Acidic bacterial exopolysaccharide having immunopotentiating activity

Abstract

The present invention relates to a bacterial exopolysaccharide having a repeating structure comprising a series of repeating units represented by formula (I), and to the use of the bacterial exopolysaccharide for immunopotentiation. (In formula (I), n represents an integer of 0 or 1, independently for each repeating unit.)

Description

Acidic bacterial exopolysaccharides with immunostimulatory activity

[0001] The present invention relates to an acidic bacterial exopolysaccharide having immunostimulatory activity.

[0002] It is known that various microorganisms, including lactic acid bacteria, produce exopolysaccharides (EPS) and secrete them extracellularly. EPS is also contained in fermented milk, including yogurt, which is produced by fermenting milk using the lactic acid bacteria Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. In recent years, EPS has attracted attention due to its various physiologically active functions, such as immunostimulatory activity.

[0003] The EPS produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 strain is known to have a repeating structure with many repeating units each consisting of a main chain consisting of two glucose residues and two galactose residues and a side chain consisting of one galactose residue, and to contain both acidic polysaccharides with phosphate groups attached to the repeating structure and neutral polysaccharides without phosphate groups (Non-Patent Documents 1 and 2). Furthermore, such acidic EPS produced by the OLL1073R-1 strain is also known to have immunostimulatory activity (Non-Patent Documents 1 and 2).

[0004] On the other hand, there is a further demand for the acquisition of new EPSs that have immunostimulatory effects.

[0005] Marie-Rose Van Calsteren, et al. , Carbohydrate Research, 413 (2015) 115-122 Seiya Makino, Milk Science, Vol. 58, No. 2 (2009)

[0006] An objective of the present invention is to further provide polysaccharides having immunostimulatory activity.

[0007] As a result of extensive research to achieve the above object, the present inventors have succeeded in obtaining, from a culture of the OLL1073R-1 strain, an exopolysaccharide (EPS) having immunostimulatory activity, which has a novel repeating structure in which repeating units composed solely of galactose residues are linked (repeated), and have thereby completed the present invention.

[0008] That is, the present invention encompasses the following aspects: [1] An acidic bacterial exopolysaccharide having a repeating structure in which repeating units represented by the following formula (I) are linked together: (In formula (I), n independently represents an integer of 0 or 1 for each repeating unit.) [2] An isolated acidic bacterial exopolysaccharide according to [1] above. [3] The acidic bacterial exopolysaccharide according to [1] or [2] above, isolated from a lactic acid bacterial culture containing Lactobacillus delbrueckii subsp. bulgaricus. [4] The acidic bacterial exopolysaccharide according to any of [1] to [3] above, which is derived from Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain (accession number FERM BP-10741). [5] A composition for immunostimulation comprising, as an active ingredient, the acidic bacterial exopolysaccharide according to any of [1] to [4] above. [6] A food or pharmaceutical for immunostimulation comprising, as an active ingredient, the acidic bacterial exopolysaccharide according to any of [1] to [4] above. [7] A method for immunostimulation, comprising having a subject ingest or administering to the subject the acidic bacterial exopolysaccharide according to any one of [1] to [4] above, the composition for immunostimulation according to [5] above, or the food or pharmaceutical for immunostimulation according to [6] above. [8] Use of the acidic bacterial exopolysaccharide according to any one of [1] to [4] above in the production of a composition, food or pharmaceutical for immunostimulation.

[0009] This specification includes the disclosure of Japanese Patent Application No. 2022-155708, from which the present application claims priority.

[0010] According to the present invention, a novel EPS having immunopotentiating activity can be provided.

[0011] FIG. 1 shows the acidic EPS obtained in Example 1. １ H- １３2 shows the spectrum obtained by the [C]-HSQC method for the acidic EPS obtained in Example 1. １ H. １ 1 shows a spectrum obtained by the [H]-NOESY method. FIG. 3 shows the repeating structure of the acidic EPS obtained in Example 1. FIG. 4 is a graph showing the results of IFN-γ production induction from spleen cells by stimulation with the acidic EPS obtained in Example 1. In the figure, "*" indicates that a statistically significant difference was observed.

