Acidic exopolysaccharides with immune activation activity

Abstract

The invention relates to an exopolysaccharide with a repetitive structure formed by connecting repetitive units as shown in a formula (I) and an immune activation application of the exopolysaccharide. (In formula (I), n independently represents an integer of 0 or 1 in each repeating unit) # imgabs0 #

Description

Technical Field

[0001] The present invention relates to an acidic exopolysaccharide having immune activation activity. Background Art

[0002] It is known that various microorganisms represented by lactic acid bacteria produce exopolysaccharide (EPS) and secrete it extracellularly. Fermented milk represented by yogurt, which is produced by fermenting milk using Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, also contains EPS. In recent years, EPS has attracted attention due to various physiological activity functions such as an immune activation function.

[0003] The EPS produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 strain has a repeating structure, which is formed by connecting repeating units composed of a main chain consisting of multiple two glucose residues and two galactose residues and a side chain consisting of one galactose residue. It is known that there are acidic polysaccharides with a phosphate group attached to this structure and neutral polysaccharides without a phosphate group attached (Non-Patent Documents 1 and 2). Furthermore, it is also known that this acidic EPS produced by the OLL1073R-1 strain has an immune activation effect (Non-Patent Documents 1 and 2).

[0004] On the other hand, there is also a desire to obtain new EPS having an immune activation effect.

[0005] Prior Art Documents

[0006] Non-Patent Documents

[0007] Non-Patent Document 1: Marie-Rose Van Calsteren, et al., Carbohydrate Research, 413 (2015) 115-122

[0008] Non-Patent Document 2: Seiya Makino, Milk Science, Vol. 58, No. 2 (2009) Summary of the Invention

[0009] Problems to be Solved by the Invention

[0010] The subject of the present invention is to further provide a polysaccharide having immune activation activity.

[0011] Solutions to the Problems

[0012] The inventors of the present invention conducted intensive studies repeatedly to solve the above problems, and as a result, successfully obtained an extracellular polysaccharide (EPS) having a new repeating structure formed by linking repeating units in which the sugar residues are composed only of galactose residues and having immunostimulatory activity from a culture of Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain, thereby completing the present invention.

[0013] That is, the present invention includes the following aspects.

[0014] [1] An acidic extracellular polysaccharide having a repeating structure formed by linking repeating units represented by the following formula (I).

[0015] [Chemical Formula 1]

[0016]

[0017] (In formula (I), n independently represents an integer of 0 or 1 in each repeating unit)

[0018] [2] The acidic extracellular polysaccharide according to the above [1], which is obtained by isolation.

[0019] [3] The acidic extracellular polysaccharide according to the above [1] or [2], which is isolated from a lactic acid bacterium culture containing Lactobacillus delbrueckii subsp. bulgaricus.

[0020] [4] The acidic extracellular polysaccharide according to any one of the above [1] to [3], which is derived from Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain (deposit number: FERM BP-10741).

[0021] [5] An immunostimulatory composition comprising the acidic extracellular polysaccharide according to any one of the above [1] to [4] as an active ingredient.

[0022] [6] A food or drug for immunostimulation, comprising the acidic extracellular polysaccharide according to any one of the above [1] to [4] as an active ingredient.

[0023] [7] An immunostimulatory method, which includes the step of administering to a subject or giving to a subject the acidic extracellular polysaccharide according to any one of the above [1] to [4], the immunostimulatory composition according to the above [5], or the food or drug for immunostimulation according to the above [6].

[0024] [8] Use of the acidic extracellular polysaccharide according to any one of the above [1] to [4] in the manufacture of an immunostimulatory composition, food or drug.

[0025] This specification includes the disclosure of Japanese Patent Application No. 2022-155708, which is the basis of the priority of this application.

[0026] Effects of the Invention

[0027] According to the present invention, an EPS having immune activation activity can be newly provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a diagram showing the spectrum obtained by the 1 H- 13 C]-HSQC method for the acidic EPS obtained in Example 1.

[0029] Figure 2 It is a diagram showing the spectrum obtained by the 1 H, 1 H]-NOESY method for the acidic EPS obtained in Example 1.

[0030] Figure 3 It is a diagram showing the repeating structure of the acidic EPS obtained in Example 1.

[0031] Figure 4 It is a chart showing the results of inducing the production of IFN-γ by splenocytes by stimulation with the acidic EPS obtained in Example 1. "*" in the figure indicates a statistically significant difference. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be described in detail below.

