Intestinal immune activator, IgA production promoter, and gene expression promoter

Abstract

To provide an intestinal tract immunostimulator, an IgA production promoter and a gene expression promoter in which the novel properties of lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus have been clarified to provide novel use based thereupon.SOLUTION: Provided are an intestinal tract immunostimulator, an IgA production promoter and a gene expression promoter that contain lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus as an active ingredient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an intestinal immune activator, an IgA production promoter, and a gene expression promoter containing lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus as an active ingredient.

Background Art

[0002] Conventionally, it has been known that lactic acid bacteria and their fermentation products have various physiological functions. For example, Patent Document 1 describes that lactic acid bacteria belonging to Lactobacillus kunkeei have a high IgA production promoting effect.

[0003] In addition, the present applicant has found that Lactobacillus kousoi 10H strain, which is a lactic acid bacterium isolated from a vegetable molasses fermentation broth, is a fructophilic lactic acid bacterium having a novel genomic structure different from other lactic acid bacteria, and has an excellent IgA production promoting effect (and thus an immune activating effect) (see Patent Document 2. Note that "Lactobacillus kosoi" in Patent Document 2 refers to the same thing as "Lactobacillus kousoi" in the present specification.).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

[0005] By the way, although the classification of the genus Lactobacillus has been used for a long time, it has been previously pointed out that there are significant differences in physiological and biochemical characteristics among strains and species in terms of phylogenetic diversity. Therefore, in recent years, a re-evaluation of the classification of the genus Lactobacillus at the genomic level has been carried out. For example, the above-mentioned Lactobacillus kunkeei and Lactobacillus kousoi were reclassified into the genus Apilactobacillus after re-evaluation, and their scientific names became Apilactobacillus kunkeei and Apilactobacillus kosoi, respectively.

[0006] As described above, it is known that certain species of lactic acid bacteria belonging to the genus Apilactobacillus have an IgA production promoting effect (and thus an immune activating effect), but it is not known what causes this.

[0007] The present invention has been made in view of the above circumstances, and an object thereof is to clarify the novel properties of substances derived from lactic acid bacteria belonging to the genus Apilactobacillus and to provide new uses based on this.

Summary of the Invention

Means for Solving the Problems

[0008] In order to solve the above problems, the present inventors focused on lipoteichoic acid among the substances contained in lactic acid bacteria belonging to the genus Apilactobacillus, and found that the structure of lipoteichoic acid in lactic acid bacteria belonging to the genus Apilactobacillus is different from the typical structure of lipoteichoic acid in lactic acid bacteria belonging to the former genus Lactobacillus, and that it has an excellent IgA production promoting effect and immune activating effect, thereby completing the present invention. That is, the present invention includes the following embodiments.

[0009] (1) An intestinal immune activator containing lipoteichoic acid derived from lactic acid bacteria belonging to the genus Apilactobacillus as an active ingredient. (2) The intestinal immune activator according to (1), wherein the lactic acid bacterium belonging to the genus Apilactobacillus is a lactic acid bacterium belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum. (3) The intestinal immune activator according to (2), wherein the lactic acid bacterium belonging to the genus Apilactobacillus is Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum JCM30765 strain. (4) The intestinal immune activator according to any one of (1) to (3), which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be formulated therein. (5) An IgA production promoter containing lipoteichoic acid derived from a lactic acid bacterium belonging to the genus Apilactobacillus as an active ingredient. (6) The IgA production promoter according to (5), wherein the lactic acid bacterium belonging to the genus Apilactobacillus is a lactic acid bacterium belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum. (7) The IgA production promoter according to (6), wherein the lactic acid bacterium belonging to the genus Apilactobacillus is Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum JCM30765 strain. (8) The IgA production promoter according to any one of (5) to (7), which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be formulated therein. (9) A gene expression promoter containing lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus as an active ingredient, which promotes the expression of at least one factor among IL-6, IL-10, and retinal dehydrogenase 2 (RALDH2) in dendritic cells. (10) The gene expression promoter according to (9), wherein the lactic acid bacteria of the genus Apilactobacillus belong to lactic acid bacteria of Apilactobacillus kosoi. (11) The gene expression promoter according to (10), wherein the lactic acid bacteria of the genus Apilactobacillus is Apilactobacillus kosoi strain 10H. (12) The gene expression promoter according to any one of claims 9 to 11, which is in the form of a food or drink, a pharmaceutical, a feed, or an active ingredient composition to be formulated therein. (13) Use of lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus for producing a pharmaceutical, a food or drink, a feed, or an active ingredient composition to be formulated therein for activating intestinal immunity or promoting IgA production in humans or non-human animals. (14) Use of lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus for producing a pharmaceutical, a food or drink, a feed, or an active ingredient composition to be formulated therein for promoting gene expression of at least one factor among IL-6, IL-10, and retinal dehydrogenase 2 (RALDH2) in dendritic cells in humans or non-human animals.