[0012] The present invention will be described in detail below.

[0013] The present invention relates to an acidic bacterial exopolysaccharide (EPS) having immunostimulatory activity and its immunostimulatory uses.

[0014] The acidic EPS of the present invention is a polysaccharide having a repeating structure (sugar chain) in which repeating units each consisting of a main chain of three galactose residues and a side chain of two galactose residues are linked together, and at least one glycerol phosphate group is attached to the repeating unit. In the present invention, "linked repeating units" means that the repeating units are arranged in tandem.

[0015] More specifically, the acidic EPS of the present invention is an acidic exopolysaccharide having a repeating structure in which repeating units (repeating units; see also FIG. 3) represented by the following formula (I) are linked together:

[0016]

[0017] In formula (I), n independently represents an integer of 0 or 1 for each repeating unit. That is, each repeating unit represented by formula (I) constituting the acidic EPS of the present invention may or may not have one glycerol 3-phosphate group, but the acidic EPS as a whole has at least one glycerol 3-phosphate group added thereto.

[0018] The acidic EPS of the present invention may be an acidic exopolysaccharide having a repeating structure represented by the following formula (II), in which repeating units represented by formula (I) are linked together.

[0019]

[0020] In formula (II), n independently represents an integer of 0 or 1 for each repeating unit, and m represents an integer of 1 to 300, preferably an integer of 1 to 200.

[0021] In formulas (I) and (II), α-D-Galp represents a pyranose-type α-D-galactose residue, β-D-Galp represents a pyranose-type β-D-galactose residue, β-D-Galf represents a furanose-type β-D-galactose residue, and Gro3P represents a glycerol 3-phosphate group. In formulas (I) and (II), (1-2), (1-3), (1-5), and (1-6) represent the 1-2 bond (i.e., the 1-carbon-2 carbon bond), 1-3 bond, 1-5 bond, and 1-6 bond, respectively, between residues.

[0022] In the acidic EPS of the present invention, in the repeating structure in which the repeating units represented by formula (I) are linked together (for example, formula (II)), it is preferred that an average of about one glycerol 3-phosphate group be added per repeating unit (for example, one (n=1) or zero for each repeating unit, and about one per repeating unit on a weighted average for the entire acidic EPS), but this is not limitative.

[0023] The acidic EPS of the present invention preferably has the above repeating structure as a basic skeleton. In the present invention, "having a repeating structure as a basic skeleton" means that the entire length or almost the entire length of the acidic EPS (80% or more, preferably 90% or more, and 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 a repeating structure in which the repeating unit represented by formula (I) is linked together. In one embodiment, the acidic EPS of the present invention may consist solely of the above repeating structure.

[0024] The acidic EPS of the present invention may be derived from a microorganism (typically, a bacterium) such as a lactic acid bacterium, and may be produced by a microorganism (typically, a bacterium) such as a lactic acid bacterium and secreted extracellularly.

[0025] In one embodiment, the acidic EPS of the present invention may be obtained from a microbial culture or may be obtained from a lactic acid bacteria culture, for example, recovered or purified from a lactic acid bacteria culture. In one embodiment, the acidic EPS of the present invention may be isolated, for example, isolated from a lactic acid bacteria culture. With respect to the acidic EPS of the present invention, "isolated" 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.

[0026] Examples of lactic acid bacteria include, but are not limited to, bacteria of 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 delbrueckii subsp. bulgaricus), Lactobacillus casei, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus 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.

[0027] A more preferred example of the lactic acid bacterium used to produce the acidic EPS of the present invention is Lactobacillus delbrueckii subsp. bulgaricus, and a particularly preferred example of the lactic acid bacterium is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain.

