Novel acetic acid bacterium and use thereof

Abstract

To provide a novel strain of acetic acid bacterium and its use or application, in response to ongoing investigation of the effects of acetic acid bacteria on fermentation performance and physiological functions, particularly regarding application of acetic acid bacteria to foods and health foods, and to meet the demand for employing previously unidentified acetic acid bacteria as fermentation-use microorganisms or immunoactivators.SOLUTION: An acetic acid bacterium identified as Gluconacetobacter takamatsuzukensis, deposited under deposit number NITE P-04018 with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation, and a fermentation composition containing the same are provided, and furthermore, compositions including immunoactivating compositions comprising the acetic acid bacterium are also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a novel acetic acid bacteria strain belonging to Gluconacetobacter takamatsuzukensis and its use. [Background technology]

[0002] Acetic acid bacteria are fermentation microorganisms commonly used in the production of brewed vinegar (see, for example, Patent Document 1), and several reports have been published regarding the effects of acetic acid bacteria on the physiological functions of living organisms (e.g., the human body) (Patent Documents 2 to 6). Patent Document 2 discloses an immunostimulant containing acetic acid bacteria cells, demonstrating that the immunostimulant enhances antibody production. Patent Document 3 demonstrates that acetic acid bacteria promote the growth of lactic acid bacteria (intestinal bacteria). Patent Document 4 discloses a composition containing acetic acid bacteria for alleviating allergy symptoms, reporting that it reduces nasal discomfort caused by dust, house dust, and the like. Patent Document 5 demonstrates that acetic acid bacteria promote the growth of bifidobacteria (intestinal bacteria). Patent Document 6 describes an immunostimulatory composition containing acetic acid bacteria that activates plasmacytoid dendritic cells. However, acetic acid bacteria have rarely been used as a raw material in the health food market, and much remains unknown regarding the functionality of acetic acid bacteria.

[0003] Representative species of acetic acid bacteria include the genera Gluconacetobacter, Acetobacter, Gluconobacter, etc. Gluconacetobacter takamatsuzukensis (T61213-20-1a) has been reported as an acetic acid bacterium of the genus Gluconacetobacter (see Non-Patent Document 1).

[0004] Interleukin 12 (IL-12), a cytokine secreted by macrophages and dendritic cells, has been reported to have immunostimulatory effects. Based on this, it has been proposed to use specific lactic acid bacteria that enhance IL-12 expression in macrophage cells as probiotics (see Non-Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 63-059874 [Patent Document 2] Japanese Patent Publication No. 58-105923 [Patent Document 3] Patent Publication No. 2016-106528 [Patent Document 4] Patent Publication No. 2021-059522 [Patent Document 5] Patent Publication No. 2022-188416 [Patent Document 6] Patent Publication No. 2023-085212 [Non-patent literature]

[0006] [Non-Patent Document 1] International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3981~3988, Gluconacetobacter tumulisoli sp. nov., Gluconacetobacter takamatsuzukensis sp. nov. and Gluconacetobacter aggeris sp. nov., isolated from Takamatsuzuka Tumulus samples before and during the dismantling work in 2007 [Non-patent document 2] Maebashi Institute of Technology Research Bulletin Vol. 25, pp. 47-48 (2022) Discovery and analysis of probiotics that enhance IL-12 expression Summary of the Invention [Problem to be solved by the invention]

[0007] Further research is being conducted into the fermentation ability and physiological function of acetic acid bacteria, and in particular, attempts are being made to apply acetic acid bacteria to foods and health foods. Therefore, there is a demand for novel acetic acid bacteria that have not yet been discovered, for use as fermentation microorganisms or as immunostimulants. Therefore, the present invention provides novel acetic acid bacteria strains and their uses or applications. [Means for solving the problem]

[0008] The first aspect of the present invention relates to the following novel acetic acid bacteria. [1] A strain belonging to Gluconacetobacter takamatsuzukensis, deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-04018. [2] The strain described in [1] above, isolated from cherry blossom flowers. [3] The strain according to [1] or [2] above, which is orally administered or taken.