Effect of the Invention

[0010] According to the present invention, as a novel use of lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus, an intestinal immunity activator, an IgA production promoter, and a gene expression promoter containing this lipoteichoic acid can be provided.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

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Figure 6

Mode for Carrying Out the Invention

[0012] Next, each embodiment of the present invention will be described with reference to the drawings. It should be noted that each embodiment described below does not limit the invention according to the claims, and not all of the elements and combinations thereof described in each embodiment are essential for the solution means of the present invention.

[0013] (I) Lipoteichoic acid as an active ingredient The active ingredient in one embodiment of the present invention is lipoteichoic acid derived from lactic acid bacteria belonging to the genus Apilactobacillus. Preferably, the lactic acid bacteria belonging to the genus Apilactobacillus are lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum. More preferably, the lactic acid bacteria belonging to the genus Apilactobacillus are Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum JCM30765 strain.

[0014] As lactic acid bacteria of the genus Apilactobacillus, in addition to Apilactobacillus kuroiwae, Apilactobacillus kunkeei, and Apilactobacillus apinorum, Apilactobacillus micheneri, Apilactobacillus ozensis, Apilactobacillus quenuiae, and Apilactobacillus timberlakei are known.

[0015] Lactic acid bacteria of the genus Apilactobacillus are a group of lactic acid bacteria that were called the Lactobacillus kunkeei group among the former lactic acid bacteria of the genus Lactobacillus. Lactic acid bacteria of the genus Apilactobacillus are Gram-positive, rod-shaped, and heterofermentative, and generally grow in the range of 15 ~ to 37 °C, and many also grow under acidic conditions with a pH of less than 3.0. The genome size of lactic acid bacteria of the genus Apilactobacillus is about 1.42 to 1.58 Mbp, which is relatively small. The G+C content in DNA is in the range of 30.5 to 36.4. Lactic acid bacteria of the genus Apilactobacillus convert fructose to mannitol. Also, usually, fructose, glucose, and sucrose can be metabolized, but maltose and pentose cannot be metabolized (Zheng et al. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology 2020;70:2782-2858).

[0016] Apilactobacillus kuroiwae is considered to be a bacterium evolved from conventional lactic acid bacteria as a recently discovered fructophilic lactic acid bacterium (FLAB) (Filannino et al. “Fructose-rich niches traced the evolution of lactic acid bacteria toward fructophilic species” Critical Reviews in Microbiology, Vol. 45, No. 1, 2019, pp. 65-81). FLAB inhabits environments rich in fructose, such as flowers, fruits, fermented foods, and the digestive tracts of insects that mainly feed on fructose. FLAB is a heterofermentative lactic acid bacterium that prefers fructose rather than glucose as a carbon source, but it is said that the addition of an electron acceptor substrate such as oxygen promotes its growth in the presence of glucose. Apilactobacillus kuroiwae strain 10H has a relatively small genome size and a low GC content compared to other FLAB and lactic acid bacteria (see Figure 3 in Filannino et al.).

[0017] Apilactobacillus kunkeei is a lactic acid bacterium isolated from slowly fermenting wine, but is typically associated with honeybees and flowers. Apilactobacillus kunkeei strain JCM16173 is the reference strain of Apilactobacillus kunkeei and is the same as strains YH-15, ATCC700308, and DSM12361. Since the properties of this lactic acid bacterium have been well known, the detailed description thereof will be omitted.

[0018] Apilactobacillus apinorum is a lactic acid bacterium isolated from the honey stomach of honeybees. Apilactobacillus apinorum strain JCM30765 is the reference strain of Apilactobacillus apinorum and is the same as strains Fhon13N, DSM26257, and CCUG63287. Since the properties of this lactic acid bacterium have been well known, the detailed description thereof will be omitted.

[0019] Lipoteichoic acid (LTA) is a component of the cell membrane of Gram-positive bacteria. General lipoteichoic acid consists of a main chain (glycerol phosphate chain) with glycerol phosphate as a repeating unit and an anchor glycolipid containing several sugars and several residues of fatty acids. The structure of lipoteichoic acid varies depending on the bacterium from which it is derived. Compared with lipopolysaccharide (LPS) of Gram-negative bacteria, research on lipoteichoic acid has not advanced much, and its detailed structure and physiological activity are not yet well understood.

[0020] (II) IgA production promoter and intestinal immune activator In this specification, the "IgA production promoter" refers to a substance having an IgA production-inducing ability such that when added to the culture solution of Peyer's patch cells containing a large amount of IgA-producing cells and cultured for a predetermined period, the amount of secretory IgA secreted into the culture solution after culture is increased compared to the case where it is not added. The IgA production promoter of the present invention includes, as described in detail below, forms such as food and drink products, pharmaceuticals, feeds, or active ingredient compositions. The IgA production promoter can, for example, enhance the production of antibodies against the antigens contained in the vaccine when administered together with the vaccine, enhance the effect of the vaccine, and is likely to suppress the side effects of the vaccine. That is, it enhances the production of antibodies against the antigens contained in the vaccine, improves the induction of defensive immunity, and enhances the effect of the vaccine.