[0028] Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain was deposited internationally under the Budapest Treaty under the accession number FERM BP-10741 at the National Institute of Technology and Evaluation (NITE-IPOD [formerly the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary]) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) on February 22, 1999 (original deposit date). This deposited strain was transferred 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. permission to refer to strain OLL1073R-1 in this application.

[0029] In one embodiment, the acidic EPS of the present invention can be obtained from a lactic acid bacteria culture containing one or more of the above-mentioned lactic acid bacteria. The lactic acid bacteria culture may be, but is not limited to, fermented milk such as yogurt produced using lactic acid bacteria, or may be obtained by culturing lactic acid bacteria in a medium (such as a milk-containing medium). In the present invention, the "milk" that can be used to prepare the lactic acid bacteria culture is preferably milk from a mammal (e.g., cow, goat, sheep, etc.).

[0030] In one embodiment, the lactic acid bacteria culture may contain Lactobacillus bacteria as lactic acid bacteria, for example, Lactobacillus delbrueckii subsp. bulgaricus.

[0031] In one embodiment, the lactic acid bacteria culture may contain a combination of Lactobacillus and Streptococcus as lactic acid bacteria, and in a preferred embodiment, the lactic acid bacteria culture may contain Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0032] In one embodiment, the acidic EPS of the present invention may be derived from lactic acid bacteria, for example, from the lactic acid bacteria of the genus Lactobacillus and / or Streptococcus, preferably from Lactobacillus delbrueckii subsp. bulgaricus, and more preferably from Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain.

[0033] The culture conditions used for producing the acidic EPS of the present invention using lactic acid bacteria may be, for example, the same as those in the Examples described below, but are not limited thereto. Such culture conditions may include, for example, 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 (e.g., 18 hours).

[0034] The acidic EPS of the present invention can be obtained from a lactic acid bacteria culture using known polysaccharide isolation and purification techniques. For example, EPS in a lactic acid bacteria culture can be separated from proteins and lactic acid bacteria by treatment with trichloroacetic acid or the like, recovered, and then dialyzed, followed by fractionation techniques such as anion exchange chromatography to separate the acidic EPS fraction. Furthermore, the remaining proteins in the separated acidic EPS fraction can be removed using a protease or the like, followed by further purification to obtain the acidic EPS of the present invention. However, the method for isolating and purifying the acidic EPS of the present invention is not limited to this method.

[0035] Alternatively, the acidic EPS of the present invention may be chemically synthesized using a sugar chain chemical synthesis method. A chemically synthesized acidic EPS of the present invention is also included in the scope of the "isolated" acidic EPS of the present invention.

[0036] The acidic EPS of the present invention has immunostimulating activity (immunostimulating effect). The acidic EPS of the present invention having immunostimulating activity can preferably promote the production of interferon (IFN)-γ. IFN-γ is produced mainly by immune cells such as T cells and natural killer (NK) cells, and is known to contribute to antiviral activity, immunomodulatory activity, and antitumor activity, as well as to function as a potent activator of macrophages.

[0037] 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 below. 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 cultured at an appropriate cell density (e.g., 5 × 10 ５ Acidic EPS is added to the seeded cells at a predetermined concentration (for example, a final concentration of 25 to 200 μg / mL), and the cells are incubated at 37°C in 5% CO ２ After culturing for a certain period of time (e.g., 72 hours) under 5% CO₂O₂, the supernatant is recovered from the 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 spleen cells are cultured in the same manner without the addition of acidic EPS, and the amount of IFN-γ in the supernatant is quantified and compared with the amount of IFN-γ quantified as described above in a culture system with the addition of acidic EPS. If the amount of IFN-γ in the supernatant increases statistically significantly with the addition of acidic EPS compared to a control without acidic EPS, or if the amount of IFN-γ in the supernatant increases in a concentration-dependent manner with increasing amounts of acidic EPS, it can be determined that the acidic EPS of the present invention has immunostimulatory activity. Furthermore, the strength (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 with the addition of acidic EPS compared to a control without acidic EPS.