[0009] The second aspect of the present invention relates to a novel morphology of acetic acid bacteria. [4] Live or killed cells of the acetic acid bacteria strains described in [1] to [3] above, or a dried powder or crushed product thereof.

[0010] The third aspect of the present invention relates to an immunostimulatory composition containing a novel acetic acid bacterium. [5] An immunostimulatory composition containing the acetic acid bacterium strain described in [1] or [2] above. [6] The immunostimulatory composition described in [5] above, which promotes the expression of interleukin-12. [7] The immunostimulatory composition described in [5] or [6] above, which is for food use.

[0011] The fourth aspect of the present invention relates to a composition for fermentation containing a novel acetic acid bacterium, and a fermented composition obtained therefrom. [8] A fermentation composition comprising the acetic acid bacterium of the strain described in [1] or [2] above and a raw material to be fermented. [9] A fermentation composition obtained from the fermentation composition described in [8] above.

[10] The fermented composition according to [9] above, which is for food use. [Effects of the Invention]

[0012] The acetic acid bacterium of the present invention is a novel strain, which is expected to have superior effects different from those of conventional acetic acid bacteria and can be used in a wide range of applications. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a molecular phylogenetic tree of the acetic acid bacteria strain of the present invention. [Figure 2] 1 is a table comparing the physiological and biochemical properties of the acetic acid bacteria strain of the present invention with the type strain "T61213-20-1a." [Figure 3] 1 is a table showing the results of colony observation, morphological observation, and various Barrow & Feltham tests of the acetic acid bacteria strain of the present invention. [Figure 4] 1 is a graph showing changes in the expression level of IL-12p40 when mouse macrophage-like cells (J774.1) were treated with the acetic acid bacteria of the present invention or other acetic acid bacteria. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. Acetic acid bacteria of the present invention The acetic acid bacterium of the present invention is a strain deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary on November 22, 2023, under accession number NITE P-04018, and is sometimes referred to as "strain A192." The acetic acid bacterium of the present invention can be isolated from cherry blossoms. Specifically, it can be isolated from the flowers of pale pink cherry trees (e.g., transplanted pale pink cherry trees in the grounds of Kano Tenmangu Shrine, Gifu City, Gifu Prefecture).

[0015] 1-1. Identification of bacterial species The newly obtained bacterium (isolated from the cherry blossoms of a transplanted pale pink cherry tree) in this study was identified as Gluconacetobacter takamatsuzukensis by BLAST homology searches against DB-BA and international base sequence databases (DDBJ / ENA (EMBL) / GenBank) using the microbial identification system "ENKI" (Techno Suruga Lab Co., Ltd.) and by evaluation of its morphological and physiological properties (see Figures 2 and 3).

[0016] More specifically, the acetic acid bacteria of the present invention were cultured in the media shown in Table 1 below and analyzed by the methods shown in Table 2. [Table 1] [Table 2]

[0017] The catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F test) of the acetic acid bacteria of the present invention were evaluated. The results are shown in Figure 3. As shown in Figure 3, the acetic acid bacteria of the present invention are gram-negative rods, which were catalase-positive and oxidase-negative, and fermented glucose.

[0018] 1-2. Characteristics of the acetic acid bacteria of the present invention The acetic acid bacterium of the present invention is a strain belonging to G. takamatsuzukensis, but differs from its type strain, T61213-20-1a, in terms of sugar assimilation and other properties (see Figure 2). For example, the acetic acid bacterium of the present invention differs from the type strain in that it oxidizes D-mannitol and assimilates sodium malonate. Furthermore, the acetic acid bacterium of the present invention has weaker assimilation of 1-propanol, ethanol, sodium acetate, and sodium lactate than the type strain. These differences in sugar assimilation and other properties make the acetic acid bacterium of the present invention a novel strain. This characteristic may also be the cause of differences in immunostimulatory activity and other properties.

[0019] 2. Uses or applications of acetic acid bacteria The acetic acid bacteria of the present invention can be used in any manner, for example, by being administered to a living organism (particularly by oral administration or ingestion) to exert various functions in the living organism. The acetic acid bacteria of the present invention may be used in the form of either live bacteria or killed bacteria (sterilized bacteria). The acetic acid bacteria of the present invention may also be used as a fermentation product containing the acetic acid bacteria. The acetic acid bacteria contained in the fermentation product may be in the form of either live bacteria or killed bacteria (sterilized bacteria).

