Intestinal immunity stimulant and IgA production promoter

Abstract

To provide novel uses based on the novel properties of lactic acid bacteria of the genus Apilactobacillus.SOLUTION: Provided is an intestinal immunostimulant and IgA production promoter containing bacterial cells of lactic acid bacteria of the genus Apilactobacillus (Apilactobacillus kosoi) and Apilactobacillus kunkeei (excluding Apilactobacillus kunkeei), as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an intestinal immunity activator and an IgA production promoter comprising cells of lactic acid bacteria of the genus Apilactobacillus as an active ingredient. [Background technology]

[0002] 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 kunkeii have a high IgA production promoting effect.

[0003] The applicant has also discovered that Lactobacillus kosoi 10H strain, a lactic acid bacterium isolated from vegetable brown sugar fermentation liquid, is a fructophilic lactic acid bacterium with a novel genome structure distinct from other lactic acid bacteria, and that it has excellent IgA production promoting activity (and thus immunostimulating activity) (see Patent Document 2. Note that "Lactobacillus kosoi" in Patent Document 2 refers to the same thing as "Lactobacillus kosoi" in this specification.). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2014-73130 A [Patent Document 2] JP 2020-92704 A Summary of the Invention [Problem to be solved by the invention]

[0005] By the way, although the classification of Lactobacillus has been used for a long time, it has been pointed out that the diversity of lineages and the physiological and biochemical characteristics of different species are significantly different.For this reason, the classification of Lactobacillus has been reevaluated at the genome level in recent years.For example, the above-mentioned Lactobacillus kunkeei and Lactobacillus kosoi have been reclassified into the Apilactobacillus genus after reevaluation, and their scientific names are Apilactobacillus kunkeei and Apilactobacillus kosoi, respectively.

[0006] As mentioned above, it is known that certain species of Apiractobacillus lactic acid bacteria have the effect of promoting IgA production (and thus immunostimulating effect), but it is not known whether this is a general property of Apiractobacillus lactic acid bacteria.

[0007] The present invention has been made in view of the above circumstances, and an objective of the present invention is to clarify the novel properties of Apilactobacillus lactic acid bacteria and to provide new uses based on these properties. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors have intensively investigated new usefulness of Apiractobacillus lactic acid bacteria, and have found that Apiractobacillus lactic acid bacteria other than specific species have IgA production promoting action and immunostimulating action, thereby completing the present invention. That is, the present invention includes the following embodiments.

[0009] (1) An intestinal immune stimulant containing the bacterial cells of Apilactobacillus lactic acid bacteria (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei) as an active ingredient. (2) The intestinal immune stimulator according to (1), wherein the lactic acid bacteria of the genus Apilactobacillus are lactic acid bacteria belonging to Apilactobacillus apinorum. (3) The intestinal immune stimulator according to (2), wherein the lactic acid bacterium of the genus Apilactobacillus is Apilactobacillus apinorum JCM30765 strain. (4) The intestinal immunostimulant according to any one of (1) to (3), which is in the form of a food or drink, a medicine, a feed, or an active ingredient composition to be incorporated therein. (5) An IgA production promoter containing the bacterial cells of Apilactobacillus lactic acid bacteria (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei) as an active ingredient. (6) The IgA production promoter according to (5), wherein the lactic acid bacteria of the genus Apilactobacillus are lactic acid bacteria belonging to Apilactobacillus apinorum. (7) The IgA production promoter according to (6), wherein the lactic acid bacterium of the genus Apilactobacillus is 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 medicine, a feed, or an active ingredient composition to be mixed therewith. (9) Use of cells of Apilactobacillus lactic acid bacteria (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei) for the manufacture of medicines, foods, beverages, feed, or active ingredient compositions to be incorporated therein for stimulating intestinal immunity or promoting IgA production in humans or non-human animals. Effect of the Invention

[0010] According to the present invention, as a novel use of the bacterial cells of Apilactobacillus lactic acid bacteria (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei), it is possible to provide an intestinal immune stimulant and an IgA production promoter containing the bacterial cells. [Brief description of the drawings]

[0011] [Figure 1] 1 is a graph showing a phylogenetic tree based on the 16S rRNA gene sequences of the lactic acid bacteria used in the experiment and the ability to induce IgA production. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0013] (I) Lactic acid bacteria as active ingredient The active ingredient in one embodiment of the present invention is a bacterial cell of Apilactobacillus lactic acid bacteria (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei). Preferably, the Apilactobacillus lactic acid bacteria is a lactic acid bacterium belonging to Apilactobacillus apinorum. More preferably, the Apilactobacillus lactic acid bacteria is Apilactobacillus apinorum JCM30765 strain.