[0021] When the IgA production promoter of this embodiment is used together with a vaccine, it can be used as a vaccine effect enhancer that administers the IgA production promoter before and after the vaccine administration to enhance the effect. The amount of the IgA production promoter used varies depending on the type and quality of the vaccine used, or the age, symptoms, etc. of the vaccinated person. For example, for preventive use, about 0.01 to 1 g in terms of solid content per adult per dose is mentioned, and it is desirable to take it three times a day about 30 minutes before meals.

[0022] In addition, in this specification, the "intestinal immune activator" means something effective for promoting the secretion of IgA in the mucosal epithelium of the intestine and activating the host's immune system. The intestinal immune activator of the present invention includes, as will be described in detail below, forms such as food products, pharmaceuticals, feeds, or active ingredient compositions. Among these, health foods are preferred, and particularly, food compositions for maintaining and enhancing the health of subjects with reduced immunity are preferred. When used as a health food, it is appropriate to use an amount that does not adversely affect the taste and appearance of the food.

[0023] (III) Gene Expression Promoter In this specification, the "gene expression promoter" means something that promotes the expression of a specific factor (gene) from specific cells. The gene expression promoter of the present invention includes, as will be described in detail below, forms such as food products, pharmaceuticals, feeds, or active ingredient compositions. The gene expression promoter of the present invention is a gene expression promoter that promotes the expression of at least one factor among IL-6, IL-10, and retinal dehydrogenase 2 (RALDH2) in dendritic cells. IL (interleukin)-6 and IL-10 are cytokines. Retinal dehydrogenase 2 is an enzyme that metabolizes retinal to retinoic acid. The synthesis of IL-6, retinoic acid, and IL-10 in intestinal dendritic cells acts on Foxp3 + T cells and differentiates them into follicular T helper cells (Tfh cells) that interact with B cells. In addition, the synthesis of retinoic acid and IL-10 in intestinal dendritic cells promotes IgA class switch recombination and IgA production in B cells in the germinal center of Peyer's patches. In addition, dendritic cells expressing IL-6 enhance IgA production from B cells by IL-6R signaling. Furthermore, retinoic acid is necessary for the homing of IgA-producing B cells to the intestine. Thus, the expression of IL-6, IL-10, and retinal dehydrogenase 2 in dendritic cells is closely related to the promotion of IgA production.

[0024] (IV) Food products, pharmaceuticals, feeds, or active ingredient compositions to be formulated therein (Active Ingredient Composition) The enteric immune activator, IgA production promoter, and gene expression promoter of this embodiment can be used in the form of food and drink products, pharmaceuticals, feeds, or active ingredient compositions to be formulated therein. When used as an active ingredient composition, not only can the pure lipoteichoic acid derived from Apilactobacillus lactic acid bacteria, which is the active ingredient, be used as it is after being separated from the lactic acid bacteria, but also crude or purified products containing lipoteichoic acid, their freeze-dried products, or cell wall components obtained by treating the bacterial cells with enzymes or physical means can be used.

[0025] The active ingredient composition of this embodiment is preferably prepared into forms such as food and drink products, pharmaceuticals, etc. as described below through appropriate blending with suitable edible carriers (food materials), pharmaceutically acceptable carriers, etc.

[0026] (Pharmaceuticals) When the enteric immune activator, IgA production promoter, and gene expression promoter of this embodiment are in the form of pharmaceuticals, they are prepared and used in the form of a pharmaceutical composition using a suitable pharmaceutical carrier acceptable in pharmacy together with lipoteichoic acid derived from Apilactobacillus lactic acid bacteria. Examples of such pharmaceutical carriers include diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, etc., which are usually used in this field.

[0027] As the dosage unit form of pharmaceuticals, various forms can be selected, but preferably, formulations for oral administration are mentioned. Representative oral administration formulations include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, etc.

[0028] When forming into tablet form, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. as pharmaceutical carriers; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, polyvinyl pyrrolidone, etc.; disintegrants such as sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, etc.; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, etc.; humectants such as glycerin, starch, etc.; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, etc.; lubricants such as purified talc, stearate, boric acid powder, polyethylene glycol, etc. can be used. Tablets can be ordinary coated tablets as needed, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, or double tablets or multi-layer tablets.

[0029] When forming into pill form, excipients such as glucose, lactose, starch, cocoa butter, hardened vegetable oil, kaolin, talc, etc. as pharmaceutical carriers; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol, etc.; disintegrants such as laminaran, agar, etc. can be used.

[0030] Furthermore, in pharmaceuticals, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc. and other pharmaceuticals can also be contained as needed.

[0031] There are no particular restrictions on the method of administering the pharmaceutical of this embodiment, and it is determined according to the dosage form, the age, sex and other conditions of the patient, the degree of the disease, etc. Also, the dosage is appropriately selected according to the usage, the age, sex and other conditions of the patient, the degree of the disease, etc., but usually, the above active ingredient composition is preferably about 0.5 to 100 mg per kg of body weight per day. The pharmaceutical can be administered to humans 1 to 4 times a day.