[0020] For example, when the acetic acid bacteria of the present invention are used as components of an immunostimulating composition (see 2-1 below), the acetic acid bacteria may be live or killed, but enzymes produced by live bacteria may interact with other components of the immunostimulating composition and have adverse effects; and compositions containing live bacteria may have stability problems (e.g., in the case of a composition containing live bacteria, the number of live bacteria may decrease during formulation), so killed bacteria may be preferable. On the other hand, depending on the application (e.g., when used as a fermentation composition), live acetic acid bacteria of the present invention may be preferable.

[0021] Alternatively, the acetic acid bacteria may be cultured or otherwise grown before use. The method for culturing the acetic acid bacteria is not particularly limited, and for example, a method according to or similar to a known method can be employed. The medium used for culturing may be a conventional culture medium. For example, the medium may contain yeast extract, peptone, meat extract, or corn steep liquor as a nitrogen source, glucose, sucrose, maltose, or alcohol as a carbon source, and inorganic salts such as calcium salts, phosphate salts, magnesium salts, or sodium chloride. The culture conditions are not particularly limited, but aerobic culture is preferred, with the pH adjusted to a range of 6.0 to 7.2, the temperature adjusted to a range of 20 to 45°C, and the culture time approximately 8 to 100 hours. The culture method is not particularly limited, and the culture may be performed by static culture, shaking culture, aeration and agitation culture, or the like.

[0022] The acetic acid bacteria of the present invention contained in the culture medium after cultivation may be used as they are contained in the medium, or may be collected from the medium. Collection of bacteria is carried out by centrifugation or filtration using a filter press, and the collected acetic acid bacteria may be washed with water, physiological saline, Ringer's solution, or the like. Furthermore, the acetic acid bacteria may be subjected to drying treatment (e.g., freeze-drying treatment), high-temperature treatment at low pH, or the like.

[0023] The acetic acid bacteria of the present invention may be in the form of wet cells or freeze-dried to form a dry powder. Alternatively, the acetic acid bacteria (live cells) may be heat-sterilized and then formed into a dry powder, or may be formed into a disrupted cell product. The disrupted cell product is obtained by heating and then physically disrupting acetic acid bacteria obtained by culture, and disruption can be performed by ultrasonication, ball milling, French press, or other methods.

[0024] The acetic acid bacteria composition of the present invention may be a composition containing only acetic acid bacteria, or a composition containing acetic acid bacteria and any additives. The number of acetic acid bacteria contained in the acetic acid bacteria composition of the present invention may be set depending on its intended use. The number of acetic acid bacteria contained in the composition can be visualized and measured, for example, by staining the acetic acid bacteria. For visualization of acetic acid bacteria, for example, DAPI staining can be used. The acetic acid bacteria composition of the present invention can be produced using known methods as appropriate depending on its intended use.

[0025] The acetic acid bacteria composition of the present invention may be in either solid or liquid form. Solid compositions may be in any form, such as powder, granules, capsules, or tablets, while liquid compositions may be in any form, such as aqueous solutions, suspensions, pastes, or jellies. Furthermore, compositions containing acetic acid bacteria of the present invention are typically ingested orally, but are not limited to this and may also be administered parenterally.

[0026] The acetic acid bacteria of the present invention can be used in the same applications as those of conventional acetic acid bacteria. Preferably, they can be used in any of foods and beverages, pharmaceuticals, quasi-drugs, cosmetics, etc. When the acetic acid bacteria of the present invention are used in foods and beverages, they can be used in beverages such as water, soft drinks, fruit juice drinks, milk drinks, alcoholic drinks, sports drinks, and energy drinks; or in foods such as bread, noodles, rice, tofu, dairy products, soy sauce, miso, confectioneries, cream, sauces, mayonnaise, dressings, and supplements. Furthermore, supplements such as capsules (soft capsules and hard capsules) and tablets (including chewable tablets) may be preferred as foods and beverages because they are easily ingested.