[0038] The present invention also provides a composition comprising the Acidic EPS of the present invention, and a food or pharmaceutical product comprising the Acidic EPS of the present invention. The composition, food, or pharmaceutical product comprising the Acidic EPS of the present invention preferably has immunostimulatory activity, and is preferably immunostimulatory. In a preferred embodiment, the Acidic EPS of the present invention can be contained as an active ingredient (an active ingredient for conferring immunostimulatory activity) in the composition for immunostimulation and the food or pharmaceutical product for immunostimulation. The Acidic EPS of the present invention may be contained as the sole active ingredient for immunostimulation and / or as the sole EPS in the composition for immunostimulation and the food or pharmaceutical product for immunostimulation. In the present invention, "containing the Acidic EPS of the present invention as an active ingredient" means that the Acidic EPS of the present invention is contained in the composition, food, or pharmaceutical product in an amount (effective amount) and form sufficient to exert immunostimulatory activity when the composition, food, or pharmaceutical product comprising the Acidic EPS of the present invention is applied to a subject. The composition, food, or pharmaceutical product comprising the Acidic EPS of the present invention can contain, for example, the Acidic EPS of the present invention in an amount of 10 μg to 50 mg / kg body weight per application (ingestion or administration), but is not limited to this range.

[0039] In one embodiment, a composition, food, or pharmaceutical comprising an acidic EPS of the present invention may be free of neutral EPS (e.g., EPS derived from lactic acid bacteria such as Lactobacillus delbrueckii subsp. bulgaricus, including strain OLL1073R-1). In one embodiment, a composition, food, or pharmaceutical comprising an acidic EPS of the present invention may be free of acidic EPS (e.g., EPS derived from lactic acid bacteria such as Lactobacillus delbrueckii subsp. bulgaricus, including strain OLL1073R-1) having a repeating structure in which repeating units each consisting of a main chain consisting of two glucose residues and two galactose residues and a side chain consisting of one galactose residue, with a phosphate group attached to the structure (e.g., EPS derived from lactic acid bacteria such as Lactobacillus delbrueckii subsp. bulgaricus, including strain OLL1073R-1).

[0040] The composition, food, or pharmaceutical of the present invention may contain, in addition to the Acidic EPS of the present invention, additives acceptable for food processing (food additives) or pharmaceutically acceptable additives (pharmaceutical additives). Such additives include, but are not limited to, carriers, excipients, binders, lubricants, disintegrants, humectants, stabilizers, buffers, flavoring agents, preservatives, colorants, etc. In one embodiment, the composition comprising the Acidic EPS of the present invention (e.g., an immunostimulatory composition) may be, but is not limited to, a food composition or a pharmaceutical composition. The composition, food, or pharmaceutical of the present invention may further contain other pharmacological and / or functional ingredients.

[0041] In the present invention, the term "food" may refer to beverages or other foods, preferably processed foods. Examples of beverages include, but are not limited to, lactic acid bacteria beverages, milk beverages (coffee milk, fruit milk, etc.), tea beverages (green tea, black tea, oolong tea, etc.), fruit and vegetable beverages (beverages containing fruit juices such as orange, apple, grape, etc., and vegetable juices such as tomato and carrot), alcoholic beverages (beer, happoshu, wine, etc.), carbonated beverages, soft drinks, and water-based beverages. Examples of foods other than beverages include, but are not limited to, fermented milks such as yogurt, confectioneries, jams, prepared foods, instant foods, and seasonings. For information on the production methods of various foods, existing reference books on food production can be consulted.

[0042] The food of the present invention may be a functional food or other food. In the present invention, "functional food" refers to a food that has a certain functionality for the living body, and includes so-called health foods in general, such as health functional foods including foods for specified health uses (including conditional FOSHU [foods for specified health uses]) and foods with nutrient functions in Japan, foods with functional claims, foods for special dietary uses, dietary supplements, health supplements, supplements (e.g., tablets, coated tablets, sugar-coated tablets, capsules, and liquids), and beauty foods (e.g., diet foods). The functional food of the present invention also includes health foods to which a health claim based on the food standards of Codex Alimentarius (Joint FAO / WHO Food Standards Commission) is applied.

