TW202335676A - Intestine immunostimulant and IgA generation promoter for ensuring Apilactobacillus lactobacillus having IgA generation promotion effect and immunostimulation function - Google Patents

Abstract

The subject of the invention is to clarify the novel properties contained by Apilactobacillus lactobacillus and provide new purposes based on the same. The intestine immunostimulant and IgA generation promoter according to the invention contains fungus of Apilactobacillus lactobacillus (except Apilactobacillus kosoi and Apilactobacillus kunkeei) as effective ingredients.

Description

Intestinal immune activator and IgA production promoter

The present invention relates to an intestinal immune activating agent and an IgA production accelerator containing bacteria of the genus Apilactobacillus lactic acid bacteria as active ingredients.

It has been previously known that lactic acid bacteria or fermentation products thereof have various physiological functions. For example, Patent Document 1 describes that lactic acid bacteria belonging to Lactobacillus kunstii have a high IgA production-promoting effect.

Furthermore, the applicant of this case discovered that Lactobacillus fermentum 10H strain, which is a lactic acid bacterium isolated from vegetable brown sugar fermentation broth, is a fructophilic lactic acid bacterium with a novel genome structure that is different from other lactic acid bacteria, and has excellent IgA production-promoting effects (and immune activating effect) (refer to Patent Document 2; in addition, "Lactobacillus kosoi" in Patent Document 2 means the same as "Lactobacillus kosoi" in this specification). [Prior technical literature] [Patent Document]

[Patent Document 1] Japanese Patent Application Publication No. 2014-73130 [Patent Document 2] Japanese Patent Application Publication No. 2020-92704

[Problem to be solved by the invention]

However, although the classification of Lactobacillus has been used for a long time, it was previously pointed out that the diversity of the system or the physiological and biochemical characteristics of the strains are very different. Therefore, in recent years, the genus Lactobacillus has been re-evaluated at the genome level. For example, the above-mentioned Lactobacillus kunkeei and Lactobacillus kosoi were reclassified to the genus Apilactobacillus after re-evaluation, and their scientific names became Apilactobacillus kunkeei and Lactobacillus kosoi respectively.

As mentioned above, it is known that specific strains of lactic acid bacteria of the genus Lactobacillus meli have an IgA production-promoting effect (and an immune stimulating effect), but it is not known whether this is a property of all lactic acid bacteria of the genus Lactobacillus meli.

The present invention was completed in view of the above-mentioned circumstances, and its object is to clarify the novel properties of lactic acid bacteria of the genus Lactobacillus api and provide new uses based on them. [Technical means to solve problems]

In order to solve the above-mentioned problems, the present inventors conducted intensive research on the new usefulness of lactic acid bacteria of the genus Lactobacillus api and found that lactic acid bacteria of the genus Lactobacillus api other than specific strains also have IgA production-promoting effects and immune-stimulating effects. Thus, the present invention was completed. That is, the present invention includes the following embodiments.

(1) An intestinal immune stimulating agent containing bacteria of the genus Lactobacillus (Apilactobacillus) (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei) as an active ingredient. (2) The intestinal immune stimulating agent according to (1), wherein the lactic acid bacteria belonging to the genus Apilactobacillus are lactic acid bacteria belonging to the genus Apilactobacillus apinorum. (3) The intestinal immunity stimulating agent according to (2), wherein the lactic acid bacterium of the genus Apilactobacillus is Apilactobacillus apinorum JCM30765 strain. (4) The intestinal immunity stimulating agent according to any one of (1) to (3), which is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition formulated therein. (5) An IgA production accelerator containing bacteria of the genus Lactobacillus (Apilactobacillus) (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei) as an active ingredient. (6) The IgA production accelerator according to (5), wherein the lactic acid bacterium belonging to the genus Apilactobacillus is a lactic acid bacterium belonging to the genus 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 (5) to (7), which is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition formulated therein. (9) Use of a bacterial cell of the genus Lactobacillus kosoi (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei), which is used to produce intestinal immune stimulation of humans or non-human animals Pharmaceuticals, foods, and feeds used to promote IgA production, or active ingredient compositions formulated therein. [Effects of the invention]

According to the present invention, as a novel use of the bacterial cells of the genus Lactobacillus (Apilactobacillus) (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei), a bacteria containing the bacterial cells can be provided. Intestinal immune activator and IgA production promoter.

