WO2021132418A1 - 乳酸菌の発酵促進剤 - Google Patents

乳酸菌の発酵促進剤 Download PDF

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WO2021132418A1
WO2021132418A1 PCT/JP2020/048328 JP2020048328W WO2021132418A1 WO 2021132418 A1 WO2021132418 A1 WO 2021132418A1 JP 2020048328 W JP2020048328 W JP 2020048328W WO 2021132418 A1 WO2021132418 A1 WO 2021132418A1
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Prior art keywords
acid
lactic acid
lactobacillus
fermentation
fermented milk
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English (en)
French (fr)
Japanese (ja)
Inventor
恵理 山本
麻美 土屋
玲子 渡部
佳代 藤原
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Meiji Co Ltd
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Meiji Co Ltd
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Priority to CN202080090813.8A priority Critical patent/CN115135748B/zh
Priority to EP20907502.7A priority patent/EP4083187A4/en
Priority to CN202411464906.9A priority patent/CN119410524A/zh
Priority to US17/789,045 priority patent/US20230052298A1/en
Priority to JP2021567585A priority patent/JP7229395B2/ja
Publication of WO2021132418A1 publication Critical patent/WO2021132418A1/ja
Anticipated expiration legal-status Critical
Priority to JP2023020336A priority patent/JP7696938B2/ja
Priority to JP2025011979A priority patent/JP2025069251A/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to a novel fermentation accelerator for lactic acid bacteria.
  • Patent Document 1 a substance as a raw material for nucleic acid promotes fermentation of lactic acid bacteria
  • Patent Document 2 describes a microbial productivity improver containing an organic acid extract of cruciferous plant seeds as an active ingredient.
  • succinic acid is often used as a food additive because it is an umami substance but also has useful physiological functions such as an effect of suppressing weight gain, an effect of improving glucose tolerance, and an effect of suppressing cancer growth. From the viewpoint of stimulating consumer demand, it is preferable that succinic acid is also produced in fermented milk without using food additives.
  • the present invention provides a new technical means for promoting fermentation of lactic acid bacteria.
  • the present invention also provides new technical means for promoting the production of succinic acid by lactic acid bacteria in fermented milk.
  • the present inventors have recently found that at least one organic acid selected from malic acid and fumaric acid can promote fermentation of 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 such findings.
  • a fermentation accelerator for lactic acid bacteria which comprises at least one organic acid selected from malic acid and fumaric acid.
  • the fermentation accelerator according to [1] which is used in combination with a nucleic acid raw material.
  • 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 a carbon atom at the 2-position.
  • a lactic acid bacterium starter comprising lactic acid bacteria and at least one organic acid selected from malic acid and fumaric acid.
  • a method for producing fermented milk which comprises fermenting lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.
  • nucleic acid raw material is at least one selected from a compound having a purine skeleton in which a hydrogen atom is bonded to formic acid and a carbon atom at the 2-position.
  • the fermented milk contains succinic acid.
  • the succinic acid is an endogenous organic acid produced by the lactic acid bacterium.
  • a fermented milk obtained by the method according to any one of [8] to [12]. The fermented milk containing lactic acid bacteria and succinic acid.
  • [18] The method for promoting fermentation of lactic acid bacteria according to [17], which further comprises a nucleic acid raw material.
  • a method for producing a lactic acid bacterium starter which comprises culturing a lactic acid bacterium in the presence of at least one organic acid selected from malic acid and fumaric acid.
  • the method according to [20] which comprises culturing lactic acid bacteria in the coexistence of the organic acid and a nucleic acid raw material.
  • the lactic acid bacterium starter comprises a lactic acid bacterium and at least one organic acid selected from malic acid and fumaric acid.
  • the lactic acid bacterium starter further contains succinic acid.
  • the succinic acid is an endogenous organic acid produced by the lactic acid bacterium.
  • Fermented milk having a succinic acid content of 0.15 mM or more with respect to the total amount of fermented milk.
  • the fermented milk according to [25] further comprising at least one organic acid selected from malic acid and fumaric acid.
  • [30] A method for promoting the production of lactic acid bacterium fermentation metabolites, which comprises fermenting lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid (hereinafter, also referred to as "production promotion method"). To do).
  • production promotion method also referred to as "production promotion method”.
  • [31] The method according to [30], wherein the lactic acid bacterium fermentation metabolite is extracellular polysaccharide (EPS).
  • EPS extracellular polysaccharide
  • the present invention fermentation of lactic acid bacteria can be promoted. Further, according to the present invention, the amount of succinic acid produced in fermented milk can be increased. INDUSTRIAL APPLICABILITY
  • the present invention can be advantageously utilized in promoting fermentation and metabolism of lactic acid bacteria and producing fermentation metabolites such as extracellular polysaccharides (EPS) and peptides in a short time. Further, the present invention can be advantageously used in increasing the amount of succinic acid produced by lactic acid bacteria and reducing the amount of succinic acid added as a food additive.
  • EPS extracellular polysaccharides
  • FIG. 1 is a chart showing the change in EPS concentration with respect to the culture time of Test Example 9.
  • the fermentation accelerator for lactic acid bacteria of the present invention is characterized in that it contains at least one organic acid selected from malic acid and fumaric acid.
  • the content of at least one organic acid selected from malic acid and fumaric acid in the fermentation accelerator is not particularly limited, and is, for example, 0.1 to 100% by mass, preferably 50 to 100% by mass. , More preferably 80 to 100% by mass.
  • a commercially available product as a food additive may be used, or a synthetic product, a preparation containing an organic acid, or the like may be used.
  • the fermentation accelerator may contain either an organic acid of malic acid or fumaric acid, or may contain both.
  • the mass ratio of malic acid to fumaric acid is not particularly limited, but is, for example, 0.1 to 10, preferably 0.2 to 5, and more preferably 0.5 to 0.5. It is 2, and even more preferably 0.6 to 1.5.
  • the form of malic acid in the malic acid fermentation accelerator is not particularly limited as long as it does not interfere with the effects of the present invention, and may be contained in the agent in either form of free acid or 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 for culturing may be an optical isomer, but is preferably L-malic acid.
  • the amount of malic acid or a salt thereof 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, still more preferably, based on the total amount of the medium or raw milk. Can be added in an amount of 0.5-10 mM. Therefore, malic acid is preferably contained in the fermentation accelerator in the above amount.
  • the total amount of the medium or raw milk is the total amount of all the components other than the bacteria used for culturing, and is, 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.
  • HPLC high performance liquid chromatography
  • Such a 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 (TRANSGENOMIC)). More specifically, the above measurement can be performed under the following conditions.