[0014] Known examples of Apilactobacillus lactic acid bacteria include Apilactobacillus kosoi, Apilactobacillus kunkeei, and Apilactobacillus apinorum, as well as Apilactobacillus micheneri, Apilactobacillus ozensis, Apilactobacillus quenuiae, and Apilactobacillus timberlakei.

[0015] Apilactobacillus lactic acid bacteria are a group of lactic acid bacteria that were formerly known as the Lactobacillus Kunkii group among the genus Lactobacillus. Apilactobacillus lactic acid bacteria are gram-positive, rod-shaped, heterofermentative, and generally occur in the 15 ~ They grow in the range of 37°C, and many grow even under acidic conditions of less than pH 3.0. The genome size of Apilactobacillus lactic acid bacteria is relatively small, about 1.42 to 1.58 Mbp. The G+C content in DNA is in the range of 30.5 to 36.4. Apilactobacillus lactic acid bacteria convert fructose to mannitol. In addition, they can usually metabolize fructose, glucose, and sucrose, but cannot metabolize maltose and pentose (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] A. apinorum is a lactic acid bacterium isolated from the honey stomach of honeybees. The A. apinorum JCM30765 strain is the type strain of A. apinorum, and is the same as the Fhon13N strain, the DSM26257 strain, and the CCUG63287 strain. This lactic acid bacterium is well known, so detailed descriptions of its properties are omitted.

[0017] (II) IgA production promoters and intestinal immune activators In the present specification, the term "IgA production promoter" refers to a substance that has the ability to induce IgA production, such that when the substance is added to a culture solution of Peyer's patch cells containing a large amount of IgA-producing cells and cultured for a predetermined period of time, the amount of secretory IgA secreted into the culture solution after the culture is increased compared to when the substance is not added. As described in detail below, the IgA production promoter of the present invention includes the forms of food and drink, medicine, feed, and active ingredient composition. For example, when administered together with a vaccine, the IgA production promoter can enhance the production of antibodies corresponding to the antigen contained in the vaccine, enhance the effect of the vaccine, and is highly likely to suppress side effects of the vaccine. That is, the IgA production promoter enhances the production of antibodies against the antigen contained in the vaccine, and favorably induces protective immunity, thereby enhancing the effect of the vaccine.

[0018] When the IgA production promoter of the present embodiment is used together with a vaccine, the IgA production promoter can be used as a vaccine effect enhancer that enhances the effect by administering it before or after vaccine administration. The amount of the IgA production promoter used varies depending on the type and quality of the vaccine used, or the age and symptoms of the recipient, but for example, for preventive use, the amount is about 0.01 to 10 g in terms of solid content per dose for adults, and it is desirable to take it three times a day about 30 minutes before meals.

[0019] In addition, in this specification, the term "intestinal immune activator" means an agent that promotes the secretion of IgA in the mucosal epithelium of the intestinal tract and is effective in activating the immune system of the host. As described in detail below, the intestinal immune activator of the present invention includes the forms of food and drink, pharmaceuticals, feed, and active ingredient compositions. Among these, health foods are preferred, and food compositions for maintaining and promoting the health of subjects with weakened immune systems are particularly preferred. When used as a health food, it is appropriate to use an amount that does not adversely affect the taste or appearance of the food.

[0020] (III) Foods, beverages, medicines, feeds, or compositions containing active ingredients therein (Active ingredient composition) The intestinal immunity activator and IgA production promoter of the present embodiment can be used in the form of a food or drink, a medicine, a feed, or an active ingredient composition to be blended therewith. When used as an active ingredient composition, the active ingredient, Apilactobacillus lactic acid bacteria cells, are cultured according to a standard method for culturing lactic acid bacteria, and the resultant culture is separated by a bacteria collection means such as centrifugation and the like, and the resultant is used as is. In addition, the culture / fermentation liquid (including culture supernatant) of lactic acid bacteria, a crude or purified product of the culture, a freeze-dried product thereof, or a cytoplasm or cell wall fraction obtained by treating the cells with an enzyme or physical means can also be used.