[0032] (Food and drink) As used herein, "food and drink" includes all forms that are exclusively used orally for eating and drinking (including, for example, beverages), and even in the form of tablets, etc., as long as they are exclusively used for eating and drinking, they are included in the food and drink herein. For example, health foods, health supplements, foods for patients, nutritional supplements, or health functional foods (specific health foods, nutritional functional foods) defined by the Ministry of Health, Labour and Welfare, which are conceptually for infection prevention, diarrhea prevention, etc. and display the purpose as necessary, are also included in the food and drink herein. A health food means a food for the purpose of health care, health maintenance and promotion, etc. in a more positive sense than ordinary foods.

[0033] When the enteral immune activator, IgA production promoter and gene expression promoter of this embodiment are used as food and drink, examples include fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, fermented soy milk beverages, etc. "Fermented milk" refers to a paste or liquid obtained by fermenting milk or dairy products with lactic acid bacteria or yeast. Therefore, the fermented milk includes both the beverage form and the yogurt form. Also, "lactic acid bacteria beverage" refers to a beverage obtained by diluting with water a paste or liquid obtained by fermenting milk or dairy products with lactic acid bacteria or yeast as the main raw material.

[0034] Examples of other food and drink forms include pickles, miso, fermented tea, fermented foods such as bread, baby foods such as weaning foods, powdered milk, and baby food, confectionery such as foaming preparations, gums, gummies, and puddings, noodles, nutritional supplements such as capsules, granules, powders, and tablets, and dairy products other than the above fermented milk and lactic acid bacteria beverages.

[0035] The content of the active ingredient composition in the food or drink according to this embodiment is not particularly limited and can be appropriately determined. From the viewpoint of exerting the effects of intestinal immune activation, IgA production promotion, or gene expression promotion, for each food or drink, for example, 0.001% by mass or more is preferable, 0.01% by mass or more is more preferable, and 0.1% by mass or more is more preferable, based on the total mass of each food or drink. On the other hand, the upper limit of the content of the active ingredient composition in the food or drink is not particularly limited and can usually be appropriately adjusted according to the form of the food or drink.

[0036] (Feed) When the intestinal immune activator, IgA production promoter, and gene expression promoter of this embodiment are in the form of feed, for example, for preventing infections during the non-antibiotic administration period of chickens or during the weaning period of pigs, cows, etc., they can be in an oral administration preparation form (aqueous solution, emulsion, granules, powder, capsules, tablets, etc.).

[0037] [Examples] Next, examples are given to explain the present invention in more detail, but the present invention is not limited to these examples in any way. In the following examples, the unit % of the numerical values indicating the addition amounts of various components means % by mass unless otherwise specified.

[0038] [Example 1] Obtaining and culturing lactic acid bacteria In order to obtain lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus, Apilactobacillus apinorum JCM30765 strain, Apilactobacillus kosoii 10H strain, and Apilactobacillus kunkee JCM16173 strain were prepared. Also, for comparison, lactic acid bacteria other than the genus Apilactobacillus, Lactiplantibacillus plantarum subsp. plantarum (hereinafter simply referred to as "Lactiplantibacillus plantarum") JCM1149 strain and Lacticaseibacillus rhamnosus GG strain (ATCC53103) were also prepared. The Lactiplantibacillus plantarum subsp. plantarum JCM1149 strain is a reference strain.

[0039] Among the above lactic acid bacteria, for Apilactobacillus kuroiwae 10H strain, the strain stored in the Matsuzaki Laboratory of Ishikawa Prefectural University was used; for Lactiplantibacillus rhamnosus GG strain (ATCC53103), the one obtained from the American Type Culture Collection (ATCC) in the United States was used; for the others, the ones obtained from the Microbial Material Development Laboratory (JCM) of the RIKEN BioResource Research Center, National Institute of Advanced Industrial Science and Technology were used.

[0040] For Apilactobacillus apinorum JCM30765 strain and Apilactobacillus kuroiwae 10H strain, they were cultured using MRS broth for lactobacilli supplemented with 10% fructose. For Apilactobacillus kunkeei JCM16173 strain, it was cultured using MRS broth for lactobacilli supplemented with 10% tomato juice and 0.05% L-cysteine hydrochloride, respectively. The strain of lactic acid bacteria for comparison was cultured using MRS broth for lactobacilli (Difco Laboratories). Each strain was precultured overnight at 30 °C and then cultured for one day at 30 °C.