  • Analyzer LC20 system manufactured by Shimadzu Corporation, Column: ICSep ICE-ORH-801, 6.5 mm I.D. D.
  • the form of fumaric acid in the fumaric acid fermentation accelerator is not particularly limited as long as it does not interfere with the effects of the present invention, and may be contained in the agent in either 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.
  • the content of fumaric acid or a salt thereof is, for example, 0.001 to 10 mM, preferably 0.01 to 7.5 mM, more preferably, with respect to the total amount of the medium or raw milk. It can be added in an amount of 0.1 to 5 mM, more preferably 0.1 to 2.5 mM. Therefore, it is preferable that fumaric acid is contained in the fermentation accelerator 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 malic acid.
  • the fermentation accelerator of the present invention is preferably used in combination with a nucleic acid raw material from the viewpoint of more effectively promoting fermentation of lactic acid bacteria.
  • the nucleic acid raw material may be contained as a constituent component of the fermentation accelerator or may be used as a separate substance.
  • the nucleic acid raw material is not particularly limited as long as it does not interfere with the effects of the present invention, and preferred examples include formic acid and a compound having a purine skeleton in which a hydrogen atom is bonded to a carbon atom at the 2-position.
  • Formic acid is known as a raw material that constitutes 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 may be contained in the agent in either 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.
  • the content of formic acid in the culture system of the present invention can be measured in the same manner as malic acid.
  • a compound having a purine skeleton has the following structure (purine skeleton): [The numbers in formula (I) represent the position numbers of carbon or nitrogen atoms] Indicates a substance having as a basic skeleton.
  • Compounds having a purine skeleton are also generally referred to as purines.
  • Compounds having a purine skeleton typically include purine bases, purine nucleosides, purine nucleotides, or salts thereof.
  • the compound having a purine skeleton used as a nucleic acid raw material of the present invention has a purine skeleton in which a hydrogen atom is bonded to a 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 components, purine nucleotides containing adenine or hypoxanthine as components, and salts thereof.
  • the purine nucleoside is a substance in which a purine base and a sugar (ribose, deoxyribose, etc.) are bound, and may be a ribonucleoside or a deoxyribonucleoside.
  • Purine nucleosides containing adenine or hypoxanthine as components include, for example, adenosine and inosin (ribonucleoside), and deoxyadenosine and deoxyinosine (deoxyribonucleoside).
  • a purine nucleotide is a substance in which one or more phosphate groups are bonded to a purine nucleoside, and may be a ribonucleotide or a deoxyribonucleotide.
  • Purine nucleotides may be nucleoside monophosphates (nucleoside monophosphates), nucleoside diphosphates, or nucleoside triphosphates.
  • purine nucleotides containing adenine or hypoxanthin as components include adenylic acid (adenosine monophosphate or adenosine monophosphate; AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), and deoxyadenosine monophosphate.
  • adenylic acid adenosine monophosphate or adenosine monophosphate
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • dAMP deoxyadenosine diphosphate
  • dATP deoxyadenosine triphosphate
  • inosinic acid inosinic acid or inosin monophosphate
  • IMP inosin diphosphate
  • IDP inosin triphosphate Acids
  • ITP deoxyinosinic monophosphate
  • dIDP deoxyinosin diphosphate
  • dITP deoxyinosin triphosphate
  • a compound having a purine skeleton in which a hydrogen atom is bonded to a carbon atom at the 2-position also includes a purine base, a purine nucleoside, or a derivative of a purine nucleotide.
  • the "derivative" refers to a compound in which a purine base, a purine nucleoside or a purine nucleotide portion, a sugar residue portion, and / or a phosphate group portion is chemically modified or a substituent is introduced.
  • the compound having a purine skeleton in which a hydrogen atom is bonded to a carbon atom at the 2-position may be a salt, for example, adenine, hypoxanthine, or a salt of a purine nucleoside or purine nucleotide containing adenine or hypoxanthine as a component.
  • preferred salts are alkali metal salts (eg, sodium salts, potassium salts), including, but not limited to, sodium adenylate and sodium inosinate.
  • the compound having a purine skeleton in which a hydrogen atom is bonded to a carbon atom at the 2-position is selected from the group consisting of, for example, adenine, hypoxanthine, adenosine, inosine, deoxyadenosine, deoxyadenosine, adenylic acid, inosinic acid, and salts thereof. It 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, and CELL BIOLABBS.
  • the fermentation accelerator of the present invention uses at least one compound having a purine skeleton in which a hydrogen atom is bonded to the carbon atom at the 2-position, preferably 1 to 4, for example, 1 to 3 or 1 to 2 in combination. You may.
  • the mass ratio of the organic acid to the nucleic acid raw material is not particularly limited, and is, for example, 0.005 to 500, preferably 0.05 to 200. It is more preferably 0.1 to 100.
  • the nucleic acid raw material contains an amount in which 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, and more preferably 0.1 to 10 mM with respect to the total amount of the medium or raw milk. , More preferably, it can be added in an amount of 0.5 to 2 mM.
  • the agent of the present invention can be provided as an agent containing the above-mentioned components and optionally food hygiene or pharmaceutically acceptable additives.
  • aqueous media such as water, solvents, solubilizers, lubricants, emulsifiers, tonicity agents, stabilizers, preservatives, preservatives, surfactants , Regulators, chelating agents, pH regulators, buffers, excipients, thickeners, colorants, fragrances or fragrances and the like.
  • the agent of the present invention may be in any form such as liquid, powder, granule, gel, solid, capsule inclusion body and the like.
  • the fermentation accelerator can be produced by appropriately mixing at least one organic acid selected from malic acid and fumaric acid with other optional components such as nucleic acid, and the resulting mixture is further added to a solvent. Dissolution, powdering, granulation, gelation, solidification, encapsulation and the like can be processed according to a known formulation technique.
  • the fermentation promoting action of the present invention involves culturing and fermenting lactic acid bacteria in raw milk to which a fermentation accelerator has been added, examining an index indicating the progress of the fermentation state, and as a result, a control (without adding the fermentation accelerator of the present invention). It can be confirmed by the fact that the fermentation is proceeding faster than the group).
  • the index indicating the progress of the fermentation state is not particularly limited, but for example, a decrease in the pH value of fermented milk accompanying an increase in the amount of lactic acid produced by fermentation of lactic acid bacteria can be used as an index.
  • the pH is, for example, pH 4.6.
  • pH 4.6 can be set as a pH at which fermentation is sufficiently performed in the production of normal fermented milk and fermentation is completed.