[0027] Foods and beverages to which the acetic acid bacteria of the present invention are applied may be general foods, but may also be functional health foods such as foods for specified health uses (foods approved under Article 43, Paragraph 1 of the Health Promotion Act) or functional food products (foods notified under the Food Labeling Act). These functional health foods can be labeled with their functions, so the function (e.g., immunostimulatory effect) exerted by the acetic acid bacteria of the present invention can be labeled, for example, to indicate that they are useful for maintaining the immune function of healthy individuals and those with borderline immune conditions.

[0028] When the acetic acid bacteria of the present invention are used in foods and beverages, other bacteria or yeasts may be further blended into the foods and beverages, such as lactic acid bacteria, bifidobacteria, koji mold, butyric acid bacteria, and natto bacteria.

[0029] When the acetic acid bacteria of the present invention are used in foods and beverages, they may contain other ingredients, such as dietary fiber, sweeteners such as trehalose, sucralose, aspartame, and oligosaccharides, acidulants such as citric acid, acetic acid, lactic acid, phosphoric acid, and lemon juice, amino acids such as glutamic acid, glycine, γ-aminobutyric acid (GABA), taurine, and alanine, other seasonings such as sucrose, lactose, salt, and soy sauce, herbal medicines such as turmeric, liver extract, cinnamon bark, fennel, peony, and licorice, cosmetic ingredients such as collagen, hyaluronic acid, placenta, coenzyme Q10, chondroitin, and royal jelly, thickening polysaccharides such as soy polysaccharides, guar gum, and xanthan gum, and flavorings such as grapefruit and lemon.

[0030] When the acetic acid bacteria of the present invention are applied to pharmaceuticals, quasi-drugs, cosmetics, etc., they may be oral (internal) compositions or topical compositions. Examples of topical compositions include compositions applied to skin or hair, and oral care compositions. Topical compositions as quasi-drugs may be mouth fresheners, underarm odor suppressants, hair growth agents, bath additives, medicated cosmetics, medicated toothpastes, etc.

[0031] The composition containing the acetic acid bacteria of the present invention may be administered to humans (e.g., by ingestion) or to other animals, including mammals, such as pets and livestock.

[0032] Compositions containing the acetic acid bacteria of the present invention may be, but are not limited to, immunostimulatory compositions that enhance various immune functions (see 2-1 below), fermentation compositions (e.g., compositions for fermenting alcohol components) (see 2-2 below), compositions for improving the intestinal environment (see 2-3 below), or compositions that promote the growth of various bacteria (e.g., bacteria known as intestinal bacteria, specifically lactic acid bacteria and bifidobacteria) (see 2-4 below). This may also enable the development of new applications not previously available for acetic acid bacteria.

[0033] 2-1. Immunostimulating composition The acetic acid bacteria of the present invention can be used as a component of an immunostimulatory composition. That is, the immunostimulatory composition of the present invention can contain the acetic acid bacteria of the present invention, preferably as an "active ingredient."

[0034] The immunostimulatory composition of the present invention containing acetic acid bacteria may be able to improve allergic symptoms and suppress liver dysfunction, for example. Allergic symptoms include nasal discomfort such as rhinitis, sneezing, runny nose, nasal congestion, and nasal itching, itching and foreign body sensation, eye discomfort such as eye discharge and swelling, skin symptoms such as hives and swelling, and respiratory symptoms such as cough, asthma, and difficulty breathing, and the causative agents of these symptoms include dust, house dust, hay fever, and food.

[0035] The immunostimulatory composition containing the acetic acid bacteria of the present invention is, for example, preferably prepared by injecting 1×10 acetic acid bacteria into a human body at a daily intake of 1×10 acetic acid bacteria. 8 or more, more preferably 5×10 9 or more, more preferably 2 × 10 10 The upper limit of the daily intake of acetic acid bacteria is not particularly limited, but it is preferably 1 × 10 13 1 x 10 or less 11The number of acetic acid bacteria in the composition can be measured by any method, for example, by using the DAPI staining method.