Hereinafter, each embodiment of the present invention will be described with reference to the drawings. Furthermore, each embodiment described below does not limit the invention within the scope of the patent application, and not all elements and combinations thereof described in each embodiment are necessarily necessary for the solution of the present invention.

(I) Lactic acid bacteria as active ingredient The active ingredient in one embodiment of the present invention is bacteria of the genus Lactobacillus (Apilactobacillus) (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei). Preferred are lactic acid bacteria belonging to the genus Apilactobacillus, which are lactic acid bacteria belonging to the genus Apilactobacillus apinorum. Furthermore, it is more preferable that the lactic acid bacterium of the genus Apilactobacillus is Apilactobacillus apinorum JCM30765 strain.

As the lactic acid bacteria of the genus Lactobacillus api, in addition to Lactobacillus apiensis, Lactobacillus kunstii and Lactobacillus swarming, Apilactobacillus micheneri, Apilactobacillus ozensis, Apilactobacillus quenuiae and Apilactobacillus timberlakei.

Lactobacillus apiensis is a group of lactic acid bacteria called Lactobacillus kunstii group among the lactic acid bacteria of the genus Lactobacillus genus. Lactobacillus apiensis is a lactic acid bacterium with Gram-positive, rod-shaped, and heterozygous fermentation properties. It usually grows in the range of 15 to 37°C, and mostly grows under acidic conditions with a pH value less than 3.0. The genome size of Lactobacillus api lactic acid bacteria is about 1.42-1.58 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 Lactobacillus apiensis convert fructose into mannitol. In addition, although it can generally metabolize fructose, glucose and sucrose, it 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).

Lactobacillus apiensis is a lactic acid bacterium isolated from the honey stomach of honey bees. Furthermore, the type strain of Lactobacillus swarming L. apiensis JCM30765 is the same as the Fhon13N strain, the DSM26257 strain and the CCUG63287 strain. This lactic acid bacterium has been well known for a long time, so detailed description of its properties will be omitted.

(II) IgA production promoter and intestinal immune activator In this specification, "IgA production promoter" refers to the amount of secretory IgA secreted in the culture medium after culture for a specific period of time when added to the culture medium containing a large number of IgA-producing cells. Those who have the ability to induce IgA production will have an increased risk. The IgA production accelerator of the present invention is described in detail below and may be in the form of food, drink, medicine, feed, or active ingredient composition. For example, by administering an IgA production promoter together with a vaccine, the IgA production promoter can enhance the production of antibodies corresponding to the antigens contained in the vaccine, enhance the effectiveness of the vaccine, and have a high possibility of suppressing side effects of the vaccine. That is, it enhances the production of antibodies against the antigens contained in the vaccine, thereby improving the induction of defensive immunity and enhancing the effectiveness of the vaccine.

When the IgA production promoter of the present embodiment is used together with a vaccine, the IgA production promoter can be used as an effect enhancer of the vaccine that is administered before or after vaccine administration to improve the effect. The amount of IgA production promoter used varies depending on the type and quality of the vaccine used, or the age and symptoms of the recipient. For example, when used for prevention, for adults, it can be 0.01 to 10 per time in terms of solid content. g, ideally taken three times a day about 30 minutes before meals.

In addition, in this specification, "intestinal immune activating agent" means one that is effective in promoting the secretion of IgA in the intestinal mucosal epithelium and activating the host's immune mechanism. The intestinal immune stimulating agent of the present invention is described in detail below and includes the form of food, drink, medicine, feed, or active ingredient composition, etc. Among these, a health food is preferred, and a food composition for maintaining the health of a subject with reduced immunity is 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.