  • the pH can be measured using a commercially available pH meter.
  • 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 may be of animal origin or plant origin.
  • Preferred lactic acid bacteria of the present invention include Lactobacillus spp., Streptococcus spp., Lactococcus spp., Enterococcus spp., Enterococcus spp., Leuconostock spp. Combinations can be mentioned, but lactic acid bacteria containing Lactobacillus spp. Are preferred. Examples of lactic acid bacteria containing Lactobacillus include Lactobacillus, a combination of Lactobacillus and Streptococcus, a combination of Lactobacillus and Lactococcus, and the like. These lactic acid bacteria can be obtained from, for example, a storage organization such as ATCC, or commercially available ones can be used as appropriate.
  • Lactobacillus spp. are Lactobacillus delbruecchii, Lactobacillus acidofilus, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus gasseri (Lactobacillus reuteri), Lactobacillus salivalius, Lactobacillus pentosus, Lactobacillus kefilanofacilus Lactobacillus jhonsii, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus amilovacillus lavactobacillus amyloves , And Lactobacillus sakei.
  • Lactobacillus spp. are Lactobacillus delbrucky, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus ramnosus, Lactobacillus reuteri, Lactobacillus salibarius, Lactobacillus pentosas and the like.
  • the preferred Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus ramnosus, Lactobacillus reuteri, Lactobacillus salivalius and Lactobacillus pentosas are Lactobacillus acidophilus JCM 1132T strain and Lactobacillus gasseri JCM, respectively.
  • Examples include 1131T strain, Lactobacillus ramnosus JCM 1136T strain, Lactobacillus reuteri JCM 1112T strain, Lactobacillus salivalius JCM 1231T strain, and Lactobacillus pentosas JCM 1558T strain.
  • Lactobacillus delbrucky for example, Lactobacillus delbruecchii subsp. Bulgaricus (Lactobacillus delbruckii subspecies bulgarix; Lactobacillus delbruecchii subsp.). Lactis (Lactobacillus delbrucky subspecies lactis), Lactobacillus delbruecchii subsp. Delbruecchii (Lactobacillus delbrucky subspecies delbrucky), Lactobacillus delbruecchii subsp. Indicus (Lactobacillus delbrucky Subspecies Indicators) and the like can be mentioned.
  • Lactobacillus del Brooky is Lactobacillus del Brooky subspecies bulgaricus, and more preferably Lactobacillus delbrucky subspecies bulgaricus (2038 strain, OLL 1073R-1 strain, P1902901 strain). , OLL1171 strain, OLL1255 strain, OLL1247 strain, OLL205013 strain) and the like.
  • Lactobacillus kefilanofaciens include Lactobacillus kefiranofaciens subsp.
  • Kefirgranum (Lactobacillus kefirano faciens subspecies kefir granum) and the like can be mentioned, with preference given to Lactobacillus kefirano faciens subspecies kefir granum JCM 8572T and the like.
  • Lactobacillus acidophilus JCM 1132T strain Lactobacillus gasseri JCM 1131T strain, Lactobacillus ramnosus JCM 1136T strain, Lactobacillus reuteri JCM 1112T strain, Lactobacillus salivalius JCM 1231T strain, Lactobacillus pentos Lactobacillus kefilanofaciens subspecies kefiagranum JCM 8572T strain is from Japan Collection of Microorganisms (RIKEN BRC-JCM, Japan), Bioresource Center (RIKEN BRC), Incorporated Administrative Agency. , Can be obtained under the accession numbers JCM 1132T, JCM 1131T, JCM 1136T, JCM 1112T, JCM 1231T, JCM 1558T, and JCM 8572T, respectively.
  • Lactobacillus delbrucky subspecies bulgaricus 2038 strain can be isolated from "Meiji Bulgaria Yogurt” (registered trademark) in a commercially available selective medium for the genus Lactobacillus, Meiji Innovation Center Co., Ltd. ( ⁇ 192). -0919 Meiji Innovation Center, 1-29-1, Nanakuni, Hachioji-shi, Tokyo, Japan).
  • Lactobacillus del Brooky Subspecies Bulgarics OLL 1073R-1 strain was issued on February 22, 1999 by the Incorporated Administrative Agency Industrial Technology Research Institute Patent Organism Depositary Center (1-1-1, Higashi, Tsukuba City, Ibaraki Prefecture, Japan). It was deposited in 6), then transferred to an international deposit, and the deposit number FERM BP-10741 was given.
  • Budapest Notification No. As described in 282 (http://www.wipo.int/treaties/en/notifications/budapest/treaty_budapest_282.html), the National Institute of Advanced Industrial Science and Technology (IPOD, NITE) is an independent administrative agency.
  • Lactobacillus delbrucky Subspecies Bulgalix P1902901 shares are stored by Meiji Innovation Center Co., Ltd. (Meiji Innovation Center, 1-27-1, Hachioji City, Hachioji City, Japan 192-0919).
  • Lactobacillus delbrucky Subspecies Bulgarics OLL1171 strain was sent to the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, which is an international depositary authority based on the provisions of the Butabest Treaty, on March 13, 2013, NITE BP- It has been deposited internationally as 01569.
  • Lactobacillus delbrucky Subspecies Bulgarics OLL1255 strain was sent to the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, which is an international depositary authority based on the provisions of the Butabest Treaty, on February 10, 2005, NITE BP- It has been deposited internationally as 76.
  • Lactobacillus delbrucky Subspecies Bulgarics OLL1247 strain was sent to the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, which is an international depositary authority based on the provisions of the Butabest Treaty, on March 6, 2014, NITE BP- It has been deposited internationally as 0814.
  • Lactobacillus delbrucky Subspecies Bulgarics OLL205013 strain was sent to the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, which is an international depositary authority based on the provisions of the Butabest Treaty, on February 3, 2017, NITE BP- It has been deposited internationally as 02411.
  • Streptococcus spp examples include Streptococcus thermophilus.
  • Lactococcus spp examples include Lactococcus lactis, Lactococcus plantarum, and Lactococcus raffinolactis.
  • Lactobacillus spp. And Streptococcus spp. Is preferably Lactobacillus delbrucky subspecies bulgaricus and Streptococcus thermophilus.
  • the mixing ratio of lactic acid bacteria, at least one organic acid selected from malic acid and fumaric acid, and nucleic acid raw materials is appropriately set according to the types and properties of lactic acid bacteria, medium, raw milk, temperature conditions, and other fermentation conditions. You may.
  • the mass ratio (lactic acid bacterium / organic acid) of lactic acid bacteria to at least one organic acid selected from malic acid and fumaric acid is, for example, 0.001 to 500,000, preferably 0.01 to 5,000, and more. It is preferably 0.1 to 500.