[0041] [Example 2] Purification of Lipoteichoic Acid Purification of lipoteichoic acid was carried out with reference to known methods (Morath et al., J. Exp. Med., 193:393 - 397, 2001, and Claes et al., Microbial Cell Factories, 11:161 - 168, 2012). First, lactic acid bacteria cells after main culture were collected by centrifugation and suspended by adding 0.1 M citrate buffer (pH 4.7). Next, using a Multi-Beads Shocker (registered trademark) (manufactured by Yasui Kikai Co., Ltd.), they were disrupted on ice using 0.3 mm zirconia beads. The disruption time was 1 minute, and this was repeated 6 times. The disrupted lactic acid bacteria cells were once frozen at -80 °C, and then, to remove lipophilic cell molecules, an equal volume of butanol was added and stirred for 2 hours. This was centrifuged, and after recovering the aqueous layer, it was lyophilized. The lyophilized sample was dissolved in a column equilibration buffer (0.1 M sodium acetate buffer containing 15% n-propanol, pH 4.7), and solids were removed by centrifugation for 30 minutes, and then loaded onto an octyl-Sepharose 4 Fast Flow column (manufactured by GE Healthcare) to perform hydrophobic chromatography. Lipoteichoic acid was eluted using a linear gradient of n-propanol from 15% to 60% in 0.1 M sodium acetate buffer (pH 4.7). Fractions containing lipoteichoic acid were identified by measuring the contents of phosphate and sugar. The content of phosphate was measured by the phosphomolybdenum test. The content of sugar was measured by the phenol-sulfuric acid method using glucose as a standard. Also, regarding the absence of nucleic acid and protein in the recovered fractions, it was confirmed by measuring the UV absorption at 260 nm and 280 nm, respectively. The fractions recovered in this way were once lyophilized, suspended in 10 ml of Milli-Q water, dialyzed with Milli-Q water, and lyophilized again. The purity of lipoteichoic acid was determined by measuring the endotoxin content using an LAL reagent (<0.0001%) (manufactured by Seikagaku Corporation).

[0042] [Example 3] Measurement of IgA production-inducing ability (Preparation of Peyer's patch cells) Six-week-old male BALB / cA mice (purchased from CREA Japan) were bred using AIN-76 diet (purchased from Research Diets) as the basal diet. The AIN-76 diet is a mixture containing 20.0% milk casein, 0.3% DL-methionine, 5.0% corn oil, 50.0% sucrose, 15.0% corn starch, 5.0% cellulose powder, 1.0% AIN-76 vitamin mix, 3.5% AIN-76 mineral mix, and 0.2% choline bitartrate.

[0043] [[ID=④]]The mice were handled in accordance with the guidelines for the proper conduct of animal experiments issued by the Science Council of Japan in 2006. After breeding the mice for one week, they were euthanized with carbon dioxide gas, and the Peyer's patches of the small intestine were excised by laparotomy.

[0044] The Peyer's patches were placed in a Petri dish filled with ice-cold RPMI1640 medium (PSMF) [RPMI1640 medium (Gibco BRL) supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, 55 μmol / l 2-mercaptoethanol, and 10% heat-inactivated fetal bovine serum (FBS; GibcoBRL)] and washed three times with the medium. Subsequently, they were cultured at 37 °C for 45 minutes in RPMI1640 medium (PSMF) supplemented with 25 mmol / l HEPES, 5 mmol / l EDTA (pH 8.0), and 1 mmol / l dithiothreitol. After washing the Peyer's patches again with RPMI1640 medium (PSMF) containing 5 mmol / l EDTA (pH 8.0), they were treated at 37 °C for 50 minutes in RPMI1640 medium (PSMF) supplemented with 400 U / ml type I collagenase (Sigma) and 30 U / ml DNase I (Takara Bio Inc.). The resulting mixture was filtered through a 40-μm nylon mesh, washed twice with RPMI1640 medium (PSMF), and Peyer's patch cells used for measuring IgA production-inducing ability were obtained. The viability of the Peyer's patch cells was confirmed by trypan blue staining. The final concentration of the Peyer's patch cells was adjusted to 1.25×10 6 cells / ml and used for evaluation.

[0045] (Measurement of IgA) The lipoteichoic acid obtained in Example 2 was added to the suspension of Peyer's patch cells so that the final concentration was 50 μg / ml, and co-cultured in a 96-well T-cell activation plate (Becton Dickinson) for 5 days at 37°C under 5% CO2 conditions. Thereafter, the amount of IgA in the obtained culture supernatant was measured using a mouse IgA ELISA kit (Bethyl Laboratories).

[0046] The results are shown in Figure 1. In Figure 1, the results of saline, which is a negative control, are placed at the top. Also, the notations "a", "b", and "c" in Figure 1 indicate that there are significant differences (P < 0.05) for each group with different alphabets. As a result of the experiment, in the lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus, namely, Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain, and Apilactobacillus apinorum JCM30765 strain, a clearly higher IgA production-inducing ability was confirmed compared to the lipoteichoic acid derived from other lactic acid bacteria.

[0047] From the above results, lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus, particularly lipoteichoic acid derived from lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei, or Apilactobacillus apinorum, and further, lipoteichoic acid derived from Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain, or Apilactobacillus apinorum JCM30765 strain, can be expected as an immunostimulant having a remarkable effect.

[0048] [Example 4] Gene Expression Analysis in Dendritic Cells (Generation of bone marrow-derived dendritic cells from mouse bone marrow cells) Bone marrow-derived dendritic cells were generated from bone marrow cells of the femurs and tibias of 4-week-old female BALBc / A mice (purchased from CREA Japan). After washing the bone marrow cells collected from the mice, the cell count was adjusted to 1×10 6 cells / ml, and the cells were suspended in RPMI1640 medium (PSMF) supplemented with granulocyte macrophage colony-stimulating factor (manufactured by PeproTech) at a concentration of 20 ng / mL, and cultured under conditions of 37°C and 5% CO2. On the 3rd and 5th days of culture, half of the medium was replaced with fresh medium. On the 6th day of culture, the cells containing dendritic cells were collected, magnetically labeled with anti-CD11c microbeads (manufactured by Miltenyi Biotec), and dendritic cells were separated by a conventional method using AutoMACS (manufactured by Miltenyi Biotec).