[0033] The present invention relates to an acidic extracellular polysaccharide (EPS) having an immune activation effect and its use for immune activation.

[0034] The acidic EPS of the present invention is a polysaccharide having a repeating structure (sugar chain) formed by connecting repeating units each composed of a main chain consisting of 3 galactose residues and a side chain consisting of 2 galactose residues, and at least 1 glycerol phosphate group is connected to the repeating structure. In the present invention, "formed by connecting repeating units" means that the repeating units are arranged in series repeatedly.

[0035] More specifically, the acidic EPS of the present invention is an acidic extracellular polysaccharide having a repeating structure formed by connecting repeating units (repeating unit; also refer to Figure 3 ).

[0036] [Chemical Formula 2]

[0037]

[0038] In formula (I), n independently represents an integer of 0 or 1 in each repeating unit. That is, in each repeating unit represented by formula (I) that constitutes the acidic EPS of the present invention, it has one or no glycerol 3-phosphate group, and as the acidic EPS as a whole, at least one glycerol 3-phosphate group is connected.

[0039] The acidic EPS of the present invention can be an acidic exopolysaccharide having a repeating structure shown in the following formula (II) formed by connecting the repeating units shown in formula (I).

[0040] [Chemical formula 3]

[0041]

[0042] In formula (II), n independently represents an integer of 0 or 1 in each repeating unit. In formula (II), m represents an integer of 1 to 300, preferably an integer of 1 to 200.

[0043] In formulas (I) and (II), α-D-Galp represents an α-D-galactose residue in pyranose form, β-D-Galp represents a β-D-galactose residue in pyranose form, β-D-Galf represents a β-D-galactose residue in furanose form, and Gro3P represents a glycerol 3-phosphate group. (1-2), (1-3), (1-5), and (1-6) in formulas (I) and (II) respectively represent 1-2 bonds (i.e., 1-position carbon - 2-position carbon bond), 1-3 bonds, 1-5 bonds, and 1-6 bonds between residues.

[0044] Regarding the acidic EPS of the present invention, it is preferred that in the repeating structure (such as formula (II)) formed by connecting the repeating units shown in formula (I), each repeating unit is connected with an average of about one (for example, one (n = 1) or zero relative to each repeating unit, and about one in each repeating unit based on the weighted average of the acidic EPS as a whole), but it is not limited thereto.

[0045] The acidic EPS of the present invention preferably has the above repeating structure as the basic backbone. In the present invention, "having... repeating structure as the basic backbone" means that the entire length or almost the entire length (more than 80%, preferably more than 90%, more preferably 95% or 98% or more of the total number of sugar residues constituting the main chain of the acidic EPS) is composed of the repeating structure formed by connecting the repeating units shown in formula (I). In one embodiment, the acidic EPS of the present invention can be composed only of the above repeating structure.

[0046] The acidic EPS of the present invention can be an acidic EPS derived from microorganisms (typically, bacteria) represented by lactic acid bacteria. The acidic EPS of the present invention can be an acidic EPS produced by microorganisms (typically, bacteria) represented by lactic acid bacteria and secreted extracellularly.

[0047] In one embodiment, the acidic EPS of the present invention can be the acidic EPS obtained from a microbial culture, can be the acidic EPS obtained from a lactic acid bacteria culture, for example, can be the EPS recovered or purified from a lactic acid bacteria culture. In one embodiment, the acidic EPS of the present invention can be obtained by isolation, for example, can be obtained by isolation from a lactic acid bacteria culture. Regarding the acidic EPS of the present invention, "obtained by isolation" means that the purity of the acidic EPS of the present invention is 80% by mass or more, preferably 90% by mass or more, and more preferably 98% by mass or more.

[0048] Examples of lactic acid bacteria include, but are not limited to, bacteria belonging to the genus Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Enterococcus, and Pediococcus. Examples of lactic acid bacteria include, but are not limited to, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus kefiranofaciens, Lactobacillus sake, Streptococcus thermophilus, Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Leuconostoc mesenteroides, Enterococcus faecalis, and Pediococcus pentosaceus.

[0049] Examples of more preferred lactic acid bacteria used for producing the acidic EPS of the present invention include Lactobacillus delbrueckii subsp. bulgaricus, and examples of particularly preferred lactic acid bacteria include Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1.