  • the mass ratio of the lactic acid bacterium to the nucleic acid raw material is, for example, 0.1 to 11000, preferably 1 to 11000, more preferably 10 to 1100, and further preferably 50 to 50. It is 550.
  • the amount of lactic acid bacteria is, for example, 0.001% by mass to 5% by mass, preferably 0.01% by mass to 2.5% by mass, and more preferably 0.01% by mass to 2% by mass, based on the total amount of the medium or raw milk. Even more preferably, it can be added in an amount of 0.1% by mass to 1% by mass.
  • Lactic Acid Bacteria Starter The organic acid selected from malic acid and fumaric acid as described above can be used as a lactic acid bacterium starter by using lactic acid bacteria together. Therefore, according to a preferred embodiment of the present invention, there is provided a lactic acid bacterium starter comprising a lactic acid bacterium and at least one organic acid selected from malic acid and fumaric acid.
  • the lactic acid bacterium starter includes a lactic acid bacterium prepared by culturing the lactic acid bacterium in a medium (for example, an activation medium) and undergoing intermediate fermentation.
  • the lactic acid bacterium starter preferably contains a lactic acid bacterium and a medium in which the lactic acid bacterium is cultured as a component. Therefore, the lactic acid bacterium starter may further contain succinic acid.
  • the succinic acid is preferably an endogenous organic acid produced by the lactic acid bacterium.
  • the lactic acid bacterium starter In addition to the lactic acid bacterium starter that is directly inoculated into the raw milk that is the source of fermented milk, the lactic acid bacterium starter is inoculated into another medium to further grow (scale up) the lactic acid bacterium. Is included.
  • Lactic acid bacteria starter is basically used to ferment raw milk to obtain fermented milk.
  • the lactic acid bacterium starter obtained by the present invention is further cultured in a medium at least once and then cultured. It also includes inoculating the raw milk with a later next-generation lactic acid bacterium starter.
  • the lactic acid bacterium starter may be used in combination with the nucleic acid raw material. Therefore, according to another aspect of the present invention, there is provided a lactic acid bacterium starter comprising a lactic acid bacterium and at least one organic acid selected from malic acid and fumaric acid for use in combination with a nucleic acid raw material.
  • the lactic acid bacterium starter is preferably used in combination with the nucleic acid raw material.
  • the nucleic acid raw material may be added to the medium or raw milk as a separate substance together with the lactic acid bacterium starter, and the nucleic acid raw material is mixed with the lactic acid bacterium starter as a constituent component. And may be used integrally.
  • the lactic acid bacterium starter may contain a nucleic acid raw material.
  • Each aspect of malic acid, fumaric acid, nucleic acid raw material, and lactic acid bacterium in the lactic acid bacterium starter can be the same as the description regarding the fermentation accelerator of the present invention.
  • the viable number of lactic acid bacteria in the lactic acid bacterium starter is not particularly limited, but is, for example, 1.0 ⁇ 10 4 to 1.0 ⁇ 10 13 cfu / g, preferably 1.0 ⁇ 10 5 to 1.0 ⁇ 10. It is 12 cfu / g, more preferably 1.0 ⁇ 10 6 to 1.0 ⁇ 10 11 cfu / g.
  • the mass ratio of lactic acid bacteria to organic acid in the lactic acid bacterium 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 organic acid are the mass ratios in the above fermentation accelerator. It can be similar to the ratio.
  • the lactic acid bacterium starter can be produced from the lactic acid bacterium and an arbitrary component such as the above-mentioned organic acid, a nucleic acid raw material, and a medium component.
  • an arbitrary component such as the above-mentioned organic acid, a nucleic acid raw material, and a medium component.
  • a preferred method for producing a lactic acid bacterium starter includes a medium preparation step, a medium sterilization step, a lactic acid bacterium inoculation step, a culture step (medium fermentation step), and an organic acid addition step.
  • the medium preparation step is a step of preparing a medium for inoculating lactic acid bacteria (for example, an activation medium).
  • the medium is not particularly limited as long as it does not interfere with the effect of the present invention, and examples thereof include a medium containing a milk component, for example, skim milk, skim milk concentrate, skim milk powder (reduced skim milk), and proteins of these skim milk components.
  • a medium containing skim milk, skim milk powder, whey, whey powder and the like is even more preferable.
  • the said medium may be the same as the raw material milk described later.
  • the medium preferably further contains yeast extract.
  • the medium can be prepared by a known method such as mixing, dissolving, dispersing, and suspending each of the above components.
  • the medium sterilization step is a step of sterilizing the medium prepared in the medium preparation step by, for example, heating.
  • the heating temperature and the heating time may be adjusted to the extent that various germs in the medium can be sterilized.
  • a known method can be used for heat sterilization.
  • heat treatment may be performed by a plate type heat exchanger, a tube type heat exchanger, a steam injection type heating device, a steam infusion type heating device, an energization type heating device, an autoclave device, etc.
  • Heat treatment may be performed by the tank.
  • the sterilization of the medium is not limited to heating, and can also be performed by a known method such as irradiation with ultraviolet rays.
  • the lactic acid bacterium addition (inoculation) step is a step of adding (inoculating) lactic acid bacteria to the sterilized medium.
  • frozen bacteria for example, freeze-concentrated bacteria, frozen pellets, freeze-dried powder, etc.
  • the lactic acid bacterium addition step the lactic acid bacterium is preferably added in an amount of 0.05% by mass or more, more preferably 0.05 to 10% by mass, and 0.1 to 5% by mass with respect to the medium. It is more preferable to add it.
  • the culturing step is a step of culturing lactic acid bacteria in a medium, growing the lactic acid bacteria, and obtaining a lactic acid starter.
  • the culture time of the lactic acid bacterium 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.
  • the above-mentioned culturing time means one culturing time.
  • the temperature of the medium is preferably maintained in the fermentation temperature range of 30 ° C. or higher.
  • the temperature of the medium is preferably maintained at 30 to 50 ° C, more preferably 35 to 50 ° C.
  • "standing” means not stirring the medium. For example, even when moving the container containing the medium, if the inside of the medium is not stirred, "standing". Corresponds to. In this way, by allowing the medium to stand during the culturing step, the growth of lactic acid bacteria can be promoted and the time until the end of culturing can be shortened.
  • the organic acid addition step 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 organic acid addition step may be carried out at any time before the culturing step, during the culturing step, or after the culturing step, but from the viewpoint of shortening the culturing time, it should be carried out before the culturing step or during the culturing step. Is preferable.