(III) Food, drink, medicine, feed, or active ingredient compositions formulated therein (active ingredient composition) The intestinal immunity stimulating agent and the IgA production promoter according to the present embodiment can be used in the form of food, drink, medicine, feed, or an active ingredient composition formulated therein. When used in the form of an active ingredient composition, not only the cells of the lactic acid bacteria of the genus Lactobacillus apiensis as the active ingredient can be cultured according to the usual method of culturing lactic acid bacteria, but also the culture obtained from the culture obtained by bacterial collection methods such as centrifugation. The isolated product can be used directly, and the culture of lactic acid bacteria, the fermentation liquid (including the culture supernatant), the crude purified product or purified product of the culture, the freeze-dried product, or the use of enzymes or physical methods can be used. The differentiated part of the cytoplasm or cell wall obtained by processing the bacterial cells.

The culture solution containing the active ingredient lactic acid bacteria can be obtained, for example, by using a culture medium suitable for the active ingredient lactic acid bacteria, such as an MRS medium, and culturing it at 18 to 39° C. for about 16 to 48 hours. The cultured bacterial cells can be obtained by centrifuging the culture solution at 3,000 rpm, 4°C, and 10 minutes for 10 minutes after culturing, and collecting the bacteria. These can be purified according to conventional methods. Furthermore, the bacterial cells can also be made into a dried product by freeze drying, spray drying, vacuum drying, drum drying, or the like.

In addition, the bacterial cells may be not only live bacterial cells but also dead bacterial cells sterilized by ordinary general heat sterilization operations. The immune induction activity derived from lactic acid bacteria such as proteinaceous components and nucleic acids that are susceptible to thermal denaturation is reduced by heat treatment. However, according to the results of Example 3 described below, it is also known that the IgA production induction ability of lactic acid bacteria of the genus Lactobacillus apiensis is even after heat treatment. It will not attenuate, so it is believed that the immune-enhancing ingredients contained in Lactobacillus api lactic acid bacteria are heat-resistant. The heat treatment is preferably at 75°C for 1 minute or more, more preferably at 85°C for 1 minute or more. Even if the bacteria are heat-treated, an immune stimulating effect based on the ability to induce IgA can be expected. Not only that, but also in the case of raw bacteria, there is a possibility that morphological changes will occur during distribution or display after the product is manufactured. Therefore, dead bacterial cells that do not undergo further morphological changes can be suitably used.

The active ingredient composition of this embodiment may optionally contain an appropriate amount of nutrients suitable for the maintenance, growth, etc. of lactic acid bacteria of the genus Lactobacillus api. Specific examples of the nutritional components include carbon sources such as fructose, glucose, sorbose, ribose, lyxose, xylose, arabinose, lactulose, and sucrose used in culture media for culturing microorganisms. For example, yeast extract, proteinaceous and other nitrogen sources, vitamins, minerals, trace metal elements, other nutrients and other ingredients. Examples of vitamins include vitamin B, vitamin D, vitamin C, vitamin E, vitamin K, and the like. Examples of trace metal elements include zinc, selenium, and the like. Examples of other nutritional components include various oligosaccharides such as lactulooligosaccharide, soybean oligosaccharide, lactulose, lactitol, fructooligosaccharide, and galactooligosaccharide. The compounding amount of these oligosaccharides is not particularly limited, but is generally preferably selected from the range of approximately 1 to 30% by weight in the composition of the present embodiment.

The amount of lactic acid bacteria of the genus Lactobacillus api that is added to the active ingredient composition of the present embodiment can usually be appropriately selected from an amount such that the number of bacteria in 100 g of the composition is about 10 ⁸ to 10 ¹³ (it does not need to be the number of biotic bacteria). The bacterial count can be measured, for example, by a limiting dilution culture method using an MRS liquid medium supplemented with fructose. In this case, since the number of bacteria is related to the turbidity, if the correlation between the number of bacteria and the turbidity is determined in advance, the number of bacteria can be counted by measuring the turbidity instead of measuring the number of bacteria. The active ingredient composition of the present embodiment is preferably prepared in the form of foods, drinks, pharmaceuticals, etc. as described below by appropriately blending a suitable edible carrier (food material), a pharmaceutically acceptable carrier, etc.