[0050] Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1 was internationally deposited under the Budapest Treaty with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NITE-IPOD [formerly: Patent Microorganisms Depositary, National Institute of Advanced Industrial Science and Technology]) (Room 120, 2-5-8 Kamigoura, Kisarazu-shi, Chiba 292-0818, Japan) on February 22, 1999 (original deposit date) under the deposit number FERM BP-10741. It should be noted that this deposited strain was converted from a domestic deposit (original deposit) to an international deposit under the Budapest Treaty on November 29, 2006. The current depositor of Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1 is Meiji Co., Ltd. Meiji Co., Ltd. has granted Meiji Holdings Co., Ltd. the right to mention strain OLL1073R-1 in this application.

[0051] In one embodiment, the acidic EPS of the present invention can be obtained from a lactic acid bacterium culture containing one or more of the above lactic acid bacteria. The lactic acid bacterium culture can be fermented milk such as yogurt produced using lactic acid bacteria, or can be obtained by culturing lactic acid bacteria in a medium (such as a milk-containing medium), and is not limited to these. The "milk" that can be used when preparing the lactic acid bacterium culture in the present invention is preferably milk of mammals (such as cows, goats, sheep, etc.).

[0052] In one embodiment, the lactic acid bacterium culture can contain bacteria of the genus Lactobacillus as lactic acid bacteria, for example, it can contain Lactobacillus delbrueckii subsp. bulgaricus.

[0053] In one embodiment, the lactic acid bacterium culture can contain, as lactic acid bacteria, a combination of bacteria of the genus Lactobacillus and bacteria of the genus Streptococcus. In a preferred embodiment, the lactic acid bacterium culture can contain Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0054] In one embodiment, the acidic EPS of the present invention can be derived from lactic acid bacteria, for example, from the above lactic acid bacteria such as bacteria of the genus Lactobacillus and / or bacteria of the genus Streptococcus, preferably from Lactobacillus delbrueckii subsp. bulgaricus, and more preferably from Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1.

[0055] The culture conditions used to produce the acidic EPS of the present invention using lactic acid bacteria can be, for example, the same culture conditions as those described in the examples below, but are not limited thereto. Such culture conditions can be, for example: culture conditions including culturing in a milk-containing medium such as a skim milk medium at 35 to 39 °C (typically 37 °C) for 12 to 24 hours (such as 18 hours).

[0056] In order to obtain the acidic EPS of the present invention from a lactic acid bacterium culture, well-known polysaccharide isolation and purification techniques can be used. For example, EPS in the lactic acid bacterium culture can be separated from proteins and lactic acid bacteria and recovered by treatment with trichloroacetic acid or the like, and the acidic EPS fraction can be separated using fractionation techniques such as anion exchange chromatography after dialysis. Furthermore, residual proteins in the separated acidic EPS fraction are removed with a proteolytic enzyme or the like for further purification, whereby the acidic EPS of the present invention can be obtained. However, the method for isolating and purifying the acidic EPS of the present invention is not limited to this method.

[0057] Alternatively, the acidic EPS of the present invention can also be chemically synthesized using a sugar chain chemical synthesis method. The acidic EPS of the present invention obtained by chemical synthesis can also be included within the scope of the "isolated" acidic EPS of the present invention.

[0058] The acidic EPS of the present invention has immunostimulatory activity (immunostimulatory effect). The acidic EPS of the present invention having immunostimulatory activity preferably can promote the production of interferon (IFN)-γ. It is known that IFN-γ is mainly produced by immune cells such as T cells and natural killer (NK) cells, contributes to antiviral activity, immunomodulatory activity, and antitumor activity, and also acts as a potent activating factor for macrophages.