  • the step of adding an organic acid or the like can be carried out, for example, by collecting a predetermined amount of lactic acid bacteria or a lactic acid bacterium-containing medium thereof after the culturing step, and adding an organic acid and a nucleic acid raw material to the lactic acid bacterium or the lactic acid bacterium-containing medium as desired. it can. Further, the step of adding the organic acid or the like may be carried out, for example, by adding the organic acid and, if desired, the nucleic acid raw material to the medium before or during the culturing step.
  • the period before the culturing step is not particularly limited, and may be any time before the medium sterilization step, after the medium sterilization step, before the lactic acid bacterium addition step, after the lactic acid bacterium addition step, and at the same time as the lactic acid bacterium addition. May be good. It is preferable that the organic acid to be added is dissolved in water and the pH is adjusted to 6.0 to 7.0.
  • fermented milk can be efficiently produced by using the above-mentioned fermentation accelerator or lactic acid bacterium starter.
  • “fermented milk” includes “fermented milk”, “dairy product lactic acid bacteria beverage”, and “lactic acid bacteria beverage” defined by the ministry ordinance (Ministry Ordinance on Milk, etc.) concerning component standards of milk and dairy products.
  • fermented milk includes raw milk, milk, special milk, raw goat milk, sterilized goat milk, raw noodle milk, ingredient-adjusted milk, low-fat milk, non-fat milk, processed milk, etc.
  • Milk containing milk solids is fermented with lactic acid bacteria or yeast to make it paste-like or liquid, or frozen, and these include hard yogurt, soft yogurt (paste-like fermented milk), or drink yogurt (paste-like fermented milk). Liquid fermented milk) is included.
  • hard yogurt such as plain yogurt is produced by filling a container with raw materials and then fermenting (post-fermentation) (also referred to as “set yogurt”).
  • soft yogurt and drink yogurt are produced by atomizing or homogenizing fermented fermented milk (pre-fermented) and then filling it in a container (also referred to as “stirring yogurt”).
  • succinic acid is contained in fermented milk in a high content by fermenting lactic acid bacteria in the presence of malic acid, fumaric acid, and optionally a nucleic acid raw material, without using food additives.
  • a fermented milk containing lactic acid bacteria and succinic acid, wherein the succinic acid is an endogenous organic acid produced by the lactic acid bacteria. ..
  • the content of succinic acid 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, still more preferably 1 mM or more, still more preferably 3 mM or more with respect to the total amount of fermented milk. It is said that.
  • the preferred 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 preferred upper limit is 50 mM, more preferably 20 mM, even more preferably 15 mM.
  • the succinic acid content in the fermented milk is preferably 1 mM or more with respect to the total amount of the fermented milk, for example. Is 1.5 mM or more, more preferably 3 mM or more.
  • the succinic acid content in the fermented milk is, for example, 0.5 mM or more, preferably 1 mM or more with respect to the total amount of fermented milk.
  • 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.
  • HPLC high performance liquid chromatography
  • 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 (TRANSGENOMIC)). The measurement can be performed under the following conditions, for example.
  • Analyzer LC20 system manufactured by Shimadzu Corporation, Column: ICSep ICE-ORH-801, 6.5 mm I.D. D. ⁇ 300 mm, used by connecting two, mobile phase: 7.5 mM p-toluenesulfonic acid, reaction solution: 7.5 mM p-toluenesulfonic acid, 150 ⁇ M EDTA (2NA), 30 mM Bis Tris, flow velocity: 0.5 ml / min, injection volume: 10 ⁇ l, oven temperature: 55 ° C., detection: electrical conductivity detector.
  • Fermented milk may contain malic acid, fumaric acid, and a nucleic acid raw material because it is suitably produced in the presence of an organic acid such as malic acid or fumaric acid, and if desired, a nucleic acid raw material.
  • 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.
  • 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.
  • the content of the nucleic acid raw material in the fermented milk is, for example, 0.0001 to 5% by mass, preferably 0.0001 to 1.5% by mass.
  • the fermented milk of the present invention contains more than twice as much succinic acid as the fermented milk to which at least one organic acid selected from malic acid and fumaric acid is not added at the time of production, and is preferably 2. It contains 5 times or more, more preferably 5 times or more. The upper limit is 30 times, preferably 20 times.
  • fermented milk can be produced using the above fermentation accelerator or lactic acid bacterium starter in the presence of organic acids such as malic acid and fumaric acid, and nucleic acid raw materials. Therefore, there is provided a method for producing fermented milk comprising 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 ferment 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 milk preparation step, a raw milk sterilization step, a lactic acid bacterium starter inoculation step, and a fermentation step.
  • the raw milk preparation process is a step of preparing raw milk to be inoculated with a lactic acid bacterium starter.
  • "Raw 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, known raw milk can be used as appropriate.
  • Raw milk includes both pre-sterilized and post-pasteurized milk.
  • raw milk Specific raw materials for raw milk include raw milk, sterilized milk, skim milk, full-fat milk powder, skim milk powder, buttermilk, butter, cream, whey protein concentrate (WPC), and whey protein isolate (WPI).
  • WPC whey protein concentrate
  • WPI whey protein isolate
  • ⁇ (alpha) -lactoalbumin (La), ⁇ (beta) -lactoglobulin (Lg) and the like may be included. Pre-warmed gelatin or the like may be added as appropriate.
  • Raw milk is known and can be prepared according to known methods.
  • Preferred raw milk includes, but is not limited to, raw milk, skim milk, skim milk powder, and cream. More preferable raw material milks include skim milk and skim milk powder.
  • the content of non-fat milk solids in the raw milk used in the present invention is not particularly limited as long as it does not interfere with the effects of the present invention, and is preferably 6 to 11% by mass, more preferably 7 to 10% by mass. is there.
  • the fat content in the raw milk is not particularly limited as long as it does not interfere with 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. Can exemplify 0.1 to 3% by mass, but is not limited thereto.
  • the raw milk sterilization step is a step of sterilizing the raw milk prepared in the raw milk preparation step by, for example, heating.
  • the raw milk is preferably sterilized. Examples of such sterilization include sterilization by heating.
  • the sterilization the heating temperature and the heating time can be adjusted to the extent that various germs in the raw material milk can be sterilized. For example, it is preferable to sterilize the raw milk at a temperature of 80 ° C. or higher, preferably 90 ° C. or higher.
  • a known method can be used for the heat treatment.
  • the lactic acid bacterium starter inoculation step is a step of inoculating (adding) the lactic acid bacterium starter to the above raw material milk.