[0036] The acetic acid bacteria of the present invention preferably have a high effect of promoting the expression of interleukin-12 (IL-12). Interleukin-12 is a cytokine generally expressed by macrophages and dendritic cells and has been reported to have immunostimulatory effects. Specifically, expressed interleukin-12 differentiates naive T cells into Th1 cells and enhances the production of interferon-γ, which is known to activate natural killer (NK) cells, thereby enhancing their attack against virus-infected cells, cancer cells, and other cells, thereby enhancing the host's defense capabilities. In other words, the acetic acid bacteria of the present invention can exert immunostimulatory effects by promoting the expression of interleukin-12.

[0037] The effect of the acetic acid bacteria of the present invention in promoting the expression of interleukin-12 can be confirmed, for example, by a test according to the procedures in the Examples described below.

[0038] 2-2. Fermentation composition The acetic acid bacteria of the present invention can be used as a fermenting microorganism in a fermentation composition. Acetic acid bacteria can ferment alcohol (ethanol) to produce acetic acid (brewed vinegar), and can also ferment various fermentation raw materials, such as carbon sources such as glucose, sorbitol, and various polysaccharides, nitrogen sources such as various peptides and proteins, and other biologically derived materials containing trace components. Therefore, the fermentation composition of the present invention contains at least the acetic acid bacteria and the fermentation raw material.

[0039] The fermented composition obtained from the fermentation composition of the present invention is, for example, a fermented food, typically brewed vinegar. Fermented foods are foods to which microorganisms have imparted a special flavor, such as a unique taste, aroma, or texture, not present in the original food. Brewed vinegar is an acidic seasoning made from grains, sugars, and ethanol as ingredients, and the main component is fermented acetic acid produced by the action of acetic acid bacteria. Depending on the ingredients, brewed vinegar can be classified into grain vinegar (made from grains such as rice, barley, corn, and sake lees), fruit vinegar (made from fruits such as grapes, apples, and persimmons), and alcohol vinegar (made primarily from brewing ethanol).

[0040] The acetic acid bacteria of the present invention can be used as a fermentation microorganism for the production of any brewed vinegar. Brewed vinegar is produced by alcoholic fermentation of a raw material containing carbohydrates, followed by the addition of the acetic acid bacteria of the present invention to carry out acetic acid fermentation. Acetic acid fermentation techniques can be broadly divided into "surface fermentation (static fermentation)" and "total fermentation (submerged fermentation)." In the surface fermentation method, acetic acid bacteria are added to alcohol, mixed, and allowed to stand. The liquid naturally circulates in the container, allowing acetic acid fermentation to proceed, and brewed vinegar is generally obtained in one to three months. In the total fermentation method, the liquid is stirred to introduce air and promote acetic acid fermentation. Therefore, acetic acid is usually produced in a shorter time than in the surface fermentation method. The acetic acid bacteria of the present invention can be used as a fermentation microorganism for the production of brewed vinegar, regardless of the acetic acid fermentation method.

[0041] 2-3. Composition for improving intestinal environment The acetic acid bacteria of the present invention may be used in compositions for improving the intestinal environment in vivo. Improving the intestinal environment includes improving the intestinal flora and increasing the number of intestinal bacteria (particularly beneficial bacteria such as bifidobacteria and lactic acid bacteria). Improvement of the intestinal environment is also expected to have effects such as regulating immune function, ameliorating allergic symptoms, improving bowel movements, improving brain function, skin beauty, oral care, and reducing body odor. The relationship between intestinal bacteria and these effects has been reported in publicly known literature.

[0042] 2-4. Bacterial growth promoting composition The acetic acid bacteria of the present invention may be used as a growth promoter when culturing other bacteria, particularly bacteria known as enterobacteria. That is, the enterobacteria growth-promoting composition of the present invention contains at least the acetic acid bacteria of the present invention and other bacteria (particularly bacteria known as enterobacteria). The enterobacteria are preferably lactic acid bacteria or bifidobacteria. In the enterobacteria growth-promoting composition of the present invention, the ratio of the number of cells of the acetic acid bacteria to the enterobacteria is not particularly limited, but it is preferable that the ratio of the number of cells of the acetic acid bacteria to the enterobacteria is 10 6 :1~1:10 3 Preferably, the range is 10 4 It is more preferable that the ratio is in the range of 1:1 to 1:1. It has been reported in the above-mentioned Patent Documents 2 and 4 that acetic acid bacteria have the effect of increasing the proliferation of intestinal bacteria. [Example]

[0043] Below, with reference to examples, it will be shown that the acetic acid bacteria of the present invention are useful as a component of an immunostimulatory composition.