[0059] The immunostimulatory activity of the acidic EPS of the present invention can be evaluated using the amount of IFN-γ produced as an index. The immunostimulatory activity of the acidic EPS of the present invention can be evaluated, for example, using the same test method as in Example 3 described later. Specifically, first, mouse spleen cells are suspended in a mammalian cell culture medium (e.g., RPMI 1640 medium) containing fetal bovine serum (e.g., 10% (vol / vol) fetal bovine serum), and then seeded in a plate or the like at an appropriate cell density (e.g., 5×10 5 cells / well). To the seeded cells, acidic EPS is added at a specified concentration (e.g., a final concentration of 25 to 200 μg / mL), and incubated at 37°C and 5% CO 2After culturing for a certain period of time (e.g., 72 hours), the supernatant is recovered from the culture medium, and the amount of IFN-γ contained in the recovered supernatant is quantified by ELISA (enzyme-linked immunosorbent assay) using an anti-IFN-γ antibody. As a control, mouse splenocytes are cultured in the same manner without adding acidic EPS, the amount of IFN-γ in the supernatant is quantified, and compared with the amount of IFN-γ quantified as described above in the culture system supplemented with acidic EPS. When the amount of IFN-γ in the supernatant is significantly increased statistically or the amount of IFN-γ in the supernatant increases concentration-dependently with the increase in the added amount of acidic EPS compared with the control without adding acidic EPS, it can be determined that the acidic EPS of the present invention has immunostimulatory activity. In addition, the intensity (level) of the immunostimulatory activity of the acidic EPS of the present invention can be evaluated based on the increase in the amount of IFN-γ in the supernatant when acidic EPS is added compared with the control without adding acidic EPS.

[0060] The present invention further provides a composition containing the acidic EPS of the present invention, and a food or drug containing the acidic EPS of the present invention. The composition, food or drug containing the acidic EPS of the present invention preferably has immunostimulatory activity and is preferably used for immunostimulation. In a preferred embodiment, the acidic EPS of the present invention can be included as an active ingredient (an active ingredient for bringing about immunostimulatory activity) in a composition for immunostimulation, and a food or drug for immunostimulation. In the composition for immunostimulation and the food or drug for immunostimulation, the acidic EPS of the present invention can be included as the sole active ingredient for immunostimulation and / or the sole EPS. In the present invention, "including" the acidic EPS of the present invention "as an active ingredient" means that when the composition, food or drug containing the acidic EPS of the present invention is used for a subject, the acidic EPS of the present invention is included in the composition, food or drug in an amount (effective amount) and form sufficient to exert immunostimulatory activity. The composition, food or drug containing the acidic EPS of the present invention can contain the acidic EPS of the present invention in an amount of 10 μg to 50 mg / kg body weight per application (ingestion or administration), for example, but is not limited to this range.

[0061] In one embodiment, a composition, food, or drug containing the acidic EPS of the present invention may be free of neutral EPS (e.g., neutral EPS derived from lactic acid bacteria such as Lactobacillus delbrueckii subsp. bulgaricus represented by strain OLL1073R-1). In one embodiment, a composition, food, or drug containing the acidic EPS of the present invention may be free of the following acidic EPS, which has a repeating structure and a phosphate group attached thereto, and the repeating structure is formed by connecting repeating units each composed of a main chain consisting of 2 glucose residues and 2 galactose residues and a side chain consisting of 1 galactose residue (e.g., acidic EPS derived from lactic acid bacteria such as Lactobacillus delbrueckii subsp. bulgaricus represented by strain OLL1073R-1).

[0062] In the composition, food, or drug of the present invention, in addition to the acidic EPS of the present invention, it may further contain additives acceptable in food processing (food additives) or pharmaceutically acceptable additives (drug additives). Examples of such additives include, but are not limited to, carriers, excipients, binders, lubricants, disintegrants, wetting agents, stabilizers, buffers, flavoring agents, preservatives, coloring agents, etc. In one embodiment, a composition containing the acidic EPS of the present invention (e.g., a composition for immune activation) may be a food composition or a pharmaceutical composition, but is not limited thereto. The composition, food, or drug of the present invention may further contain other pharmacological components and / or functional components.

[0063] In the present invention, the "food" may be a beverage or other foods, preferably processed foods. As beverages, but not limited to the following, there may be mentioned lactic acid bacteria beverages, milk beverages (such as coffee milk, fruit milk, etc.), tea-based beverages (such as green tea, black tea, and oolong tea), fruit / vegetable-based beverages (beverages containing fruit juices such as oranges, apples, grapes, etc., and vegetable juices such as tomatoes, carrots, etc.), alcoholic beverages (such as beer, sparkling wine, wine, etc.), carbonated beverages, soft drinks, water-based beverages, etc. As foods other than beverages, but not limited to the following, there may be mentioned processed foods such as fermented milk such as yogurt, confectionery, jam, semi-finished dishes, convenience foods, seasonings, etc. Regarding the manufacturing methods of various foods, etc., existing reference books related to food manufacturing can be referred to.