(Pharmaceuticals) When the intestinal immunity stimulating agent or the IgA production accelerator of the present embodiment is in the form of a pharmaceutical, a pharmaceutical composition is prepared by using an appropriate preparation carrier acceptable in pharmaceuticals together with lactic acid bacteria of the genus Lactobacillus meli. The form is actually used. Examples of the preparation carrier include diluents or excipients such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, and lubricants commonly used in this field.

As the dosage unit form of the pharmaceutical, various forms can be selected, and a preferred example is a preparation for oral administration. Representative oral administration preparations include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, and the like.

When it is formed into a tablet, excipients such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. can be used as the preparation carrier; water , ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone and other binding agents; carboxymethylcellulose sodium , carboxymethyl cellulose calcium, low-substitution hydroxypropyl cellulose, dry starch, sodium alginate, agar powder, lamina sugar powder, sodium bicarbonate, calcium carbonate and other disintegrants; polyoxyethylene sorbitan fatty acid ester Surfactants such as surfactants, sodium lauryl sulfate, monoglyceryl stearate, etc.; disintegration inhibitors such as sugar, stearin, cocoa butter, hydrogenated oil, etc.; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate, etc.; glycerin, Starch and other humectants; starch, lactose, kaolin, bentonite, colloidal silicic acid and other adsorbents; refined talc, stearate, boric acid powder, polyethylene glycol and other lubricants. Tablets may be coated with a conventional coating as needed, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, or double-layered tablets or multi-layered tablets.

When forming into pills, as preparation carriers, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc can be used; combined with gum arabic powder, tragacanth powder, gelatin, ethanol, etc. Agents; disintegrating agents such as lamina sugar, agar, etc.

Furthermore, pharmaceuticals may optionally contain coloring agents, preservatives, spices, flavoring agents, sweeteners, etc. or other pharmaceuticals.

The method of administering the pharmaceutical according to this embodiment is not particularly limited and is determined based on the form of the preparation, conditions such as age and gender of the patient, the degree of the disease, and the like. In addition, the dosage is appropriately determined depending on the usage, conditions such as age and gender of the patient, the degree of the disease, etc. Generally, the active ingredient composition can be about 0.5 to 100 mg per day per kg of body weight. Pharmaceuticals can be administered to humans 1 to 4 times a day.

(Food and drink) The term "food and drink" in this manual includes all forms (for example, drinks) that are used orally specifically for eating and drinking. Even if they are in the form of tablets, etc., as long as they are specifically used for eating and drinking, they are also included in the food and drink in this specification. Good quality. For example, health foods, health supplements, patient foods, nutritional supplements, or health functional foods (foods for specific health uses, foods for specific health uses, Nutritional functional foods) are also included in the food and beverages in this manual. Health food refers to food with a more positive meaning than ordinary food for the purpose of health care, maintenance/enhancement of health, etc.

When the intestinal immune stimulating agent or IgA production accelerator of the present embodiment is used as a food or drink, examples thereof include fermented milk, lactic acid bacteria beverage, fermented vegetable beverage, fermented fruit beverage, fermented soy milk beverage, and the like. "Fermented milk" refers to a paste or liquid obtained by fermenting milk or dairy products using lactic acid bacteria or yeast. Therefore, this fermented milk includes a beverage form and a yogurt form. In addition, "lactic acid bacteria drink" refers to a drink obtained by diluting a paste or liquid obtained by fermenting milk or dairy products with lactic acid bacteria or yeast as the main raw material.

Examples of other food and drink forms include fermented foods such as pickles, bean paste, fermented tea, and bread, foods for infants and young children such as weaning foods, milk powder, and baby food, foaming preparations, chewing gum, gummy candies, and puddings Nutritional supplements such as snacks, noodles, capsules, granules, powders, and tablets, and dairy products other than the above-mentioned fermented milk and lactic acid bacteria drinks. The intestinal immune stimulating agent or the IgA production promoter according to the present embodiment contains an active ingredient that maintains functionality even after being heated, so it can also be in the form of a processed food that requires a heating step. A particularly preferred form is processed food that requires heating for hygienic management, such as nursing food. The food and drink of this embodiment can provide an intestinal immune activator or an IgA production promoter that is stable even when heated at 75°C, which is effective in preventing food poisoning.