  • a lactic acid bacterium starter obtained through the method for producing the lactic acid bacterium starter, a lactic acid bacterium starter prepared by a usual method and frozen, or a lactic acid bacterium starter dried after freezing can be used.
  • At least one organic acid selected from malic acid and fumaric acid and a nucleic acid raw material as an optional component are preferably added to the raw material milk as a constituent component in the lactic acid bacterium starter, but separately from the lactic acid bacterium starter. It may be contained in raw milk as an additive or as an intrinsic component.
  • the amount of each component of the lactic acid bacterium, the organic acid, and the nucleic acid raw material added to the raw material milk can be the same as the amount described in the above fermentation accelerator.
  • the fermentation process is a process of fermenting raw milk with a lactic acid bacterium starter.
  • the raw milk inoculated with the lactic acid bacterium starter is fermented while maintaining the fermentation temperature range to obtain fermented milk.
  • 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), the type and flavor of the fermented milk to be prepared, and the like.
  • the fermentation temperature is about 30 to 50 ° C. If the temperature is within this range, lactic acid bacteria are generally easy to act, so that 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.
  • the fermentation time may be appropriately adjusted according to the lactic acid bacteria (lactic acid bacteria starter) used, the fermentation temperature, and the like.
  • the fermentation time can be adjusted by using the pH in the fermented milk as an index of 4.6.
  • the fermentation time is not limited, but may be, for example, 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.
  • the apparatus for producing fermented milk and the production conditions known ones can be used.
  • the post-fermented product has a fermentation chamber for fermenting after filling, and the pre-fermented product has a fermentation tank for fermenting and a line filter or homogenizer for crushing fermented milk curds.
  • a deoxidizer or the like can be appropriately adopted as a manufacturing condition.
  • succinic acid contained in fermented milk is an umami substance and is an organic acid having useful physiological functions such as weight gain suppressing action, glucose tolerance improving action, and cancer growth suppressing action. Therefore, according to one embodiment, the fermented milk of the present invention has a composition for improving umami, suppressing weight gain, improving glucose tolerance, or suppressing cancer growth. Provided as a thing.
  • At least one organic acid selected from malic acid and fumaric acid can be optionally combined with a nucleic acid raw material to promote fermentation of lactic acid bacteria. Therefore, according to another aspect of the present invention, a medium (for example, a medium containing a milk component) or raw milk is fermented by lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.
  • a method for promoting fermentation of lactic acid bacteria is provided. Further, according to another preferred embodiment, the method for promoting fermentation of lactic acid bacteria includes fermenting lactic acid bacteria in the coexistence of the organic acid and the nucleic acid raw material.
  • a medium for example, a medium containing a milk component
  • a raw material milk or the like is fermented with lactic acid bacteria in the presence of at least one organic acid selected from malic acid and fumaric acid.
  • a method for producing or promoting the production of a lactic acid bacterium fermentation metabolite, which comprises the above, is provided. Further, according to another preferred embodiment, the method comprises fermenting lactic acid bacteria in the coexistence of the organic acid and the nucleic acid raw material.
  • the fermentation metabolite include succinic acid, extracellular polysaccharide (EPS), peptide and the like, and more preferably extracellular polysaccharide (EPS).
  • At least one organic acid selected from malic acid and fumaric acid for promoting fermentation of lactic acid bacteria is provided.
  • the organic acid is fermented by lactic acid bacteria in the coexistence of a nucleic acid raw material.
  • the use of at least one organic acid selected from malic acid and fumaric acid is provided in the production of a fermentation accelerator for lactic acid bacteria.
  • the said organic acid is used in combination with a nucleic acid raw material.
  • the use of at least one organic acid selected from malic acid and fumaric acid in the production of a lactic acid bacterium starter is provided.
  • the said organic acid is used in combination with a nucleic acid raw material.
  • the use of at least one organic acid selected from malic acid and fumaric acid in the production of fermented milk, in which lactic acid bacteria are fermented in the presence of the organic acid is provided that includes.
  • the said organic acid is used in combination with a nucleic acid raw material.
  • the fermented milk comprises succinic acid, which may be an endogenous organic acid produced by lactic acid bacteria.
  • Test Example 1 Lactobacillus delbruecchii subsp. Of malic acid or fumaric acid. Examination of fermentation promoting effect on bulgaricus (hereinafter, also referred to as "L. bulgaricus”) First, the following Lactobacillus delbruecchii subsp. Frozen bacteria of the bulgaricus strain were prepared. (1) Lactobacillus delbruecchii subsp. bulgaricus 2038 (hereinafter also referred to as "2038”) (2) Lactobacillus delbruecchii subsp. bulgaricus OLL1073R-1 (hereinafter, also referred to as "OLL 1073R-1”) (3) Lactobacillus delbruecchii subsp.
  • bulgaricus P1902901 (hereinafter, also referred to as “P1902901”) (4) Lactobacillus delbruecchii subsp. bulgaricus OLL1171 (hereinafter, also referred to as “OLL1171”) (5) Lactobacillus delbruecchii subsp. bulgaricus OLL1255 (hereinafter, also referred to as “OLL1255") (6) Lactobacillus delbruecchii subsp. bulgaricus OLL1247 (hereinafter, also referred to as “OLL1247”) (7) Lactobacillus delbruecchii subsp. bulgaricus OLL205013 (hereinafter, also referred to as "OLL205013”)
  • the above strain was 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.
  • the 10% by mass reduced skim milk powder is skim milk powder (fat content 1% by mass, protein 34% by mass, lactose 54% by mass, ash content 8% by mass, non-fat milk solid content 96% by mass) (manufactured by Meiji Co., Ltd.). ) Is a 10% by mass aqueous solution (in the above medium, lactose content is 5.4% by mass, non-fat milk solid content is 9.6% by mass).
  • Frozen bacteria were added in an amount of 0.1% by mass (relative to the amount of the activation medium) to the above activation medium, and the mixture was allowed to stand at 37 ° C. for 16 hours to obtain an activation solution. 0.1% by mass (relative to the amount of activation medium) of the obtained activation solution was added to another activation medium, and the mixture was allowed to stand at 37 ° C. for 16 hours to obtain a lactic acid bacterium starter.
  • fermentation was carried out using a fermentation medium as the raw material milk.
  • a fumaric acid aqueous solution or a malic acid aqueous solution adjusted to pH about 6.5 with NaOH before sterilization was added so as to have a final concentration of 1 mM.
  • the pH of the fermentation medium was about 6.4.
  • the lactic acid bacterium starter of each strain obtained above was inoculated into the fermentation medium in an amount of 0.5% by mass (relative to the amount of the fermentation medium), statically cultured at 40 ° C., and fermented.
