fold with pure water, 20 μL of Caretz Reagent I (53.5% (w / v) zinc sulfate) was added and vortexed, and 20 μL of Caretz Reagent II (17.2% (w / v) potassium ferrocyanide) was added and vortexed. The mixture was centrifuged at 4°C, 20,620 g for 10 minutes, and the supernatant was filtered through a 0.22 μm filter and analyzed under the conditions in Table 2.

[0103] [Table 2]

[0104] The results are shown in Tables 3 to 5. From Tables 3 and 4, it was confirmed that L. bulgaricus 2038 strain had a fermentation promoting effect at 0.01 to 2.5 mM fumaric acid and 0.01 to 50 mM malic acid. In addition, 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, while the concentration of succinic acid increased (Table 5). In other words, 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 in L. delbrueckii subsp. bulgaricus when malic acid or fumaric acid was added The strains shown in Tables 6 and 7 were activated in the activation medium before use. As the activation medium, a 10% by mass reduced skim milk medium containing 0.1% by mass yeast extract was sterilized at 121°C for 7 minutes and used. Here, the 10% by mass reduced skim milk medium is a 10% by mass aqueous solution of skim milk powder (fat 1% by mass, protein 34% by mass, lactose 54% by mass, ash 8% by mass, non-fat milk solids 96% by mass) (manufactured by Meiji Co., Ltd.) (lactose content 5.4% by mass, non-fat milk solids 9.6% by mass in the above medium). 0.1% by mass (relative to the amount of the activation medium) of frozen bacteria was added to the above activation medium and statically cultured at 37°C for 16 hours to obtain an activation liquid. 0.1% by mass (relative to the amount of the activation medium) of the obtained activation liquid was added to another activation medium and statically cultured at 37°C for 16 hours to obtain a lactic acid bacteria starter.

[0109] Thereafter, fermentation was carried out using a fermentation medium as the raw milk. As the fermentation medium, a 10% by mass reduced skim milk medium to which formic acid was added 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 final concentration of about 6.5 with NaOH before sterilization was added to the fermentation medium to a final concentration of 1 mM. The pH of the fermentation medium was about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated into the fermentation medium at 2.5% by mass (relative to the amount of the fermentation medium), and the fermentation was carried out by static culture at 40°C. 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 6 and 7. From Table 6, in all L. delbrueckii subsp. bulgaricus strains, the concentration of succinic acid increased by the addition of malic acid or fumaric acid. Furthermore, as can be seen from Table 7, similarly to Test Example 1, the addition of malic acid or fumaric acid promoted the fermentation of all seven strains of L. bulgaricus.

[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 L. delbrueckii subsp. bulgaricus First, type 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 strains were activated in an activation medium before use. The activation medium used was a 10% reduced skim milk medium containing 0.1% yeast extract, sterilized at 121°C for 7 minutes. Frozen bacteria were added to the activation medium at 1% (relative to the volume of the activation medium) and cultured at 37°C for 24 hours to obtain an activation liquid. 1% of the obtained activation liquid (relative to the volume of the activation medium) was added to another activation medium and cultured at 37°C for 24 hours to obtain a lactic acid bacteria starter.

[0114] Thereafter, fermentation was carried out using the fermentation medium (raw milk). The fermentation medium used was a 10% reduced skim milk medium to which formic acid was added to a final concentration of 1 mM, which was sterilized at 95°C. In addition, an aqueous fumaric acid solution adjusted to about pH 6.5 with NaOH before sterilization was added to the fermentation medium to a final concentration of 1 mM. The lactic acid bacteria starter of each strain activated above was inoculated into the fermentation medium at 1% (relative to the amount of the fermentation medium), and the mixture was statically cultured at 37°C to carry out fermentation. The fermentation time was the time required to reach pH 4.6. The results 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 L. delbrueckii subsp. bulgaricus First, the following type strains were prepared: (12)Lactobacillus salivarius JCM 1231T (13)Lactobacillus pentosus JCM 1558T (14) Lactobacillus kefiranofaciens subsp. kefirgranum JCM 8572T