The content of the active ingredient composition in the food and drink of this embodiment is not particularly limited and can be determined appropriately. From the viewpoint of exerting the effect of activating intestinal immunity or promoting IgA production, for example, it is preferably 0.001 mass% or more, more preferably 0.01 mass% or more, and still more preferably 0.1 mass% or more relative to the total mass of each food and drink. On the other hand, the upper limit of the content of the active ingredient composition in food and beverages is not particularly limited and can usually be appropriately adjusted according to the form of the food and beverages.

(feed) When the intestinal immune stimulating agent or IgA production promoter of the present embodiment is in the form of feed, it can be prepared for preventing infectious diseases during the non-antibiotic administration period of chickens or the weaning period of pigs, cows, etc., for example. In the form of preparations for oral administration (aqueous solution, emulsion, granules, powder, capsules, tablets, etc.).

[Example] Hereinafter, the present invention will be described in further detail with reference to examples, but the present invention is not limited by these examples in any way. In addition, in the following examples, the unit % which represents the numerical value such as the addition amount of each component means mass % unless otherwise stated.

[Example 1] Acquisition and culture conditions of lactic acid bacteria In order to measure the IgA production inducing ability produced by the lactic acid bacteria of the genus Lactobacillus api, the swarming Lactobacillus apis strain JCM30765, the enzyme Lactobacillus apis 10H strain and the Lactobacillus kunstii strain JCM16173 were prepared. In addition, in order to compare with the IgA production inducing ability of lactic acid bacteria of the genus Lactobacillus api, lactic acid bacteria other than the genus Lactobacillus api were also prepared, namely, Lactobacillus gasseri JCM1131 strain and Lactobacillus johnsonii JCM2012 strain. , Lactobacillus delbrueckii subsp. bulgaricus JCM1002, Lacticaseibacillus casei JCM1134, Lacticaseibacillus rhamnosus JCM1136, Lacticaseibacillus casei subsp. paracasei subsp. paracasei) JCM8130 strain, Latilactobacillus sakei subsp. sakei JCM1157 strain, Latilactobacillus curvatus JCM1096 strain, Apple liquid Lactobacillus (Liquorilactobacillus mali) JCM1116 strain, aqueous liquid emulsion Liquorilactobacillus aquaticus JCM16869, Liquorilactobacillus oeni JCM18036, Ligilactobacillus salivarius JCM1231, Ligilactobacillus animalis JCM5670, Lactiplantibacillus plantarum subsp. plantarum) JCM1149 strain, Lactiplantibacillus pentosus JCM1558 strain, Limosilactobacillus reuteri subsp. reuteri strain JCM1112, Limosilactobacillus fermentum JCM1173 strain, Fructilactobacillus sanfranciscensis JCM5668 strain, Lentilactobacillus buchneri strain JCM1115, Lentilactobacillus kefir JCM5818 strain, Lactococcus lactis subsp. lactis )JCM5805 strain, Enterococcus durans strain JCM8725, Enterococcus faecium strain JCM5804, Enterococcus faecalis strain JCM5803, Leuconostoc mesenteroides subsp. cremoris JCM16943 strain, Leuconostoc mesenteroides subsp. mesenteroides strain JCM6124 and Lacticaseibacillus rhamnosus GG strain (ATCC53103). In addition, the above-mentioned lactic acid bacteria except Lactobacillus rhamnosus GG strain (ATCC53103) are type strains.

Among the above-mentioned lactic acid bacteria, Lactobacillus apis 10H strain is a strain kept by the Matsuzaki Laboratory of Ishikawa Prefectural University, and Lactobacillus rhamnosus GG strain (ATCC53103) is a strain obtained from the American Type Culture Collection (ATCC). For external use, it was obtained from the Microbial Materials Development Laboratory (JCM), Bioresources Research Center, National Research Institute of RIKEN, Japan.

