Novel Acetic Acid Bacteria and Their Applications

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] This 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 Patent Document 1, etc.), but there are also several reports on the effects of acetic acid bacteria on the physiological functions of living organisms (e.g., the human body) (Patent Documents 2-6). Patent Document 2 discloses an immunostimulant containing acetic acid bacteria cells, and it has been shown that this immunostimulant enhances antibody production. Patent Document 3 has been shown that acetic acid bacteria promote the growth of lactic acid bacteria (intestinal bacteria). Patent Document 4 has been disclosed a composition for improving allergy symptoms containing acetic acid bacteria, and it has been reported that it reduces nasal discomfort caused by dust and house dust. Patent Document 5 has been shown that acetic acid bacteria promote the growth of bifidobacteria (intestinal bacteria). Patent Document 6 has been described as an immunostimulant composition containing acetic acid bacteria that activates plasmacytoid dendritic cells. However, acetic acid bacteria are hardly used as raw materials in the health food market, and much remains unknown about the functionality of acetic acid bacteria.

[0003] Acetobacteria include representative genera such as Gluconacetobacter, Acetobacter, and Gluconobacter. Furthermore, Gluconacetobacter takamatsuzukensis (T61213-20-1a) has been reported as an acetic acid bacterium of the Gluconacetobacter genus (see Non-Patent Document 1).

[0004] Furthermore, interleukin-12 (IL-12) is a cytokine secreted by macrophages and dendritic cells, and its immunostimulatory effects have been reported. 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 Literature 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] Japanese Patent Publication No. 2016-106528 [Patent Document 4] Japanese Patent Publication No. 2021-059522 [Patent Document 5] Japanese Patent Publication No. 2022-188416 [Patent Document 6] Japanese 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) Exploration and Analysis of Probiotics that Enhance IL-12 Expression [Overview of the project] [Problems that the invention aims to solve]

[0007] Further research is underway on the fermentation capabilities and physiological effects 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 need to utilize novel acetic acid bacteria that have not been discovered to date as fermentation microorganisms or as immunostimulants. Accordingly, this invention provides a novel strain of acetic acid bacteria and offers its uses or applications. [Means for solving the problem]

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

[0009] The second aspect of this invention relates to a novel morphology of acetic acid bacteria. [4] Live or dead acetic acid bacteria of the strains described in [1] to [3] above, or dried powder or crushed material 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 bacteria strain described in [1] or [2] above. [6] The immunostimulatory composition according to [5], which promotes the expression of interleukin-12. [7] The immunostimulatory composition according to [5] or [6] above, which is for food use.

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

[10] The fermentation composition described in [9] for food use.

Effects of the Invention

[0012] The acetic acid bacterium of the present invention is a novel strain, and it is expected that the strain has excellent effects different from those of conventional acetic acid bacteria and can be applied to a wide range of uses.

Brief Description of the Drawings

[0013] [Figure 1] It is a molecular phylogenetic tree of the acetic acid bacterium strain of the present invention. [Figure 2] It is a comparison table regarding the physiological and biochemical properties between the acetic acid bacterium strain of the present invention and the reference strain "T61213-20-1a". [Figure 3] It is a table showing the results of colony observation, morphological observation, and various tests of Barrow & Feltham of the acetic acid bacterium strain of the present invention. [Figure 4] It is a graph showing the change in the expression level of IL-12p40 when the acetic acid bacterium of the present invention or other acetic acid bacteria is treated with mouse macrophage-like cells (J774.1).

Modes for Carrying Out the Invention

[0014] 1. Acetic acid bacteria of the present invention The acetic acid bacteria of the present invention is a strain deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE) on November 22, 2023, under accession number NITE P-04018, and is sometimes referred to as "strain A192". Furthermore, the acetic acid bacteria of the present invention can be isolated from cherry blossoms. Specifically, it can be isolated from the blossoms of the Usuzumi cherry tree (for example, a transplanted Usuzumi cherry tree on the grounds of Kano Tenmangu Shrine (Gifu City, Gifu Prefecture)).