[0117] The same procedure as in Test Example 3 was repeated except that malic acid was added instead of fumaric acid. The results 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 malic acid, fumaric acid and succinic acid concentrations in L. rhamnosus and L. salivarius In Test Example 3 or 4, the organic acid concentrations after the completion of fermentation of L. rhamnosus and L. salivarius were measured in the same manner as in Test Example 2-1. The results 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 an activation medium before use. The activation medium used was a 10% reduced skim milk medium containing 0.1% yeast extract, sterilized at 121°C for 7 minutes. Frozen bacteria were added to the activation medium at 1% (relative to the volume of the activation medium) and cultured at 37°C for 24 hours to obtain an activation liquid. 1% of the obtained activation liquid (relative to the volume of the activation medium) was added to another activation medium and cultured at 37°C for 24 hours to obtain a lactic acid bacteria starter.

[0123] Thereafter, fermentation was carried out using the 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 was sterilized at 95°C. In addition, an aqueous fumaric acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added to the fermentation medium to a final concentration of 1 mM. The lactic acid bacteria starter of each strain activated above was inoculated into the fermentation medium at 1% (relative to the amount of the fermentation medium), and the mixture was 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. As can be seen from Table 11, the addition of fumaric acid promoted the fermentation of all seven strains of L. bulgaricus.

[0124] [Table 11]

[0125] Test Example 7: Examination of the fermentation promotion effect of malic acid on L. 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 same procedure as in Test Example 6 was repeated except that malic acid was added instead of fumaric acid. The results are shown in Table 12. The addition of malic acid promoted fermentation in the L. bulgaricus 2038, OLL1073R-1, P1902901, OLL1171, and OLL1247 strains.

[0127] [Table 12]

[0128] Test Example 8: Effect of malic acid or fumaric acid on succinic acid production by L. delbrueckii subsp. bulgaricus when inosinic acid was added The strains shown in Tables 13 and 14 were activated in the activation medium before use. As the activation medium, a 10% by mass reduced skim milk medium containing 0.1% by mass yeast extract was sterilized at 121°C for 7 minutes and used. Here, the 10% by mass reduced skim milk medium is a 10% by mass aqueous solution of skim milk powder (fat 1% by mass, protein 34% by mass, lactose 54% by mass, ash 8% by mass, non-fat milk solids 96% by mass) (manufactured by Meiji Co., Ltd.) (lactose content 5.4% by mass, non-fat milk solids 9.6% by mass in the above medium). 0.1% by mass (relative to the amount of the activation medium) of frozen bacteria was added to the above activation medium and statically cultured at 37°C for 16 hours to obtain an activation liquid. 0.1% by mass (relative to the amount of the activation medium) of the obtained activation liquid was added to another activation medium and statically cultured at 37°C for 16 hours to obtain a lactic acid bacteria starter.

[0129] Thereafter, fermentation was carried out using a fermentation medium as the raw milk. As the fermentation medium, a 10% by mass reduced skim milk medium to which inosinic acid was added 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 final concentration of about 6.5 with NaOH before sterilization was added to the fermentation medium to a final concentration of 1 mM. The pH of the fermentation medium was about 6.4. The lactic acid bacteria starter of each strain obtained above was inoculated into the fermentation medium at 2.5% by mass (relative to the amount of the fermentation medium), and the fermentation was carried out by static culture at 40°C. 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, the concentration of succinic acid increased in all L. delbrueckii subsp. bulgaricus strains by adding malic acid or fumaric acid. Therefore, it was shown that the amount of succinic acid produced by L. delbrueckii subsp. bulgaricus was increased by adding malic acid or fumaric acid not only to the formic acid-added medium but also to the inosinic acid-added medium. Also, as shown in Table 14, the fermentation of all seven strains of L. bulgaricus was promoted by adding malic acid or fumaric acid, as in Test Example 6.