Each strain of lactic acid bacteria was cultured using Lactobacillus MRS culture medium (Difco Laboratories), with some exceptions. Colonial Lactobacillus apis strain JCM30765 and enzyme Lactobacillus apiensis 10H strain were cultured using Lactobacillus MRS culture medium supplemented with 10% fructose. Lactobacillus kunstii JCM16173 strain was cultured using Lactobacillus MRS culture medium supplemented with 10% tomato juice and 0.05% L-cysteine hydrochloride. Lactobacillus sanfrancisco JCM5668 strain uses a modified sourdough medium (pH value adjusted to 5.6 with 2.0% maltose, 0.3% yeast extract, 0.03% polysorbate 80 and 0.6% trypsin) (trypticase peptone)) for culture.

Each bacterial strain was cultured at 30°C for 24 to 48 hours, and the bacterial cells were collected by centrifugation, washed with physiological saline, and then suspended in physiological saline again. Moreover, a part was separated and heat-processed at 70 degreeC for 30 minutes.

[Example 2] Systematic analysis of lactic acid bacteria The sequences of the 16S rRNA genes of 30 species of lactic acid bacteria used in the experiment were obtained from the National Center for Biotechnology Information (NCBI). ClustalW was used to align the sequences and construct a phylogenetic tree. The evolutionary analysis system uses the neighbor-joining method and is implemented by MEGA X. The evolutionary history is inferred by the nearest neighbor method. Tree topology was evaluated using bootstrap analysis based on 1,000 replicates.

[Example 3] Measurement of IgA production inducing ability (Preparation of Peyer's lymph node cells) Six-week-old male BALB/cA mice (purchased from CREA Japan) were raised using AIN-76 diet (purchased from Research Diets) as a basal feed. AIN-76 diet contains 20.0% casein, 0.3% DL-methionine, 5.0% corn oil, 50.0% sucrose, 15.0% corn starch, 5.0% cellulose powder, 1.0% AIN A mixture of -76 mixed vitamins, 3.5% AIN-76 mixed minerals and 0.2% choline bitartrate.

The mice were operated in accordance with the guidelines for the appropriate conduct of animal experiments issued by the Japanese Academic Council in 2006. After the mice were raised for 1 week, they were euthanized using carbon dioxide, and Peyer's lymph nodes in the small intestine were removed through laparotomy.

Peyer's lymph nodes were placed in ice-cold RPMI 1640 medium (PSMF) [RPMI 1640 medium (Gibco BRL) added with 100 U/ml penicillin, 100 μg/ml streptomycin, 55 μmol/l 2-mercaptoethanol and 10% inactivated fetal bovine serum (FBS; GibcoBRL)], and washed three times with this culture medium. Thereafter, RPMI 1640 medium (PSMF) supplemented with 25 mmol/l HEPES, 5 mmol/l EDTA (pH 8.0), and 1 mmol/l dithiothreitol was used to culture at 37°C for 45 minutes. Peyer's lymph nodes were washed again with RPMI 1640 medium (PSMF) containing 5 mmol/l EDTA (pH 8.0), and then washed with type I collagenase (Sigma) supplemented with 400 U/ml and 30 U/ml. Treat with RPMI 1640 medium (PSMF) of DNaseI (Takara Bio Co., Ltd.) at 37°C for 50 minutes. The obtained mixture was filtered through a 40 μm nylon mesh and washed twice with RPMI 1640 medium (PSMF) to obtain Peyer's lymph node cells used for measurement of IgA production induction ability. The survival rate of Peyer's lymph node cells was confirmed by trypan blue staining. It was prepared so that the final concentration of Peyer's lymph node cells would be 1.25×10 ⁶ cells/ml, and used for evaluation.

(Measurement of IgA) The suspension of physiological saline containing live or dead bacteria obtained in Example 1 was added to the suspension of Peyer's lymph node cells so that the final absorbance (OD ₆₀₀ ) became 0.01. , co-cultured in a 96-well T cell activation plate (Becton Dickinson) for 5 days at 37°C and 5% _CO2 . Thereafter, the amount of IgA in the obtained culture supernatant was measured using a mouse IgA ELISA kit (Bethyl Laboratories).

Statistical analysis was performed using Excel Statistics (SSRI Co., Ltd.). The measurement results were analyzed using one-way configured ANOVA and Dunnett's post-hoc analysis.