  • 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 results obtained are shown in Table 1. From Table 1, by adding malic acid or fumaric acid, L. It can be seen that the fermentation of all 7 bulgaricus strains was promoted. L. The fermentation promoting effects of malic acid and fumaric acid on bulgaricus were almost the same.
  • the fermentation shortening time varied from strain to strain and ranged from 40 minutes to 12 hours and 15 minutes.
  • Test Example 2-1 L. Examination of the concentrations of malic acid and fumaric acid showing a fermentation promoting effect on the bulgaricus 2038 strain 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 was sterilized at 95 ° C. was used. Further, to the fermentation medium, a fumaric acid aqueous solution or a malic acid aqueous solution adjusted to pH about 6.5 with NaOH before sterilization was added so as to have the final concentrations shown in Tables 3 and 4. In the above fermentation medium, L.
  • the lactic acid bacterium starter of the bulgaricus 2038 strain was inoculated at 0.5% (relative to the amount of fermentation medium), statically cultured at 40 ° C., and fermented.
  • the fermentation time was defined as the time required to reach pH 4.6.
  • 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, and 20 ⁇ L of Calet's Reagent I (53.5% (w / v) zinc sulfate) was added and vortexed, and Calet's Reagent II (17) was added. .2% (w / v) potassium ferrocyanide) was added in an amount of 20 ⁇ l and vortexed. Centrifugated at 4 ° C., 20620 g for 10 minutes, and the supernatant was filtered through a 0.22 ⁇ m filter and analyzed under the conditions shown in Table 2.
  • Test Example 2-2 L. delbruecchii subsp.
  • the strains shown in Tables 6 and 7 were activated before use in a succinic acid production amount activating medium when malic acid and fumaric acid were added in bulgaricus.
  • 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.
  • the 10% by mass reduced skim milk powder is skim milk powder (fat content 1% by mass, protein 34% by mass, lactose 54% by mass, ash content 8% by mass, non-fat milk solid content 96% by mass) (manufactured by Meiji Co., Ltd.).
  • Is a 10% by mass aqueous solution in the above medium, lactose content is 5.4% by mass, non-fat milk solid content is 9.6% by mass.
  • Frozen bacteria were added to the above-mentioned activation medium in an amount of 0.1% by mass (relative to the amount of the activation medium) and statically cultured at 37 ° C. for 16 hours to obtain an activation solution. 0.1% by mass (relative to the amount of activation medium) of the obtained activation solution was added to another activation medium, and the mixture was allowed to stand at 37 ° C. for 16 hours to obtain a lactic acid bacterium starter.
  • fermentation was carried out using a fermentation medium as the raw material milk.
  • a fumaric acid aqueous solution or a malic acid aqueous solution adjusted to pH about 6.5 with NaOH before sterilization was added so as to have a final concentration of 1 mM.
  • the pH of the fermentation medium was about 6.4.
  • the lactic acid bacterium starter of each strain obtained above was inoculated into the fermentation medium in an amount of 2.5% by mass (relative to the amount of the fermentation medium), statically cultured at 40 ° C., and fermented.
  • the fermentation time was defined as the time required to reach pH 4.6.
  • the pH was measured using a commercially available pH meter.
  • Tables 6 and 7. From Table 6, any L. Delbruecchii subsp. Also in the bulgaricus strain, the concentration of succinic acid was increased by the addition of malic acid and fumaric acid. Further, from Table 7, as in Test Example 1, by adding malic acid or fumaric acid, L. Fermentation of all 7 bulgaricus strains was promoted.
  • Test Example 3 L. delbruecchii subsp. Examination of the fermentation promoting effect of fumaric acid on lactic acid bacteria species other than bulgaricus First, reference strains of the following strains other than Lactobacillus delbrucky subspecies bulgaricus were prepared. (8) Lactobacillus acidofilus JCM 1132T (9) Lactobacillus gasseri JCM 1131T (10) Lactobacillus rhamnosus JCM 1136T (11) Lactobacillus reuteri JCM 1112T (12) Lactobacillus salivalius JCM 1231T (13) Lactobacillus pentosus JCM 1558T
  • the above strain was activated in the activation medium before use.
  • As the activation medium a 10% reduced skim milk medium containing 0.1% yeast extract was sterilized at 121 ° C. for 7 minutes. 1% of frozen bacteria was added to the above-mentioned activation medium (relative to the amount of the activation medium), and the cells were allowed to stand at 37 ° C. for 24 hours to obtain an activation solution. 1% of the obtained activation solution (relative to the amount of activation medium) was added to another activation medium, and the mixture was allowed to stand at 37 ° C. for 24 hours to obtain a lactic acid bacterium starter.
  • fermentation was performed using a fermentation medium (raw milk).
  • an aqueous fumaric acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added to the fermentation medium so as to have a final concentration of 1 mM.
  • the fermentation medium was inoculated with 1% of the lactic acid bacteria starter of each strain activated above (relative to the amount of the fermentation medium), statically cultured at 37 ° C., and fermented.
  • the fermentation time was defined as the time required to reach pH 4.6.
  • Table 8 By adding fumaric acid, L. Acidofilus, L. Gasseri, L. Rhamnosus, L. et al. Reuteri, L. et al. Salivalius, and L. Fermentation was promoted by pentosus.
  • Test Example 4 L. delbruecchii subsp. Examination of the fermentation promoting effect of malic acid on lactic acid bacteria species other than bulgaricus First, reference strains of the following bacterial species were prepared. (12) Lactobacillus salivalius JCM 1231T (13) Lactobacillus pentosus JCM 1558T (14) Lactobacillus kefiranofaciens subsp. kefirgranum JCM 8572T
  • Test Example 5 L. rhamnosus, L. et al. Measurement of Malic Acid, Fumaric Acid and Succinic Acid Concentrations in Salivalius L. rhamnosus, L. et al. The measurement of the organic acid concentration after the completion of fermentation of salivalius was carried out in the same manner as in Test Example 2-1. The results obtained are shown in Table 10. By adding malic acid or fumaric acid, L. rhamnosus, L. et al. The production of succinic acid was promoted in salivalius.
  • Test Example 6 Lactobacillus delbruecchii subsp. Of fumaric acid when inosinic acid is added. Examination of fermentation promoting effect on bulgaricus Lactobacillus delbruecchii subsp. Frozen bacteria of the bulgaricus strain were prepared. (1) Lactobacillus delbruecchii subsp. bulgaricus 2038 (2) Lactobacillus delbruecchii subsp. bulgaricus OLL1073R-1 (3) Lactobacillus delbruecchii subsp. bulgaricus P1902901 (4) Lactobacillus delbruecchii subsp.