[0021] A culture solution containing the lactic acid bacteria as an active ingredient can be obtained, for example, by culturing the lactic acid bacteria as an active ingredient in a medium suitable for the lactic acid bacteria, such as MRS medium, at 18 to 39°C for about 16 to 48 hours. After the culture, the cultured cells can be obtained, for example, by centrifuging the culture solution at 3,000 rpm, 4°C, for 10 minutes to collect the cells. These can be purified according to a conventional method. Furthermore, the cells can be dried by freeze drying, spray drying, vacuum drying, drum drying, etc.

[0022] The bacteria may be not only live bacteria, but also killed bacteria sterilized by a normal general heat sterilization operation. Heat treatment reduces the immune-inducing activity derived from lactic acid bacteria, such as proteinaceous components and nucleic acids that are susceptible to heat denaturation. However, as is clear from the results of Example 3 described below, the IgA production-inducing ability of Apilactobacillus lactic acid bacteria is not attenuated by heat treatment, so the immune-enhancing components contained in Apilactobacillus lactic acid bacteria are considered to be heat-resistant. Heat treatment is preferably performed at 75°C for 1 minute or more, and more preferably at 85°C for 1 minute or more. Even if the bacteria are heat-treated, not only can the immune-activating effect due to the IgA production-inducing ability be expected, but live bacteria may undergo morphological changes during delivery or display after product production, so killed bacteria that do not undergo further morphological changes can be preferably used.

[0023] The active ingredient composition of this embodiment may further contain an appropriate amount of nutritional components suitable for the maintenance, proliferation, etc. of Apilactobacillus lactic acid bacteria, if necessary. Specific examples of the nutritional components include carbon sources such as fructose, glucose, sorbose, ribose, lyxose, xylose, arabinose, lactulose, sucrose, etc., nitrogen sources such as yeast extract, peptone, etc., vitamins, minerals, trace metal elements, other nutritional components, etc., which are used in culture media for culturing microorganisms. Examples of vitamins include vitamin B, vitamin D, vitamin C, vitamin E, vitamin K, etc. Examples of trace metal elements include zinc, selenium, etc. Examples of other nutritional components include various oligosaccharides such as lactose oligosaccharides, soybean oligosaccharides, lactulose, lactitol, fructooligosaccharides, and galactooligosaccharides. The amount of these oligosaccharides to be added is not particularly limited, but is preferably selected from the range of about 1 to 30% by weight in the composition of this embodiment.

[0024] The amount of Apilactobacillus lactic acid bacteria to be blended in the active ingredient composition of this embodiment is generally set to 10 8 ~10 13The amount of the active ingredient composition of the present embodiment can be appropriately selected from the range of amounts that will result in about 1000 viable bacteria (not necessarily the number of viable bacteria). The viable bacteria count can be measured, for example, by a limiting dilution culture method using a fructose-added MRS liquid medium. In this case, since there is a correlation between the viable bacteria count and the turbidity, if the correlation between the viable bacteria count and the turbidity is determined in advance, the viable bacteria count can be counted by measuring the turbidity instead of measuring the viable bacteria count. The active ingredient composition of the present embodiment is preferably prepared in the form of a food or drink, a pharmaceutical product, etc., as described below, through appropriate blending of an appropriate edible carrier (food material), a pharma- ceutical acceptable carrier, etc.

[0025] (Drugs) When the intestinal immunity activator or IgA production promoter of the present embodiment is in the form of a pharmaceutical product, it is prepared in the form of a pharmaceutical composition using a suitable pharmaceutical acceptable carrier together with the Apilactobacillus lactic acid bacteria and used in practice. Examples of the pharmaceutical carrier include diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and lubricants that are commonly used in this field.

[0026] The dosage unit form of a pharmaceutical product may be selected from a variety of forms, and is preferably a formulation for oral administration, such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, etc.