[0043] The functional food of the present invention may be a food for special uses such as a food for the sick, powdered milk for pregnant and nursing women, infant formula, food for the elderly, or food for caregivers.

[0044] The functional food of the present invention may be in the form of a solid preparation such as a tablet, granule, powder, pill, or capsule; a liquid preparation such as a solution, suspension, or syrup; or a gel or paste; or may be in the form of a conventional food (e.g., a beverage, yogurt, or confectionery).

[0045] The food of the present invention may contain any food ingredient in addition to the acidic EPS of the present invention, and is not particularly limited. The food of the present invention may contain water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. Examples of proteins include animal and plant proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, β-lactoglobulin, α-lactalbumin, lactoferrin, soy protein, egg protein, and meat protein, as well as hydrolysates thereof, butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, and various milk-derived components such as lactose. Examples of carbohydrates include common sugars, modified starches (dextrin, soluble starch, British starch, oxidized starch, starch esters, starch ethers, etc.), and dietary fiber. Examples of lipids include animal fats and oils such as lard, fish oil, and their fractionated oils, hydrogenated oils, and interesterified oils; and vegetable fats and oils such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, and their fractionated oils, hydrogenated oils, and interesterified oils. Examples of vitamins include vitamin A, carotenes, B vitamins, vitamin C, D vitamins, vitamin E, K vitamins, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of organic acids include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used alone or in combination of two or more, and synthetic products and / or ingredients containing large amounts of these can also be used as raw materials. The food of the present invention can be produced by a conventional method, for example, by adding the Acidic EPS of the present invention or a raw material containing the Acidic EPS of the present invention to other food ingredients in any step of food production.

[0046] The pharmaceutical of the present invention may be in any dosage form, such as a solid preparation such as a tablet, granule, powder, pill, or capsule, a gel, or a liquid preparation such as a solution, suspension, or syrup. The pharmaceutical of the present invention is preferably, but is not limited to, a pharmaceutical for oral administration.

[0047] The composition, food, or pharmaceutical product of the present invention can provide an immunostimulating effect based on the immunostimulating activity (immunostimulating effect) of the acidic EPS of the present invention. For example, the composition, food, or pharmaceutical product of the present invention can promote the production of interferon (IFN)-γ. The composition, food, or pharmaceutical product of the present invention can promote (stimulate) the immune response in a subject ingested or administered with the composition, food, or pharmaceutical product.

[0048] The present invention also provides a method for producing an immunostimulatory effect in a subject (immunostimulatory method), which comprises having the subject ingest or administer the acidic EPS of the present invention, or a composition, food, or pharmaceutical of the present invention containing the same.

[0049] The subject to which the acidic EPS of the present invention or the composition, food, or pharmaceutical of the present invention containing the same is ingested or administered is preferably a mammal, including primates such as humans, livestock, pets, laboratory (test) animals, etc., and more preferably a human. Such subjects may be subjects requiring immunostimulation or subjects for which immunostimulation is desired, including, but not limited to, pregnant women, infants, elderly people, sick people, those recovering from illness, the weak, subjects with weakened immune systems, athletes, and subjects in need of prevention of infection or other diseases (e.g., medical professionals, caregivers, students taking exams, etc.).

[0050] The acidic EPS of the present invention, or the composition, food, or pharmaceutical of the present invention containing the same, is preferably taken or administered to a subject orally, but may also be taken or administered via a tube such as a nasal tube, an oral esophageal tube, a gastrostomy tube, or an enterostomy tube, although the route of administration to a subject in the present invention is not limited to these.