[0064] The food of the present invention can be a functional food or a food other than a functional food. In the present invention, "functional food" refers to a food having certain functions for organisms, including, for example, health functional foods such as foods for specified health use in Japan (including conditional foods for specified health use), nutritional functional foods, functional labeled foods, foods for special purposes, nutritional supplementary foods, health supplementary foods, supplements (such as various dosage forms of foods including tablets, coated tablets, sugar-coated tablets, capsules and liquids), and beauty foods (such as weight loss foods), etc., that is, all so-called health foods. The functional food of the present invention further includes health foods applying health claims based on food standards of the Codex Alimentarius (Joint FAO / WHO Food Standards Programme).

[0065] The functional food of the present invention can be foods for special purposes such as foods for patients, milk powder for pregnant women and lactating women, formula milk for infants and young children, foods for the elderly, and foods for nursing care.

[0066] The functional food of the present invention can be solid preparations such as tablets, granules, powders, pills, and capsules; liquid preparations such as liquids, suspensions, and syrups; or gels, pastes, etc., and can also be in the form of ordinary foods (such as beverages, yogurts, snacks, etc.).

[0067] In addition to the acidic EPS of the present invention, the food of the present invention may contain any food ingredients, without particular limitation. The food of the present invention may contain water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavoring agents, etc. As proteins, examples include animal and plant proteins such as whole milk powder, skim milk powder, partially skimmed milk powder, casein, β-lactoglobulin, α-lactalbumin, lactoferrin, soy protein, egg protein, meat protein, hydrolysates thereof, butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, lactose and other various milk-derived components. As carbohydrates, examples include general sugars, modified starches (such as dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), dietary fiber, etc. As lipids, examples include animal fats such as lard, fish oil, fractionated oils thereof, hardened oils, interesterified oils; vegetable oils such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hardened oils, interesterified oils, etc. As vitamins, examples include vitamin A, carotenoids, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid, etc. As minerals, examples include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, whey minerals, etc. As organic acids, examples include malic acid, citric acid, lactic acid, tartaric acid, etc. These components can be used alone, or two or more of them can be used in combination, and synthetic products and / or food materials rich in these can also be used as raw materials. The food of the present invention can be manufactured by conventional methods. For example, in any process of food manufacturing, the acidic EPS of the present invention or a raw material containing the acidic EPS of the present invention can be added to other food ingredients for manufacturing.

[0068] The medicine of the present invention can be in any dosage form such as solid preparations including tablets, granules, powders, pills, capsules, etc., liquid preparations such as gel agents or liquid agents, suspensions, syrups, etc. The medicine of the present invention is preferably a medicine for oral administration, but is not limited thereto.

[0069] The composition, food or medicine of the present invention can bring about an immune activation effect based on the immune activation activity (immune activation effect) of the acidic EPS of the present invention. For example, the composition, food or medicine of the present invention can promote the production of interferon (IFN)-γ. The composition, food or medicine of the present invention can promote (stimulate) the immune response in the subject after ingestion or administration.

[0070] The present invention also provides a method (immune activation method) for bringing about an immune activation effect to a subject, which includes the step of making the subject ingest or administering to the subject the acidic EPS of the present invention or the composition, food or medicine of the present invention containing the acidic EPS.

[0071] The subjects for ingesting or administering the acidic EPS of the present invention or the composition, food or medicine of the present invention containing the acidic EPS are preferably mammals including primates such as humans, domestic animals, pet animals, experimental (test) animals, etc., and more preferably humans. Such subjects are not limited to the following, and may be subjects in need of or desiring immune activation, and examples thereof include pregnant and lying-in women, infants and young children, the elderly, the sick, convalescents, the weak, subjects with decreased immunity, athletes, subjects in need of preventing diseases such as infectious diseases (e.g., medical practitioners, nursing practitioners, examinees, etc.).

[0072] The ingestion or administration of the acidic EPS of the present invention or the composition, food or medicine of the present invention containing the acidic EPS to a subject is preferably oral ingestion or administration, and may be ingestion or administration by means of a tube such as a nasal tube, an oral-esophageal tube, a gastric fistula, an intestinal fistula, etc. However, the route of administration to a subject in the present invention is of course not limited to these.