[0027] When forming into a tablet form, the following may be used as pharmaceutical carriers: excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, and potassium phosphate; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone; sodium carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, dry starch, alginate, and the like. Disintegrants such as sodium phosphate, powdered agar, powdered laminaran, sodium hydrogen carbonate, calcium carbonate, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearic acid, etc.; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, etc.; moisturizers such as glycerin, starch, etc.; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, etc.; lubricants such as purified talc, stearates, powdered boric acid, polyethylene glycol, etc. Tablets can be made into tablets with a normal coating, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, and film-coated tablets, or can be made into double- or multi-layered tablets, if necessary.

[0028] When forming into pills, the following may be used as pharmaceutical carriers: excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oil, kaolin, talc, etc.; binders such as powdered gum arabic, powdered tragacanth, gelatin, ethanol, etc.; disintegrants such as laminaran, agar, etc.

[0029] Furthermore, the medicine may contain coloring agents, preservatives, fragrances, flavorings, sweeteners, and other medicines, if necessary.

[0030] There is no particular limit to the method of administration of the pharmaceutical of this embodiment, and it is determined according to the formulation form, the age, sex and other conditions of the patient, the degree of the disease, etc. The dosage is appropriately selected according to the method of use, the age, sex and other conditions of the patient, the degree of the disease, etc., but it is usually preferable that the active ingredient composition is about 0.5 to 100 mg per kg of body weight per day. The pharmaceutical can be administered to humans in 1 to 4 divided doses per day.

[0031] (food and drink) In this specification, "food and drink" includes all forms used orally exclusively for eating and drinking (including beverages, for example), and even if it is in the form of a tablet or the like, it is included in the food and drink of this specification as long as it is used exclusively for eating and drinking. For example, health foods, dietary supplements, foods for the sick, nutritional supplements, or health functional foods (specified health foods, nutritional functional foods) defined by the Ministry of Health, Labor and Welfare that are based on the concept of infection prevention or diarrhea prevention and labeled as such as necessary are also included in the food and drink of this specification. Health foods refer to foods intended for health, health maintenance, and promotion, in a more positive sense than regular foods.

[0032] When the intestinal immunity activator or IgA production promoter of this embodiment is used as a food or drink, examples of the food and drink include fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, and fermented soy milk beverages. "Fermented milk" refers to milk or dairy products fermented with lactic acid bacteria or yeast into a paste or liquid form. Thus, the fermented milk includes both beverage forms and yogurt forms. Furthermore, "lactic acid bacteria beverages" refer to beverages made by diluting milk or dairy products fermented with lactic acid bacteria or yeast into a paste or liquid form as the main ingredient with water.

[0033] Examples of other food and drink forms include fermented foods such as pickles, miso, fermented tea, and bread; foods for infants such as weaning food, powdered milk, and baby food; confectioneries such as foaming preparations, gums, gummies, and puddings; nutritional supplements such as noodles, capsules, granules, powders, and tablets; and dairy products other than the fermented milk and lactic acid bacteria beverages. Since the intestinal immune activator or IgA production promoter of this embodiment contains an active ingredient whose functionality is maintained even by heating, it can also be in the form of a processed food that requires a heating process. Particularly preferred forms are processed foods that require cooking for hygiene management, such as nursing care foods. The food and drink of this embodiment can provide an intestinal immune activator or IgA production promoter that is stable even when heated to 75°C, which is effective in preventing food poisoning.

[0034] The content of the active ingredient composition in the food and drink according to the present embodiment is not particularly limited and can be appropriately determined. From the viewpoint of exerting the effect of intestinal immunity activation or IgA production promotion, for example, it is preferably 0.001 mass% or more, more preferably 0.01 mass% or more, and more preferably 0.1 mass% or more, based on the total mass of each food and drink. Meanwhile, the upper limit of the content of the active ingredient composition in the food and drink is not particularly limited, and can usually be appropriately adjusted according to the form of the food and drink.

[0035] (feed) When the intestinal immune activator or IgA production promoter of this embodiment is in the form of feed, it can be made into a formulation form for oral administration (aqueous solution, emulsion, granules, powder, capsules, tablets, etc.) for preventing infectious diseases, for example, in chickens during the non-antibiotic administration period or in pigs, cows, etc. during the weaning period.

[0036] [Example] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. In the following examples, the unit % of the numerical values ​​showing the amounts of various components added means % by mass unless otherwise specified.