[0130] [Table 13]

[0131] [Table 14]

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

[0133] The composition of the medium was as shown in Table 15 below. All raw ingredients except for anhydrous crystalline glucose were dissolved in elix water, and the pH was adjusted to 6.65 using sodium hydroxide, after which the weight was increased to 70% of the medium charge volume. Anhydrous crystalline glucose was dissolved in elix water, and the weight was increased to 30% of the medium charge volume. When malic acid or fumaric acid was added, it was dissolved in elix water together with the raw ingredients except for anhydrous crystalline glucose to a final concentration of 4 mM and added. The raw materials other than the sugar solution and sugar were each heat sterilized in an autoclave at 110°C for 1 minute, and then mixed aseptically before use.

[0134] [Table 15]

[0135] 1.5 kg of the above medium was adjusted to pH 5.4 using 6N potassium carbonate, frozen bacteria were inoculated at 0.30% by mass (based on the amount of medium), and cultured at 37°C with stirring at 150 rpm and nitrogen overhead aeration at a flow rate of 0.5 L / min. The change in EPS concentration over time is shown in FIG. As shown in Figure 1, it was confirmed that the EPS concentration was higher when malic acid or fumaric acid was added compared to the control. This indicates that the addition of malic acid or fumaric acid can promote the production of EPS, a metabolic product of L. delbrueckii subsp. bulgaricus. Furthermore, the EPS concentration increased in a short period of time when malic acid or fumaric acid was added compared to the control, reaffirming that malic acid or fumaric acid promotes the fermentation of L. delbrueckii subsp. bulgaricus.

[0136] The EPS concentration was measured under the following conditions. 1.5 g of culture medium was weighed out and MilliQ water was added to make up to 10 g. 100% trichloroacetic acid was added and mixed well, then centrifuged (4°C, 13400g, 10 min) and the supernatant was collected. 5 ml of 10% trichloroacetic acid solution was added to the precipitate to suspend it, then centrifuged again to collect the supernatant. Chilled 99.5% ethanol was added in an amount twice that of the supernatant, mixed by inversion, and left to stand overnight at -20°C. The supernatant was removed by centrifugation (4°C, 13400g, 20min), and 20ml of chilled 66% ethanol was added to suspend the precipitate. The supernatant was removed by centrifugation (4°C, 13400g, 20min), and the mixture was air-dried to remove the ethanol, and then MilliQ water was added to make a constant volume of 10ml to prepare the analytical sample. The EPS concentration of the analytical sample was measured by the phenol-sulfuric acid method.

Claims

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 claim 1 for use in combination with a nucleic acid source.

3. The fermentation promoter according to claim 2, wherein the nucleic acid source 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 carbon atom at the second position.

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

5. The fermentation promoter according to claim 4, wherein the Lactobacillus bacterium 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 malic acid and fumaric acid.

7. The lactic acid bacteria starter according to claim 6, further comprising a nucleic acid source.

8. A method for producing fermented milk, comprising lactic acid fermentation 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 the organic acid with a nucleic acid source in the presence of the lactic acid bacteria.

10. 10. The method according to claim 8 or 9, wherein the fermented milk comprises 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 comprises 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 succinic acid content is 0.15 mM or more based on the total amount of the fermented milk.

15. Fermented milk according to any one of claims 12 to 14, further comprising at least one organic acid selected from malic acid and fumaric acid.

16. A method for promoting lactic acid bacterium fermentation, comprising carrying out lactic acid bacterium fermentation in the presence of at least one organic acid selected from malic acid and fumaric acid.

17. The method for promoting lactic acid bacterium fermentation according to claim 16, further comprising a nucleic acid source.

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 a nucleic acid source.

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

21. The method of claim 20, wherein the lactic acid bacteria starter further comprises succinic acid.

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

23. Fermented milk having a succinic acid content of 0.15 mM or more based on the total amount of the fermented milk.

24. 24. The fermented milk of claim 23, further comprising at least one organic acid selected from malic acid and fumaric acid.

25. 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 a lactic acid bacteria fermentation metabolite, comprising fermenting with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.

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