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

[0016] More specifically, the acetic acid bacteria of the present invention were cultured in the culture medium shown in Table 1 and analyzed using the method shown in Table 2. [Table 1] [Table 2]

[0017] The catalase reaction, oxidase reaction, acid / gas production from glucose, and oxidation / fermentation of glucose (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, positive for the catalase reaction, negative for the oxidase reaction, and fermented glucose.

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

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

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

[0021] Alternatively, acetic acid bacteria may be cultured or otherwise treated to increase their population before use. The method of culturing acetic acid bacteria is not particularly limited, and for example, a method according to or similar to a known method can be employed. The culture medium used for cultivation can be a normal, general-purpose 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 may contain inorganic salts such as calcium salts, phosphates, magnesium salts, or sodium chloride. The culture conditions are not particularly limited, but aerobic culture is preferred, with the pH adjusted to the range of 6.0 to 7.2, the temperature adjusted to the range of 20 to 45°C, and the culture time being approximately 8 to 100 hours. The culture method is not particularly limited and can be cultured by static culture, shaking culture, aerated stirring culture, etc.

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

[0023] The acetic acid bacteria of the present invention may be in the form of wet cells or as a dried powder by freeze-drying. Alternatively, the acetic acid bacteria (live cells) may be heat-sterilized and then dried as a powder, or as a cell lysate. Cell lysates are obtained by physically crushing acetic acid bacteria obtained by culture after heating, and crushing can be carried out by methods such as ultrasonic methods, ball milling methods, or French press 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 along with any additive. The number of acetic acid bacteria contained in the acetic acid bacteria composition of the present invention may be set according to 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, the DAPI staining method can be used. The acetic acid bacteria composition of the present invention can be manufactured using any known method as appropriate, depending on its intended use.

[0025] The acetic acid bacteria composition of the present invention may be in solid or liquid form. The solid composition may be in any form such as powder, granules, capsules, or tablets, and the liquid composition may be in any form such as aqueous solution, suspension, paste, or jelly. Furthermore, while the acetic acid bacteria composition of the present invention is typically taken orally, it is not limited to this and may also be administered parenterally.

[0026] The acetic acid bacteria of the present invention can be applied to the same uses as conventional acetic acid bacteria. Preferably, it can be used in any of the following applications: food and beverages, pharmaceuticals, quasi-drugs, cosmetics, etc. When the acetic acid bacteria of the present invention is applied to food and beverages, for example, it may be in beverages such as water, soft drinks, fruit juices, milk drinks, alcoholic beverages, sports drinks, and nutritional drinks; or in foods such as bread, noodles, rice, tofu, dairy products, soy sauce, miso, confectionery, cream, sauces, mayonnaise, dressings, and supplements. Furthermore, supplements such as capsules (soft capsules and hard capsules) and tablets (including chewable tablets, etc.) may be preferred as food and beverages because they are easy to take.

[0027] The foods and beverages to which the acetic acid bacteria of the present invention are applied may be general foods, but may also be health functional foods such as Foods for Specified Health Uses (foods approved under Article 43, Paragraph 1 of the Health Promotion Act) or Foods with Function Claims (foods notified under the Food Labeling Act). Since these health functional foods can display their functions, the functions performed by the acetic acid bacteria of the present invention (for example, immune-boosting effects) may be displayed, and for example, it may be stated that they are useful in maintaining the immune function of healthy individuals and those with borderline conditions.

[0028] When the acetic acid bacteria of the present invention are applied to food and beverages, other bacteria or yeasts may be further added to the food or beverage. Examples of other bacteria include lactic acid bacteria, bifidobacteria, koji mold, butyric acid bacteria, and natto bacteria.

[0029] When the acetic acid bacteria of the present invention are applied to food and beverages, any other ingredients may be included. Examples of any other ingredients include: 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; crude drugs such as turmeric, liver extract, cinnamon, fennel, peony, and licorice; beauty ingredients such as collagen, hyaluronic acid, placenta, coenzyme Q10, chondroitin, and royal jelly; thickening polysaccharides such as soybean 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 used as oral (internal) compositions or topical compositions. Examples of topical compositions include compositions applied to the skin and hair, and compositions for oral care. Topical compositions used as quasi-drugs may include mouth fresheners, deodorants, hair growth agents, bath additives, medicated cosmetics, and medicated toothpastes.