[0044] (Acetate bacteria tested) The following three strains of acetic acid bacteria were prepared. Strain 1 (Example): Gluconacetobacter takamatsuzukensis (A192, the acetic acid bacterium of the present invention) Strain 2 (comparative example): Komagataeibacter saccharivorans (JCM 25288) Strain 3 (comparative example): Komagataeibacter xylinus (NBRC 13773)

[0045] (Preparation of acetic acid bacteria powder) A medium was prepared by adding water to 30 g of yeast extract, 60 g of glucose, and 3 g of magnesium sulfate heptahydrate to a total volume of 3 kg. Each acetic acid bacterium was cultured in this medium at 30°C for 72 hours. The medium was then sterilized by heating in an 85°C water bath for 40 minutes. After sterilization, the culture medium was centrifuged to collect the bacterial cells. The collected bacterial cells were freeze-dried to obtain acetic acid bacteria powder.

[0046] (Preparation of test sample solution) The various acetic acid bacteria powders obtained were added to RPMI1640 culture medium (containing 10% FBS (fetal bovine serum), 100 U / mL penicillin, and 100 μg / mL streptomycin) to a concentration of 50 μg / mL.

[0047] (Interleukin (IL)-12 expression and measurement) 7.5×10 5 Mouse macrophage-like cells (J774.1) prepared at 1000 cells / mL were seeded in a 24-well plate (450 μL) and pre-cultured at 37°C and 5% CO2 for 6 hours. Then, 50 μL of test sample solution or negative control medium was added, followed by 24 hours of culture at 37°C and 5% CO2. After incubation, the culture supernatant was collected, and the amount of IL-12p40 in the supernatant was measured by ELISA. The measurement results are shown in the graph in Figure 4.

[0048] IL-12p40 is one of the two subunits that make up IL-12 (IL-12Ap35 and IL-12Bp40). Increased expression of IL-12p40 indicates increased secretion of IL-12.

[0049] As shown in Figure 4, strain 1, an acetic acid bacterium of the present invention, promotes the expression of IL-12 from mouse macrophage-like cells compared to strains 2 and 3. Since the promotion of IL-12 expression is thought to activate natural killer cells, which are important for immune function and host defense, it is clear that the acetic acid bacterium of the present invention is useful as a component of an immunostimulatory composition. [Industrial Applicability]

[0050] The acetic acid bacteria of the present invention have superior functions that differ from conventional acetic acid bacteria, and by applying them to the uses of conventional acetic acid bacteria, it is expected that they will exhibit even greater performance than before, or that new uses for acetic acid bacteria will be developed. In particular, the acetic acid bacteria of the present invention are expected to enhance the immune system of animals (especially humans) when used as an active ingredient in an immunostimulatory composition.

Claims

1. The strain belongs to Gluconacetobacter takamatsuzukensis, deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-04018.

2. The strain of claim 1 isolated from cherry blossoms.

3. The strain of claim 1 or 2, which is administered or taken orally.

4. 3. A live or killed acetic acid bacterium of the strain according to claim 1 or 2, or a dried powder or crushed product thereof.

5. An immunostimulatory composition comprising the acetic acid bacterium of the strain described in claim 1 or 2.

6. The immunostimulatory composition of claim 5, which promotes the expression of interleukin-12.

7. The immunostimulatory composition according to claim 5, which is for food use.

8. A fermentation composition comprising the acetic acid bacterium of the strain according to claim 1 or 2 and a raw material to be fermented.

9. A fermentation composition obtained from the fermentation composition according to claim 8.

10. The fermented composition according to claim 9, which is for food use.

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