[0073] The ingestion or administration amount of the acidic EPS of the present invention or the composition, food or medicine of the present invention containing the acidic EPS only needs to be an amount effective for immune activation, and can be changed within a wide range in consideration of the species, age, weight, administration route, administration frequency, etc. of the subject and according to the judgment of those skilled in the art. In one embodiment, the ingestion or administration amount may be an amount of the acidic EPS of the present invention reaching 10 μg to 50 mg / kg body weight per application (ingestion or administration) based on the body weight of the subject, but is not limited to this range.

[0074] The ingestion or administration of the acidic EPS of the present invention or the composition, food or medicine of the present invention containing the acidic EPS may be carried out only once (one time), or may be carried out multiple times. The acidic EPS of the present invention or the composition, food or medicine of the present invention containing the acidic EPS may be continuously ingested by a subject or continuously administered to a subject, and may be ingested or administered every day, for example, at a frequency of 1 day, 2 days, 3 days, 4 days, 5 days or 6 days per week. When continuously ingesting or administering, it is preferred to continuously ingest by a subject or continuously administer to a subject for at least 3 days, preferably 1 week or more, more preferably 2 weeks or more, further preferably 4 weeks or more, for example, 3 months or more.

[0075] The present invention further relates to the acidic EPS of the present invention for immune activation (immune response promotion). The present invention further relates to the use of the acidic EPS of the present invention for immune activation (immune response promotion). Furthermore, the present invention relates to the use of the acidic EPS of the present invention in the manufacture of a composition, food or medicine for immune activation (immune response promotion).

[0076] Examples

[0077] The present invention will be specifically described below using examples. However, the technical scope of the present invention is not limited by these examples.

[0078] [Example 1] Production and purification of acidic EPS

[0079] Lactobacillus delbrueckii ssp. bulgaricus strain OLL1073R-1 (deposit number FERM BP-10741) was cultured in a 10% (wt / vol) skim milk medium at 37°C for 18 hours. Trichloroacetic acid was added to the resulting culture at a final concentration of 10% by mass to precipitate the denatured protein and lactic acid bacteria produced, followed by centrifugation (12,000×g, 20 minutes). The obtained supernatant was recovered, 3 times the amount of cold ethanol was added, and it was left standing overnight at 4°C. The resulting precipitate was recovered by centrifugation (12,000×g, 20 minutes), and dialysis was performed using a dialysis membrane (MWCO [molecular weight cut-off] 6,000 to 8,000) with distilled water. Thus, crude EPS (exopolysaccharide) was obtained.

[0080] Acidic EPS was fractionated from the obtained crude EPS. During fractionation, DEAE-Sepharose (R) (diethylaminoethyl-Sepharose (R) ) Fast Flow was used, and a concentration gradient extraction method based on 0 to 0.5 M NaCl was used in 0.02 M Tris-HCl buffer (pH 8.6). The obtained acidic EPS was dissolved in 0.05 M Tris-HCl buffer (pH 8.0) containing 1 mM MgCl 2 , and treated with 2 mg / mL DNase and 2 mg / mL RNase at 37°C for 6 hours. Thereafter, a protease K solution was added at a final concentration of 0.1 mg / mL, and treated at 37°C for 16 hours to decompose the protein, and then heated at 90°C for 10 minutes to inactivate the enzyme. For the obtained reaction solution, 3 times the amount of cold ethanol was added in the same manner as above, left standing overnight at 4°C, the resulting precipitate was recovered by centrifugation (12,000×g, 20 minutes), and dialysis was performed using the above dialysis membrane with distilled water, and the above operation was repeated to purify acidic EPS and prepare an acidic EPS solution.

[0081] [Example 2] Structural analysis of acidic EPS

[0082] For the purified acidic EPS, structural analysis was performed by two-dimensional NMR. The two-dimensional NMR used in the analysis is nuclear magnetic resonance (NMR) spectroscopy capable of elucidating the correlation between signals and performing structural analysis of complex compounds. Specifically, it is1 H, 1 H]-COSY method ( 1 H, 1 H-correlation spectroscopy, 1 H, 1 H-correlation magneto-resonance spectrum), TOCSY (total correlation spectroscopy), [ 1 H- 13 C]-High-resolution two-dimensional NMR method ([ 1 H- 13 C]-heteronuclear multiple quantum correlation:[ 1 H- 13 C]-HSQC) and [ 1 H, 1 H]-NOESY method ([ 1 H, 1 H]-nuclear overhauser enhancement spectroscopy).