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

[0032] The compositions containing acetic acid bacteria of the present invention may preferably be immunostimulatory compositions that enhance various immune functions (see 2-1 below), fermentation compositions (for example, 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 (for example, bacteria known as intestinal bacteria (specifically, lactic acid bacteria and bifidobacteria)) (see 2-4 below), but are not particularly limited. It is also possible to develop new applications that have not been seen with conventional acetic acid bacteria.

[0033] 2-1. Immunostimulant Compositions The acetic acid bacteria of the present invention can be used as a component of an immunostimulatory composition. In other words, the immunostimulatory composition of the present invention may contain the acetic acid bacteria of the present invention, preferably "as an active ingredient."

[0034] The immunostimulatory composition containing acetic acid bacteria of the present invention may, for example, improve allergic symptoms or suppress the decline in liver function. Allergic symptoms include nasal discomfort such as rhinitis, sneezing, runny nose, nasal congestion, and nasal itchiness; eye discomfort such as itchiness, foreign body sensation, eye discharge, and swelling; skin symptoms such as hives and swelling; and respiratory symptoms such as cough, asthma, and difficulty breathing. The causative substances include dust, house dust, pollen, and food.

[0035] The immunostimulatory composition of the present invention, which contains acetic acid bacteria, preferably has a daily intake of acetic acid bacteria of 1 × 10⁶. 8 More than one, comfortable 5x10 9 One or more, more preferably 2 × 10 10 It can contain acetic acid bacteria in quantities of 1 × 10 or more. There is no particular upper limit on the daily intake of acetic acid bacteria, but 1 × 10 13 It is less than or equal to 1 × 10 11The number may be less than or equal to 1. The number of acetic acid bacteria in the composition can be measured by any method, for example, using the DAPI staining method.

[0036] The acetic acid bacteria of the present invention are preferably highly effective in 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, further enhances the production of interferon-γ, and interferon-γ activates natural killer (NK) cells, thereby strengthening the attack against virus-infected cells and cancer cells, and increasing the body'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 present invention on promoting interleukin 12 expression by acetic acid bacteria can be confirmed, for example, by testing the procedure described in the examples below.

[0038] 2-2. Compositions for Fermentation The acetic acid bacteria of the present invention can be used as fermentation microorganisms in fermentation compositions. These acetic acid bacteria can ferment alcohol (ethanol) to produce acetic acid (brewed vinegar), and can also ferment various raw materials, including bio-derived materials containing carbon sources such as glucose, sorbitol, and various polysaccharides, nitrogen sources such as various peptides and proteins, and other trace components. Therefore, the fermentation composition of the present invention contains at least acetic acid bacteria and raw materials to be fermented.

[0039] The fermentation composition obtained from the fermentation composition of the present invention is, for example, a fermented food, typically brewed vinegar. Fermented food is a food that has been given a unique taste, aroma, texture, and other special flavors not present in the original food by microorganisms. Brewed vinegar is an acidic seasoning whose main component is fermented acetic acid produced by the action of acetic acid bacteria, using grains, sugars, and ethanol as raw materials. Brewed vinegar can be classified according to its raw materials 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), alcohol vinegar (made mainly from brewing ethanol), etc.