[0037] [Example 1] Acquisition of lactic acid bacteria and culture conditions In order to measure the ability of Apilactobacillus lactic acid bacteria to induce IgA production, Apilactobacillus apinorum JCM30765 strain, Apilactobacillus kosoi 10H strain, and Apilactobacillus kunkeii JCM16173 strain were prepared. In addition, to compare with the IgA production induction ability of Apilactobacillus lactic acid bacteria, lactic acid bacteria other than Apilactobacillus lactic acid bacteria, namely, Lactobacillus gasseri JCM1131 strain, Lactobacillus johnsonii JCM2012 strain, Lactobacillus delbrueckii subsp. bulgaricus JCM1002 strain, Lacticaseibacillus casei JCM1134 strain, Lacticaseibacillus rhamnosus JCM1136 strain, Lacticaseibacillus paracasei subsp. paracasei subsp. paracasei) strain JCM8130, Latilactobacillus sakei subsp. sakei) strain JCM1157, Latilactobacillus curvatus) strain JCM1096, Liquorilactobacillus mali) strain JCM1116, Liquorilactobacillus aquaticus) strain JCM16869, Liquorilactobacillus oeni) strain JCM18036, Ligilactobacillus salivarius) strain JCM1231, Ligilactobacillus animalis) strain JCM5670, Lactipranchibacillus Lactiplantibacillus plantarum subsp.plantarum strain JCM1149, Lactiplantibacillus pentosus strain JCM1558, Limosilactobacillus reuteri subsp. reuteri strain JCM1112, Limosilactobacillus fermentum strain JCM1173, Fructilactobacillus sanfranciscensis strain JCM5668, Lentilactobacillus buchneri strain JCM1115, Lentilactobacillus kefir strain JCM5818, Lactococcus lactis subsp. Lactococcus lactis subsp. lactis JCM5805 strain, Enterococcus durans JCM8725 strain, Enterococcus faecium JCM5804 strain, Enterococcus faecalis JCM5803 strain, Leuconostoc mesenteroides subsp. cremoris JCM16943 strain, Leuconostoc mesenteroides subsp. mesenteroides JCM6124 strain, and Lacticaseibacillus The Lactic Acid Bacteria GG strain (ATCC53103) was also prepared. All of the above lactic acid bacteria were type strains except for the Lactic Acid Bacteria GG strain (ATCC53103).

[0038] Of the above lactic acid bacteria, the Apilactobacillus kosoi 10H strain was a strain stored in the Matsuzaki Laboratory of Ishikawa Prefectural University, and the Lacticaceibacillus rhamnosus GG strain (ATCC53103) was obtained from the American Type Culture Collection (ATCC) in the United States. The rest were obtained from the Japan Microbial Materials Division of the RIKEN BioResource Research Center.

[0039] Each strain of lactic acid bacteria was cultured in MRS broth for Lactobacillus (Difco Laboratory) with some exceptions. A. apinorum JCM30765 and A. kousoi 10H were cultured in MRS broth for Lactobacillus supplemented with 10% fructose. A. kunkeii JCM16173 was cultured in MRS broth for Lactobacillus supplemented with 10% tomato juice and 0.05% L-cysteine ​​hydrochloride. F. sanfranciscensis JCM5668 was cultured in modified sourdough medium (2.0% maltose, 0.3% yeast extract, 0.03% polysorbate 80, and 0.6% trypticase peptone adjusted to pH 5.6).

[0040] Each strain was cultured at 30°C for 24 to 48 hours, and the cells were collected by centrifugation, washed with physiological saline, and then resuspended in physiological saline. A portion of the cells was then heat-treated at 70°C for 30 minutes.

[0041] [Example 2] Phylogenetic analysis of lactic acid bacteria The sequences of 16S rRNA genes of the 30 species of lactic acid bacteria used in the experiment were obtained from the National Center for Biotechnology Information (NCBI). These sequences were aligned using ClustalW to construct a phylogenetic tree. Evolutionary analysis was performed with MEGA X using the neighbor-joining method. Evolutionary history was inferred by the neighbor-joining method. Bootstrap analysis based on 1,000 replications was applied to evaluate the tree topology.