[0051] The intake or administration amount of the Acidic EPS of the present invention, or the composition, food, or pharmaceutical of the present invention containing the same, may be any amount effective for immunostimulation, and can be varied over a wide range at the discretion of those skilled in the art, taking into consideration the species, age, body weight, administration route, number of administrations, etc. of the subject. In one embodiment, the intake or administration amount may be an amount that provides 10 μg to 50 mg / kg body weight of the Acidic EPS of the present invention per application (ingestion or administration) based on the body weight of the subject, but is not limited to this range.

[0052] The ingestion or administration of the acidic EPS of the present invention, or the composition, food, or medicine of the present invention containing the same, may be carried out once (once) or multiple times. The acidic EPS of the present invention, or the composition, food, or medicine of the present invention containing the same, may be continuously ingested or administered to a subject, may be ingested or administered daily, or may be ingested or administered, for example, 1, 2, 3, 4, 5, or 6 days per week. When ingested or administered continuously, it is preferable that the subject ingest or administer the acidic EPS for at least 3 days, preferably 1 week or more, more preferably 2 weeks or more, even more preferably 4 weeks or more, for example, 3 months or more.

[0053] The present invention also relates to the Acidic EPS of the present invention for use in immunostimulation (promoting an immune response). The present invention also relates to the use of the Acidic EPS of the present invention for immunostimulation (promoting an immune response). Furthermore, the present invention relates to the use of the Acidic EPS of the present invention in the manufacture of a composition, food, or pharmaceutical for immunostimulation (promoting an immune response).

[0054] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0055] Example 1 Production and Purification of Acidic EPS Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 strain (accession 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 to a final concentration of 10% by mass, and the resulting denatured proteins and lactic acid bacteria were precipitated, followed by centrifugation (12,000 x g, 20 minutes). The resulting supernatant was collected, and a three-fold volume of cold ethanol was added. The mixture was then allowed to stand overnight at 4°C. The resulting precipitate was collected by centrifugation (12,000×g, 20 minutes) and dialyzed against distilled water using a dialysis membrane (MWCO [molecular weight cutoff] 6,000 to 8,000), thereby obtaining crude EPS (exopolysaccharide).

[0056] Acidic EPS was fractionated from the obtained crude EPS. The fractionation was carried out using DEAE-Sepharose, an anion exchange carrier. （Ｒ） (Diethylaminoethyl-Sepharose （Ｒ） ) Using Fast Flow, a concentration gradient extraction method was performed using 0 to 0.5 M NaCl in 0.02 M Tris-HCl buffer (pH 8.6). The obtained acidic EPS was extracted with 1 mM MgCl ２ The resulting solution was dissolved in 0.05 M Tris-HCl buffer (pH 8.0) containing PEG-1000 and treated with 2 mg / mL DNase and 2 mg / mL RNase at 37°C for 6 hours. Proteinase K solution was then added at a final concentration of 0.1 mg / mL, and the mixture was treated at 37°C for 16 hours to decompose the protein. The enzyme was then inactivated by heating at 90°C for 10 minutes. A three-fold volume of cold ethanol was added to the resulting reaction solution, and the mixture was left standing overnight at 4°C. The resulting precipitate was collected by centrifugation (12,000 × g, 20 minutes). This procedure was repeated, and the acidic EPS was purified by dialysis against distilled water using the dialysis membrane described above, to prepare an acidic EPS solution.

[0057] [Example 2] Structural analysis of acidic EPS The purified acidic EPS was subjected to structural analysis by two-dimensional NMR. The two-dimensional NMR used in the analysis is a nuclear magnetic resonance (NMR) spectroscopy method that clarifies the correlation between signals and enables structural analysis of complex compounds. Specifically, １ H. １ H]-COSY method ( １ H. １ H-correlation spectroscopy), TOCSY method (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. １H]-nuclear overhauser enhancement spectroscopy).