The results are shown in Figure 1 . Furthermore, in FIG. 1 , the lactic acid bacteria are arranged and displayed based on the phylogenetic tree constructed in Example 2, and the results of saline as a negative control are arranged at the top. In Figure 1, there are two bar graphs for each strain of lactic acid bacteria. The upper graph is a graph showing the IgA production induction ability produced by unheated bacteria (bacterial cells), and the lower graph is a graph showing the ability to induce IgA produced at 70 A graph showing the IgA production inducing ability of bacterial cells (dead bacterial cells) that have been heated at ℃. Furthermore, in FIG. 1 , bootstrap values are shown at branch points of the phylogenetic tree.

As a result of the experiment, it was confirmed that IgA was clearly and meaningfully higher in the lactic acid bacteria of the genus Lactobacillus apis, namely, Lactobacillus apiensis strain JCM30765, Lactobacillus apiensis 10H strain, and Lactobacillus kunstii JCM16173 strain compared to other lactic acid bacteria. Produce inducible ability. Furthermore, it was confirmed that the IgA production-inducing ability produced by the lactic acid bacteria of the genus Lactobacillus meli was maintained even after the sterilization treatment by heating.

Based on the above results, it can be expected that lactic acid bacteria belonging to the genus Lactobacillus, particularly lactic acid bacteria belonging to Apilactobacillus apinorum, and furthermore, Apilactobacillus apinorum JCM30765 strain, can be used as immune activation agents with high heat resistance. agent.

Figure 1 is a diagram showing the phylogenetic tree based on the 16S rRNA gene sequence and the IgA production inducing ability of the lactic acid bacteria used in the experiment.

Claims (14)

An intestinal immune activator, which contains bacteria of the genus Lactobacillus (Apilactobacillus) (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei) as an active ingredient. As claimed in claim 1, the intestinal immune stimulating agent is a lactic acid bacterium belonging to the genus Apilactobacillus (Apilactobacillus apinorum). As claimed in claim 2, the intestinal immune stimulating agent is characterized in that the above-mentioned lactic acid bacterium of the genus Apilactobacillus is Apilactobacillus apinorum JCM30765 strain. The intestinal immune stimulating agent according to any one of claims 1 to 3 is in the form of a food, drink, medicine, feed, or an active ingredient composition formulated therein. An IgA production accelerator, which contains bacteria of the genus Lactobacillus (Apilactobacillus) (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei) as an active ingredient. An IgA production promoter according to claim 5, wherein the lactic acid bacteria belonging to the genus Apilactobacillus are lactic acid bacteria belonging to the genus Apilactobacillus apinorum. An IgA production promoter according to claim 6, wherein the lactic acid bacterium belonging to the genus Apilactobacillus is Apilactobacillus apinorum JCM30765 strain. The IgA production promoter according to any one of claims 5 to 7 is in the form of a food, drink, medicine, feed, or an active ingredient composition formulated therein. A use of bacterial cells of the genus Lactobacillus kosoi (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei), which are used to produce medicines for stimulating intestinal immunity of humans or non-human animals. foods, beverages, feeds or active ingredient compositions formulated therein. Such as the use of bacteria according to claim 9, wherein the lactic acid bacteria belonging to the genus Apilactobacillus are lactic acid bacteria belonging to the genus Apilactobacillus apinorum. Such as the use of bacterial cells in claim 10, wherein the lactic acid bacteria of the genus Apilactobacillus are Apilactobacillus apinorum JCM30765 strain. A use of bacteria of the genus Lactobacillus kosoi (excluding Apilactobacillus kosoi and Apilactobacillus kunkeei), which is used to produce medicine for promoting IgA production in humans or non-human animals. foods, beverages, feeds or active ingredient compositions formulated therein. Such as the use of bacterial cells in claim 12, wherein the lactic acid bacteria belonging to the genus Apilactobacillus are lactic acid bacteria belonging to the genus Apilactobacillus apinorum. Such as the use of bacterial cells in claim 13, wherein the above-mentioned lactic acid bacteria of the genus Apilactobacillus are Apilactobacillus apinorum JCM30765 strain.