[0040] The acetic acid bacteria of the present invention can be used as a fermentation microorganism for the production of any type of brewed vinegar. The production of brewed vinegar is carried out by fermenting a raw material containing carbohydrates into alcohol, and then adding the acetic acid bacteria of the present invention to perform acetic acid fermentation. Acetic acid fermentation methods can be broadly classified into "surface fermentation method (static fermentation method)" and "whole-surface fermentation method (deep fermentation method)". In the surface fermentation method, acetic acid bacteria are added to alcohol, mixed, and left to stand. The liquid circulates naturally in the container, and acetic acid fermentation proceeds, and brewed vinegar is generally obtained in 1 to 3 months. In the whole-surface fermentation method, air is introduced by stirring the liquid to promote acetic acid fermentation. Therefore, acetic acid is usually produced in a shorter period 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 applied to compositions for improving the intestinal environment in living organisms. Improving the intestinal environment includes improving the gut flora and increasing the number of bacteria in the intestines (especially beneficial bacteria such as bifidobacteria and lactic acid bacteria). Furthermore, improving the intestinal environment is expected to have effects such as regulating immune function, improving allergy symptoms, improving bowel movements, improving brain function, improving skin beauty, oral care, and improving body odor. The relationship between intestinal bacteria and these effects has been reported in publicly available literature.

[0042] 2-4. Bacterial Growth Promoting Compositions The acetic acid bacteria of the present invention may also be used as a growth promoter when culturing other bacteria, particularly bacteria known as intestinal bacteria. In other words, the intestinal bacteria 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 intestinal bacteria). Intestinal bacteria are preferably lactic acid bacteria or bifidobacteria. In the intestinal bacteria growth promoting composition of the present invention, the cell number ratio of acetic acid bacteria to intestinal bacteria is not particularly limited, but when acetic acid bacteria are added, 10 6 :1~1:10 3 It is preferable that it be within the range of 10 4 A ratio in the range of 1 to 1:1 is more preferable. The effect of acetic acid bacteria on increasing intestinal bacteria has been reported in the aforementioned Patent Documents 2 and 4, among others. [Examples]

[0043] The following examples illustrate the usefulness of the acetic acid bacteria of the present invention as a component of an immunostimulatory composition.

[0044] (Acetic acid 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 culture medium was prepared by adding water to 30 g of yeast extract, 60 g of glucose, and 3 g of magnesium sulfate heptahydrate, making a total volume of 3 kg. Each type of acetic acid bacteria was cultured in this medium at 30°C for 72 hours. After that, the culture was sterilized by heating in an 85°C bath for 40 minutes. The culture solution after sterilization was centrifuged to collect the bacterial cells. The collected bacterial cells were freeze-dried to obtain acetic acid bacteria powder.

[0046] (Preparation of the test sample solution) Each of the obtained acetic acid bacteria powders was added to RPMI 1640 culture medium (containing 10% FBS (fetal bovine serum), 100 U / mL of penicillin, and 100 μg / mL of streptomycin), and the concentration of the acetic acid bacteria powder was set to 50 μg / mL.

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

[0048] IL-12p40 is one of the two subunits (IL-12Ap35 and IL-12Bp40) that make up IL-12. An increase in the expression level of IL-12p40 indicates an increase in the secretion amount of IL-12.

[0049] As shown in Fig. 4, it can be seen that strain 1, which is the acetic acid bacterium of the present invention, promotes the expression of IL-12 from mouse macrophage-like cells as compared with strain 2 and strain 3. Since the promotion of IL-12 expression is considered to activate natural killer cells, which are important for immune function and biological defense ability, it can be seen that the acetic acid bacterium of the present invention is useful as a component of an immunostimulating composition.

Industrial applicability

[0050] The acetic acid bacterium of the present invention has excellent functions different from those of conventional acetic acid bacteria; by applying it to the uses of acetic acid bacteria heretofore, it is expected to exhibit performance better than before, or the development of new uses of acetic acid bacteria is expected. In particular, by being used as an active ingredient of an immunostimulating composition, the acetic acid bacterium of the present invention is expected to enhance the immunity of animals (especially humans).

Claims

1. A strain belonging to Gluconacetobacter takamatsuzukensis, deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE) under accession number NITE P-04018.

2. The strain according to claim 1, isolated from cherry blossoms.

3. The strain according to claim 1 or 2, which is administered orally.

4. Live or dead acetic acid bacteria of the strain described in claim 1 or 2, or dried powder or crushed material thereof.

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

6. The immunostimulatory composition according to 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 an acetic acid bacterium strain according to claim 1 or 2, and a raw material to be fermented.

9. A fermentation composition obtained from the fermentation composition described in claim 8.

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

Images