[0049] (Gene expression analysis) For the bone marrow-derived dendritic cells obtained as described above, culture was performed for 6 hours at 1.0×10 9 cells / well (3 ml) using RPMI1640 medium (PSMF). This culture was performed for both a sample containing lipoteichoic acid derived from Apilactobacillus kuroiwae strain 10H at a concentration of 50 μg / ml and a sample without lipoteichoic acid (control). Thereafter, total RNA was isolated from the bone marrow-derived dendritic cells using the QuickPrep Total RNA Extraction Kit (manufactured by GE Healthcare), and cDNA was synthesized from the total RNA using the SuperScript (registered trademark) III reverse transcription kit (manufactured by Invitrogen). Real-time PCR was performed using the StepOne real-time PCR system (manufactured by Applied Biosystems) and the Power SYBR (registered trademark) Green Master Mix (manufactured by Applied Biosystems). The following primers were used to amplify DNA.

[0050] (Primer for amplifying IL-6: bone marrow-derived dendritic cell IL-6 PCR primer) Forward: 5’-AATAGTCCTTCCTACCCCAATTTC-3’ (SEQ ID NO: 1) Reverse: 5’-ATTTCAAGATGAATTGGATGGTCT-3’ (SEQ ID NO: 2) (Primer for amplifying IL-10: bone marrow-derived dendritic cell IL-10 PCR primer) Forward: 5’-ATGCAGGACTTTAAGGGTTACTTG-3’ (SEQ ID NO: 3) Reverse: 5’-GAATTCAAATGCTCCTTGATTTCT-3’ (SEQ ID NO: 4) (Primer for amplifying RALDH2: bone marrow-derived dendritic cell RALDH2 PCR primer) Forward: 5’-GACTTGTAGCAGCTGTCTTCACT-3’ (SEQ ID NO: 5) Reverse: 5’-TCACCCATTTCTCTCCCATTTCC-3’ (SEQ ID NO: 6)

[0051] As an endogenous control, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used. The following primers were used to amplify this gene.

[0052] (Primer for amplifying GAPDH: bone marrow-derived dendritic cell GAPDH PCR primer) Forward: 5’-CTACACTGAGGACCAGGTTGTCT-3’ (SEQ ID NO: 7) Reverse: 5’-ATTGTCATACCAGGAAATGAGCTT-3’ (SEQ ID NO: 8)

[0053] Statistical analysis was performed using Excel Statistics (Social Information Service Co., Ltd.). The measurement results were analyzed using one-way ANOVA, and Dunnett's Post-hoc analysis was conducted. ***p<0.001

[0054] The results are shown in Figure 2. The degree of gene expression in Figure 2 is shown as the ratio between the results of the control and the samples containing lipoteichoic acid. As a result of the experiment, it was confirmed that lipoteichoic acid derived from Apilactobacillus kosoi 10H strain has the effect of promoting the gene expression of IL-6, IL-10, and retinal dehydrogenase 2 (RALDH2) in bone marrow-derived dendritic cells.

[0055] From the above results, lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus, particularly lipoteichoic acid derived from lactic acid bacteria belonging to Apilactobacillus kosoi, and more specifically, lipoteichoic acid derived from Apilactobacillus kosoi 10H strain, can be expected as a gene expression promoter that promotes the expression of IL-6, IL-10, and retinal dehydrogenase 2 (RALDH2) in dendritic cells.

[0056] [Example 5] Analysis of the glycerophosphate chain of lipoteichoic acid Analysis of the glycerophosphate chain (repeating structure, polymer site) of lipoteichoic acid was performed 1 by obtaining an 1H-NMR spectrum. First, the lipoteichoic acid obtained in Example 2 was dissolved in 0.6 ml of 99.8% D2O (purchased from Fujifilm Wako Pure Chemical Corporation). 1 The 1H-NMR spectrum was obtained using a Varian Unity Inova 500 spectrometer (manufactured by Agilent Technologies) at 500 MHz under the condition of 25°C. As a reference substance for chemical shift, sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (purchased from Fujifilm Wako Pure Chemical Corporation) was used.

[0057] The results are shown in FIGS. 3 and 4. First, an explanation will be given based on the structure of the glycerophosphate chain in lipoteichoic acid derived from lactic acid bacteria other than lactic acid bacteria belonging to the genus Apilactobacillus.

[0058] Regarding the lipoteichoic acid derived from Lactiplantibacillus plantarum JCM1149 strain, each peak was assigned with reference to a past paper (Hatano et al. Scavenger receptor for lipoteichoic acid is involved in the potent ability of Lactobacillus plantarum strain L-137 to stimulate production of interleukin-12p40. International Immunopharmacology, 25: 321-331, 2015) (see FIG. 3(b)). The lipoteichoic acid derived from Lactiplantibacillus plantarum JCM1149 strain is considered to have a glycerophosphate chain composed of GroP units, AlaGroP units, and GlcGroP units.