[0042] [Example 3] Measurement of IgA production induction ability (Preparation of Peyer's patch cells) Six-week-old male BALB / cA mice (purchased from CREA Japan) were fed a basal diet of AIN-76 diet (purchased from Research Diets), which 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] Mice were treated in accordance with the Guidelines for Proper Conduct of Animal Experiments published by the Science Council of Japan in 2006. After being kept for one week, the mice were euthanized with carbon dioxide gas, and Peyer's patches from the small intestine were removed by laparotomy.

[0044] Peyer's patches were placed in a petri dish filled with ice-cold RPMI 1640 medium (PSMF) [RPMI 1640 medium (Gibco BRL) containing 100 U / ml penicillin, 100 μg / ml streptomycin, 55 μmol / l 2-mercaptoethanol, and 10% heat inactivated fetal bovine serum (FBS; Gibco BRL)], washed three times with the medium, and then cultured for 45 minutes at 37°C in RPMI 1640 medium (PSMF) supplemented with 25 mmol / l HEPES, 5 mmol / l EDTA (pH 8.0), and 1 mmol / l dithiothreitol. The Peyer's patches were washed again with RPMI1640 medium (PSMF) containing 5 mmol / l EDTA (pH 8.0), and then treated with RPMI1640 medium (PSMF) containing 400 U / ml type I collagenase (Sigma) and 30 U / ml DNase I (Takara Bio Inc.) for 50 minutes at 37°C. The resulting mixture was filtered through a 40 μm nylon mesh and washed twice with RPMI1640 medium (PSMF) to obtain Peyer's patch cells to be used in measuring the IgA production induction ability. The viability of Peyer's patch cells was confirmed by trypan blue staining. The final concentration of Peyer's patch cells was 1.25 × 10 6The cells / ml were adjusted and used for evaluation.

[0045] (IgA measurement) The physiological saline suspension containing the live or killed bacteria obtained in Example 1 was measured for the final absorbance (OD 600 The cells were added to the suspension of Peyer's patch cells so that the IgA concentration in the culture supernatant was 0.01, and the cells were co-cultured in a 96-well T-cell activation plate (Becton Dickinson) for 5 days at 37°C and 5% CO2. The amount of IgA in the culture supernatant was then measured using a mouse IgA ELISA kit (Bethyl Laboratories).

[0046] Statistical analysis was performed using Excel Statistics (Social Information Services Co., Ltd.) Measurement results were analyzed using one-way ANOVA, followed by Dunnett's post-hoc analysis.

[0047] The results are shown in Figure 1. In Figure 1, lactic acid bacteria are arranged based on the phylogenetic tree constructed in Example 2, and the results for physiological saline (saline), which is a negative control, are placed at the top. In Figure 1, there are two bar graphs for each lactic acid bacteria strain, with the upper graph being a graph of the IgA production induction ability of non-heated bacteria (living bacteria), and the lower graph being a graph of the IgA production induction ability of bacteria heated at 70°C (killed bacteria). In Figure 1, bootstrap values ​​are shown at the branching points of the phylogenetic tree.

[0048] As a result of the experiment, it was confirmed that the Apilactobacillus lactic acid bacteria, i.e., Apilactobacillus apinorum JCM30765 strain, Apilactobacillus kosoi 10H strain, and Apilactobacillus kunkeii JCM16173 strain, had a clearly and significantly higher IgA production induction ability than other lactic acid bacteria. It was also confirmed that the IgA production induction ability of Apilactobacillus lactic acid bacteria was maintained even after heat-killing treatment.

[0049] The above results indicate that lactic acid bacteria of the genus Apilactobacillus, particularly lactic acid bacteria belonging to Apilactobacillus apinorum, and more specifically Apilactobacillus apinorum JCM30765 strain, are promising immunostimulants with high heat resistance.

Claims

1. An intestinal immune stimulant comprising the bacterial cells of Apilactobacillus apinorum JCM30765 strain as an active ingredient.

2. 2. The intestinal immunostimulant according to claim 1, which is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition to be incorporated therein.

3. An IgA production promoter comprising the bacterial cells of Apilactobacillus apinorum JCM30765 strain as an active ingredient.

4. The IgA production promoter according to claim 3, which is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition to be incorporated therein.

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