[0058] first[ １ H. １ The structure of acidic EPS was predicted using the [H]-COSY method. However, the signal of each proton is affected by two substituents attached to the same carbon atom (geminal position) or two substituents attached to adjacent carbon atoms (vicinal position), and most of the anomeric sugars are affected by this. １ Furthermore, since the signals of oligosaccharides and polysaccharides overlap in a complex manner, the TOCSY method, which can extract correlation peaks of all protons belonging to a specific spin system from the signal, is used to [ １ H. １ On the other hand, the [H]-COSY data, which can detect correlations between two different types of nuclei, such as hydrogen and carbon atoms, was used. １ H- １３ Using the [C]-HSQC method, we analyzed which hydrogen atoms are directly bonded to which carbon atoms. Furthermore, we observed signals between protons that are spatially close to each other [ １ H. １ 1H]-NOESY techniques were used to determine which signals were from protons that were close to each other.

[0059] [ １ H- １３ The spectrum obtained by the [C]-HSQC method is shown in Figure 1. １ H. １ The spectrum obtained by the [H]-NOESY method is shown in FIG.

[0060] Two-dimensional NMR structural analysis showed that the acidic EPS has a repeating structure consisting mainly of repeating units of galactose residues (three furanose-type galactose residues and two pyranose-type galactose residues). Furthermore, it was also shown that many of the repeating units in this structure have one glycerol phosphate group bound to each repeating unit, forming an anchor to the bacterial cell wall.

[0061] Furthermore, for glycosyl bond analysis, the obtained acidic EPS was permethylated, depolymerized, and acetylated, and the resulting methylated alditol acetate was analyzed by gas chromatography-mass spectrometry (GC-MS), thereby determining the repeating structure of the acidic EPS as shown in Figure 3. In Figure 3, A, B, C, D, and E / F are symbols conveniently assigned to indicate the respective sugar residues shown in the spectra of Figures 1 and 2. In the structure shown in Figure 3, Galp represents a pyranose-type galactose residue, Galf represents a furanose-type galactose residue, and Gro3p represents a glycerol 3-phosphate group.

[0062] [Example 3] Evaluation of immunostimulatory activity of acidic EPS Mouse spleen cells were suspended in RPMI 1640 medium containing 10% (vol / vol) fetal bovine serum, and then 5 × 10 ５ The cells were seeded onto a 96-well plate at a density of 100 cells / well. A solution of the acidic EPS purified in Example 1 and whose structure was determined in Example 2 was added to the seeded cells at a final EPS concentration of 25 μg / mL, 50 μg / mL, 100 μg / mL, or 200 μg / mL. The cells were incubated at 37°C, 5% CO ２ The cells were cultured for 72 hours under acidic EPS stimulation (acidic EPS-stimulated group). As a control, mouse spleen cells were cultured in the same manner without the addition of acidic EPS (unstimulated group). After the culture, the supernatant was collected, and the amount of interferon-γ (IFN-γ) produced in the medium was quantified by ELISA.

[0063] As a result, IFN-γ production in splenocytes was induced in a concentration-dependent manner by the addition of acidic EPS (Figure 4), demonstrating that the acidic EPS obtained can activate immune cells.

[0064] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. An acidic exopolysaccharide having a repeating structure in which repeating units represented by the following formula (I) are linked together: (In formula (I), n independently represents an integer of 0 or 1 for each repeating unit.) 2. The isolated acidic bacterial exopolysaccharide of claim 1.

3. The acidic exopolysaccharide according to claim 1 or 2, isolated from a lactic acid bacteria culture containing Lactobacillus delbrueckii subsp. bulgaricus.

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

5. An immunostimulating composition comprising the acidic bacterial exopolysaccharide of claim 1 as an active ingredient.

6. An immunostimulatory food or drug containing the acidic bacterial exopolysaccharide according to claim 1 as an active ingredient.

7. A method for immunostimulation, comprising having a subject ingest or administer the acidic bacterial exopolysaccharide described in claim 1, the immunostimulating composition described in claim 5, or the immunostimulating food or pharmaceutical described in claim 6.

8. Use of the acidic bacterial exopolysaccharide according to claim 1 in the production of an immunostimulatory composition, food or pharmaceutical product.

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