[0059] Regarding the lipoteichoic acid derived from Lactiplantibacillus rhamnosus GG strain, each peak was also assigned with reference to a past paper (Claes et al. Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG. Microbial Cell Factories, 11: 161-168, 2012) (see FIG. 3(c)). The Lactiplantibacillus rhamnosus GG strain is considered to have a glycerophosphate chain composed of GroP units and AlaGroP units.

[0060] The structure of the glycerophosphate chain in the lipoteichoic acid derived from the above-mentioned Lactiplantibacillus plantarum JCM1149 strain and Lactiplantibacillus rhamnosus GG strain is a structure commonly seen as lipoteichoic acid in the former genus Lactobacillus lactic acid bacteria.

[0061] On the other hand, in the lipoteichoic acid derived from Apilactobacillus kuroiwae 10H strain, 1 the 1H-NMR spectrum is clearly different from that of the lipoteichoic acid derived from Lactiplantibacillus plantarum JCM1149 strain and Lactiplantibacillus rhamnosus GG strain (see Fig. 3(a)). The assignment of each peak is currently in progress. Considering the results of 13C-NMR and two-dimensional NMR (not shown), the lipoteichoic acid derived from Apilactobacillus kuroiwae 10H strain is likely to have a GlcGroP unit. However, since there are many other unknown peaks, it is planned to clarify them by compositional analysis and the like in the future. At least, the glycerol phosphate chain in the lipoteichoic acid derived from Apilactobacillus kuroiwae 10H strain is considered to have a unique structure that is not a general structure. 1 13 13 13

[0062] Also, in the lipoteichoic acid derived from Apilactobacillus kunkeei JCM16173 strain, 1 the 1H-NMR spectrum (see Fig. 4(b)) and the 1H-NMR spectrum in the lipoteichoic acid derived from Apilactobacillus apinorum JCM30765 strain 1 also showed results quite similar to the 1H-NMR spectrum in the lipoteichoic acid derived from Apilactobacillus kuroiwae 10H strain (see Fig. 4(a). Note that Fig. 4(a) is the same as Fig. 3(a)). Therefore, the glycerol phosphate chain in the lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus is quite different from the glycerol phosphate chain in the general lipoteichoic acid derived from lactic acid bacteria of the former genus Lactobacillus, and is considered to have a structure that is somewhat common within the genus. 1 1

[0063] [Example 6] Analysis of the anchor glycolipid of lipoteichoic acid The analysis of the anchor glycolipid of lipoteichoic acid was carried out by obtaining the MALDI-TOF MS spectrum.

[0064] (Isolation of anchor glycolipid) First, 100 μg of lipoteichoic acid was collected in a PP tube. Next, 0.1 ml of 48% (w / v) hydrofluoric acid was added and left standing at 4°C for 3 hours. After removing the hydrofluoric acid by nitrogen gas blowing in a draft, 1 ml of chloroform, 1 ml of methanol, and 0.9 ml of water were added, stirred well, and then centrifuged (20°C, 200×g, 30 seconds), and the lower organic layer was recovered. The organic solvent was removed by nitrogen gas blowing in a draft to obtain the anchor glycolipid of lipoteichoic acid.

[0065] (Obtaining MALDI-TOF MS spectrum) The anchor glycolipid was dissolved in 100 μl of chloroform / methanol (2:1, v / v), and further mixed with the same amount of matrix agent (a water / methanol (7:3, v / v) solution of 10 mg / ml 2,5-dihydroxybenzoic acid (DHBA) containing 0.1% trifluoroacetic acid (TFA)) on a target plate. After the mixture was co-crystallized, the MALDI-TOF mass spectrum was obtained in positive ion mode and reflectron mode. As the mass spectrometer, TOF / TOF 5800 system (manufactured by AB SCIEX) was used.

[0066] The results are shown in FIGS. 5 and 6. First, an explanation will be given based on the structure of the anchor glycolipid in lipoteichoic acid derived from lactic acid bacteria other than Lactobacillus apilactobacillus.

[0067] The main structure of the anchor glycolipid in lipoteichoic acid derived from Lactiplantibacillus plantarum JCM1149 strain is considered to be Hex3DAG, that is, a structure in which diacylglycerol is bound to a trisaccharide (see Fig. 5(b)). Also, although the peak intensity is weak and hidden in the background, there is a peak that is considered to be due to AcylHex3DAG. Furthermore, there is a peak (956) that is considered to be due to the structure of Hex2DAG, that is, diacylglycerol bound to a disaccharide, but this peak also has weak intensity and it is considered that it cannot be said for sure that it definitely exists.

[0068] The main structure of the anchor glycolipid in lipoteichoic acid derived from Lactobacillus rhamnosus GG strain is thought to be Hex3DAG (see Fig. 5(c)). For the peak thought to be caused by AcylHex3DAG, only one peak (1368) was observed and the peak was weak, so the anchor glycolipid in lipoteichoic acid derived from Lactobacillus rhamnosus GG strain may not have AcylHex3DAG. For the peak thought to be caused by Hex2DAG, it exists as in the case of Lactiplantibacillus plantarum JCM1149 strain (942, 956), but again the peak intensity is weak.