  • bulgaricus OLL1171 Lactobacillus delbruecchii subsp. bulgaricus OLL1255 (6) Lactobacillus delbruecchii subsp. bulgaricus OLL1247 (7) Lactobacillus delbruecchii subsp. bulgaricus OLL205013
  • the above strain was activated in the activation medium before use.
  • As the activation medium a 10% reduced skim milk medium containing 0.1% yeast extract was sterilized at 121 ° C. for 7 minutes. 1% of frozen bacteria was added to the above-mentioned activation medium (relative to the amount of the activation medium), and the cells were allowed to stand at 37 ° C. for 24 hours to obtain an activation solution. 1% of the obtained activation solution (relative to the amount of activation medium) was added to another activation medium, and the mixture was allowed to stand at 37 ° C. for 24 hours to obtain a lactic acid bacterium starter.
  • fermentation was performed using a fermentation medium (raw milk).
  • an aqueous fumaric acid solution adjusted to a pH of about 6.5 with NaOH before sterilization was added to the fermentation medium so as to have a final concentration of 1 mM.
  • the fermentation medium was inoculated with 1% of the lactic acid bacteria starter of each strain activated above (relative to the amount of the fermentation medium), statically cultured at 37 ° C., and fermented.
  • the fermentation time was defined as the time required to reach pH 4.6.
  • the pH was measured using a pH meter. The results obtained are shown in Table 11. From Table 11, by adding fumaric acid, L. Fermentation of all 7 bulgaricus strains was promoted.
  • Test Example 7 L. malic acid when inosinic acid was added. delbruecchii subsp. Examination of fermentation promoting effect on bulgaricus Lactobacillus delbruecchii subsp. Frozen bacteria of the bulgaricus strain were prepared. (1) Lactobacillus delbruecchii subsp. bulgaricus 2038 (2) Lactobacillus delbruecchii subsp. bulgaricus OLL1073R-1 (3) Lactobacillus delbruecchii subsp. bulgaricus P1902901 (4) Lactobacillus delbruecchii subsp. bulgaricus OLL1171 (5) Lactobacillus delbruecchii subsp. bulgaricus OLL1247
  • Test Example 8 When inosinic acid was added, malic acid or fumaric acid was added to L. delbruecchii subsp. Effect of bulgaricus on succinic acid production 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.
  • the 10% by mass reduced skim milk powder is skim milk powder (fat content 1% by mass, protein 34% by mass, lactose 54% by mass, ash content 8% by mass, non-fat milk solid content 96% by mass) (manufactured by Meiji Co., Ltd.).
  • Is a 10% by mass aqueous solution in the above medium, lactose content is 5.4% by mass, non-fat milk solid content is 9.6% by mass.
  • Frozen bacteria were added to the above-mentioned activation medium in an amount of 0.1% by mass (relative to the amount of the activation medium) and statically cultured at 37 ° C. for 16 hours to obtain an activation solution. 0.1% by mass (relative to the amount of activation medium) of the obtained activation solution was added to another activation medium, and the mixture was allowed to stand at 37 ° C. for 16 hours to obtain a lactic acid bacterium starter.
  • fermentation was carried out using a fermentation medium as the raw material milk.
  • a 10% by mass reduced skim milk medium to which inosinic acid was added so as to have a final concentration of 1 mM was sterilized at 95 ° C.
  • a fumaric acid aqueous solution or a malic acid aqueous solution adjusted to pH about 6.5 with NaOH before sterilization was added so as to have a final concentration of 1 mM.
  • the pH of the fermentation medium was about 6.4.
  • the lactic acid bacterium starter of each strain obtained above was inoculated into the fermentation medium in an amount of 2.5% by mass (relative to the amount of the fermentation medium), statically cultured at 40 ° C., and fermented.
  • 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 results obtained are shown in Tables 13 and 14. From Table 13, in the medium containing inosinic acid, any L. Delbruecchii subsp. Also in the bulgaricus strain, the concentration of succinic acid was increased by the addition of malic acid and fumaric acid. Therefore, not only in the formic acid-added medium but also in the inosinic acid-added medium, by adding malic acid and fumaric acid, L. Delbruecchii subsp. It was shown that the amount of succinic acid produced by bulgaricus increased. Further, from Table 14, as in Test Example 6, by adding malic acid or fumaric acid, L. Fermentation of all 7 bulgaricus strains was promoted.
  • Test Example 9 Malic acid or fumaric acid is L. delbruecchii subsp. Effects of bulgaricus on proliferative and metabolite production Lactobacillus delbruecchii subsp. Frozen bacteria of the bulgaricus OLL1073R-1 strain were prepared.
  • the composition of the medium was as shown in Table 15 below. Ingredients other than anhydrous crystalline glucose were dissolved in Elix water, adjusted to pH 6.65 with sodium hydroxide, and then weighed up to 70% of the amount charged in the medium. Anhydrous crystalline glucose was dissolved in Elix water and weighed up to 30% of the amount charged in the medium. When malic acid or fumaric acid was added, it was dissolved in Elix water to a final concentration of 4 mM together with raw material components other than anhydrous crystalline glucose. The sugar solution and the raw material components other than sugar were sterilized by heating in an autoclave treatment at 110 ° C. for 1 minute, and then aseptically mixed and used.
  • the EPS concentration was measured under the following conditions. 1.5 g of the culture solution was weighed, and MilliQ water was added to make 10 g. 100% trichloroacetic acid was added, the mixture was mixed well, and the supernatant was collected after centrifugation (4 ° C., 13400 g, 10 min). After suspending the precipitate by adding 5 ml of a 10% trichloroacetic acid solution, the mixture was centrifuged again to collect the supernatant. Twice the amount of cooled 99.5% ethanol was added to the supernatant, mixed by inversion, and then allowed to stand at ⁇ 20 ° C. overnight.
  • the supernatant was removed by centrifugation (4 ° C., 13400 g, 20 min), and 20 ml of cooled 66% ethanol was added to suspend the precipitate.
  • the supernatant was removed by centrifugation (4 ° C., 13400 g, 20 min), air-dried to remove ethanol, and MilliQ water was added to adjust the volume to 10 ml, which was used as an analysis sample.
  • the EPS concentration of the analytical sample was measured by the phenol-sulfuric acid method.

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WO2024253180A1 (ja) * 2023-06-09 2024-12-12 株式会社明治 菌体外多糖の製造方法、及びその利用

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