[0083] First, use [ 1 H, 1 The structure of acidic EPS can be inferred by the [H]-COSY method. This is because the signal of each proton is affected by the two substituents bound to the same carbon atom (geminal position) and the two substituents bound to the adjacent carbon atoms (ortho position), and almost all of them are attributed to the sugars based on the different head types. 1 H chemical shift. Furthermore, since the signals of oligosaccharides and polysaccharides overlap in a complex manner, the TOCSY method, which can extract the correlation peaks of all protons belonging to a specific spin system from the signal, is used to enhance the [ 1 H, 1 On the other hand, the [H]-COSY method, which can detect the correlation between two different types of nuclei such as hydrogen atoms and carbon atoms, is used. 1 H- 13 C]-HSQC method is used to analyze which hydrogen atom is directly bonded to which carbon atom. Furthermore, the [ 1 H, 1 H]-NOESY method to determine which signal originates from protons that are close to each other.

[0084] Will use [ 1 H- 13 The spectrum obtained by the [C]-HSQC method is shown in Figure 1 , will use [1 H, 1 The spectrum obtained by the [H, Figure 2 .

[0085] The results of two-dimensional NMR structural analysis showed that the obtained acidic EPS mainly had a repeating structure formed by repeating units composed of galactose residues (3 furanose-type galactose residues and 2 pyranose-type galactose residues) connected together. Furthermore, it was also shown that in this repeating structure, most of the repeating units were connected with 1 glycerol phosphate group in each repeating unit, forming an anchor for the bacterial cell wall.

[0086] Furthermore, for glycosyl linkage analysis, the obtained acidic EPS was subjected to full methylation, depolymerization, and acetylation treatments, and the resulting methylated alditol acetates were analyzed using gas chromatography-mass spectrometry (GC-MS). Thus, the repeating structure of the acidic EPS was determined as Figure 3 shown. Figure 3 Among them, A, B, C, D, E / F are symbols marked for conveniently indicating Figure 1 and 2 each sugar residue shown in the spectrum of Figure 3 . In the structure shown, Galp represents a pyranose-type galactose residue, Galf represents a furanose-type galactose residue, and Gro3p represents a glycerol 3-phosphate group.

[0087] [Example 3] Evaluation of the Immunoactivating Activity of Acidic EPS

[0088] After suspending mouse spleen cells in RPMI 1640 medium containing 10% (vol / vol) fetal bovine serum, they were seeded in a 96-well plate at a density of 5 × 10 5 cells / well. To the seeded cells, a solution of the acidic EPS purified in Example 1 and with its structure determined in Example 2 was added at final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, or 200 μg / mL of EPS, and the cells were cultured at 37 °C and 5% CO 2 for 72 hours (acidic EPS-stimulated group). Additionally, as a control, mouse spleen cells were cultured without adding acidic EPS in the same manner (unstimulated group). After culturing, the supernatant was recovered, and the amount of interferon γ (IFN-γ) produced in the medium was quantified by ELISA.

[0089] As a result, the production of IFN-γ by spleen cells was induced in an amount dependent on the acidic EPS addition concentration ( Figure 4 ). This result indicated that the obtained acidic EPS was capable of activating immune cells.

[0090] All publications, patents, and patent applications cited in this specification are hereby incorporated by reference directly into this specification.

Claims

1. An acidic extracellular polysaccharide having a repeating structure formed by linking repeating units represented by the following formula (I), In formula (I), n independently represents an integer of 0 or 1 in each repeating unit.

2. The acidic extracellular polysaccharide according to claim 1, which is obtained by isolation.

3. The acidic extracellular polysaccharide according to claim 1 or 2, which is isolated from a lactic acid bacterium culture containing Lactobacillus delbrueckii subsp. bulgaricus.

4. The acidic extracellular polysaccharide according to claim 1, which is derived from Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1 (deposit number FERM BP-10741).

5. An immunostimulatory composition comprising the acidic extracellular polysaccharide according to claim 1 as an active ingredient.

6. A food or drug for immunostimulation, comprising the acidic extracellular polysaccharide according to claim 1 as an active ingredient.

7. An immunostimulation method comprising the step of administering to a subject or causing the subject to ingest the acidic extracellular polysaccharide according to claim 1, the immunostimulatory composition according to claim 5, or the food or drug for immunostimulation according to claim 6.

8. Use of the acidic extracellular polysaccharide according to claim 1 in the manufacture of an immunostimulatory composition, food or drug.

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