[0069] In any case, the structure of the anchor glycolipid in lipoteichoic acid derived from the above-mentioned Lactiplantibacillus plantarum JCM1149 strain and Lactobacillus rhamnosus GG strain is thought to be Hex3DAG. The trisaccharide anchor glycolipid is a structure commonly found as lipoteichoic acid in lactic acid bacteria of the former genus Lactobacillus.

[0070] On the one hand, the main structure of the anchor glycolipid in lipoteichoic acid derived from Apilactobacillus kuroiwae 10H strain is considered to be Hex2DAG(942,956,982) (see Fig. 5(a)). The diglycosyl anchor glycolipid is a structure commonly found in lipoteichoic acid derived from lactic acid bacteria of the genera Enterococcus, Lactococcus, and Leuconostock among lactic acid bacteria. On the other hand, the generally observed anchor glycolipids in lipoteichoic acid derived from former Lactobacillus genus lactic acid bacteria are trisaccharide or tetrasaccharide anchor glycolipids. However, although Apilactobacillus genus lactic acid bacteria belong to the former Lactobacillus genus, it was confirmed that the anchor glycolipid in lipoteichoic acid is mainly diglycosyl. In addition, the anchor glycolipid in lipoteichoic acid derived from Apilactobacillus kuroiwae 10H strain does not have an anchor glycolipid to which a three-residue fatty acid is bound, which is commonly found in lipoteichoic acid derived from many former Lactobacillus genus lactic acid bacteria and Lactococcus genus lactic acid bacteria. Therefore, the structure of the anchor glycolipid in lipoteichoic acid derived from Apilactobacillus kuroiwae 10H strain is characterized by being significantly different from the structure of the anchor glycolipid common to representative lactic acid bacteria revealed so far.

[0071] In addition, the MALDI-TOF MS spectrum of the anchor glycolipid in lipoteichoic acid derived from Apilactobacillus kunkeei JCM16173 strain (see Fig. 6(b)) and the MALDI-TOF MS spectrum of the anchor glycolipid in lipoteichoic acid derived from Apilactobacillus apinorum JCM30765 strain (see Fig. 6(c)) also showed similar results in that they have Hex2DAG as the main structure to the MALDI-TOF MS spectrum of the anchor glycolipid in lipoteichoic acid derived from Apilactobacillus kuroiwae 10H strain (see Fig. 6(a). Note that Fig. 6(a) is the same as Fig. 5(a)). Therefore, it is considered that the anchor glycolipid in lipoteichoic acid derived from Apilactobacillus genus lactic acid bacteria is quite different from the anchor glycolipid in general lipoteichoic acid derived from former Lactobacillus genus lactic acid bacteria and has a structure that is somewhat common within the genus.

[0072] From Examples 5 and 6 above, it was found that lipoteichoic acid derived from Lactobacillus apilactobacillus has a structure that is entirely different from that of general lipoteichoic acid derived from the former Lactobacillus genus. The difference in the structure between lipoteichoic acid derived from Lactobacillus apilactobacillus and general lipoteichoic acid derived from the former Lactobacillus genus is considered to be related to the high IgA production-inducing ability exhibited by lipoteichoic acid derived from Lactobacillus apilactobacillus.

Claims

1. An intestinal immune activator containing, as an active ingredient, purified lipoteichoic acid derived from lactic acid bacteria belonging to the genus Apilactobacillus.

2. The intestinal immune activator according to claim 1, wherein the lactic acid bacteria belonging to the genus Apilactobacillus are lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum.

3. The intestinal immune activator according to claim 2, wherein the lactic acid bacteria belonging to the genus Apilactobacillus are Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum JCM30765 strain.

4. The intestinal immune activator according to any one of claims 1 to 3, which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be formulated therein.

5. An IgA production promoter containing, as an active ingredient, purified lipoteichoic acid derived from lactic acid bacteria belonging to the genus Apilactobacillus.

6. The IgA production promoter according to claim 5, wherein the lactic acid bacteria belonging to the genus Apilactobacillus are lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum.

7. The IgA production promoter according to claim 6, wherein the lactic acid bacteria belonging to the genus Apilactobacillus are Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum JCM30765 strain.

8. The IgA production promoter according to any one of claims 5 to 7, which is in the form of a food or drink, a pharmaceutical product, a feed, or an active ingredient composition to be formulated therein.

9. A gene expression promoter containing purified lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus as an active ingredient, which promotes the expression of at least one factor among IL-10 and retinal dehydrogenase 2 (RALDH2) in dendritic cells.

10. The gene expression promoter according to claim 9, wherein the lactic acid bacterium of the genus Apilactobacillus belongs to Apilactobacillus kosoi.

11. The gene expression promoter according to claim 10, wherein the lactic acid bacterium of the genus Apilactobacillus is Apilactobacillus kosoi strain 10H.

12. The gene expression promoter according to any one of claims 9 to 11, which is in the form of a food or drink, a pharmaceutical, a feed, or an active ingredient composition to be formulated therein.

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