TW202335675A - Intestinal immune activating agent, IgA production promoter and gene expression promoter which clarify the novel property of lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus - Google Patents

Abstract

An object of the present invention is to clarify the novel property of lipoteichoic acid derived from the lactic acid bacteria of the genus Apilactobacillus, and provide new uses based on this. The intestinal immune activating agent, IgA production promoter and gene expression promoter of the present invention contain lipoteichoic acid derived from the lactic acid bacteria of the genus Apilactobacillus as an active ingredient. The lactic acid bacteria of the genus Apilactobacillus are the lactic acid bacteria of Apilactobacillus kosoi, Apilactobacillus kunkeei, or Apilactobacillus apinorum. The lactic acid bacteria of the genus Apilactobacillus are Apilactobacillus kosoi strain 10H, Apilactobacillus kunkeei strain JCM16173, or Apilactobacillus apinorum strain JCM30765. The intestinal immunity activating agent is in the form of a food, a medicine, a feed, or an active ingredient composition to be mixed therein. The IgA production promoter is in the form of a food, a medicine, a feed, or an active ingredient composition to be mixed therein. The gene expression promoter contains lipoteichoic acid derived from the lactic acid bacteria of the genus Apiractobacillus as an active ingredient and promotes the expression of at least one factor among IL-6, IL-10, and retinal dehydrogenase 2 (RALDH2) in dendritic cells, wherein the lactic acid bacteria of the genus Apilactobacillus is Apilactobacillus kosoi strain 10H. The gene expression promoter is in the form of a food, a medicine, a feed, or an active ingredient composition to be mixed therein. A use of lipoteichoic acid derived from the lactic acid bacteria of the genus Apilactobacillus is to manufacture a food, a medicine, a feed, or an active ingredient composition to be mixed therein for stimulating intestinal immunity in humans or non-human animals.

Description

Intestinal immune activator, IgA production promoter and gene expression promoter

The present invention relates to an intestinal immunity stimulating agent, an IgA production promoter and a gene expression promoter containing lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api as an active ingredient.

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

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 a specific strain of lactic acid bacteria of the genus Lactobacillus meli has an IgA production-promoting effect (and an immune-stimulating effect), but the reason for this is not known.

The present invention was completed in view of the above circumstances, and its object is to clarify the novel properties of substances derived from lactic acid bacteria of the genus Lactobacillus api and provide new uses based thereon.

[Technical means to solve problems]

In order to solve the above-mentioned problems, the present inventors focused on lipoteichoic acid among substances contained in lactic acid bacteria of the genus Lactobacillus api and found that the structure of lipoteichoic acid in lactic acid bacteria of the genus Lactobacillus api is similar to that of lipoteichoic acid in lactic acid bacteria of the genus Lactobacillus The present invention was completed because teichoic acid has a different typical structure and has excellent IgA production-promoting and immune-stimulating effects. That is, the present invention includes the following embodiments.

(1) An intestinal immune stimulating agent containing lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api as an active ingredient. (2) The intestinal immune stimulating agent as described in (1), wherein the Lactobacillus genus lactobacillus belongs to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum) lactic acid bacteria. (3) The intestinal immune stimulating agent as described in (2), wherein the lactic acid bacterium of the genus Lactobacillus kosoi is Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or swarm apis milt. Apilactobacillus apinorum strain JCM30765. (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 promoter containing lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus meli as an active ingredient. (6) The IgA production accelerator according to (5), wherein the Lactobacillus genus lactobacillus belongs to the enzyme Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum) lactic acid bacteria. (7) The IgA production accelerator according to (6), wherein the lactic acid bacterium of the genus Lactobacillus kosoi is Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain, or Apilactobacillus kosoi strain JCM16173, or Apilactobacillus kosoi Apilactobacillus apinorum strain JCM30765. (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) A gene expression promoter that contains lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api and promotes IL-6, IL-10 and retinal dehydrogenase 2 (RALDH2) in dendritic cells. The performance of at least one of the factors. (10) The gene expression promoter according to (9), wherein the Lactobacillus kosoi lactobacillus is a lactic acid bacterium belonging to the enzyme Apilactobacillus kosoi. (11) The gene expression accelerator according to (10), wherein the lactic acid bacterium of the genus Lactobacillus apiensis 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, drink, medicine, feed, or an active ingredient composition blended therein. (13) Use of lipoteichoic acid derived from lactobacilli of the genus Lactobacillus api, for use in the manufacture of pharmaceuticals, food and beverages, feeds for stimulating intestinal immunity or for promoting IgA production in humans or non-human animals, or to be formulated in the same The active ingredient composition among others. (14) Use of lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api and used to produce IL-6, IL-10 and retinal dehydrogenase in dendritic cells in humans or non-human animals 2 (RALDH2), pharmaceuticals, foods, feeds, or active ingredient compositions formulated therein for promoting the expression of genes that express at least one factor among 2 (RALDH2). [Effects of the invention]

According to the present invention, as a novel use of lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus, it is possible to provide an intestinal immune activator, an IgA production promoter and a gene expression promoter including the lipoteichoic acid.

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) Active ingredient lipoteichoic acid The active ingredient in one embodiment of the present invention is lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus. Preferable lactobacilli belonging to the genus Lactobacillus are lactobacilli belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum. Furthermore, it is more preferable that the lactic acid bacteria of the genus Lactobacillus are Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain, or 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 recently discovered fructophilic lactic acid bacteria (FLAB) and is believed to have evolved from previous lactic acid bacteria (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 survives in fructose-rich environments such as flowers or fruits, fermented foods, and the digestive tracts of insects that feed on fructose. FLAB is thought to be a heterofermentative lactic acid bacterium that prefers fructose rather than glucose as a carbon source, but it promotes growth in the presence of glucose by adding electron acceptors such as oxygen. The Lactobacillus apiensis 10H strain has a relatively smaller genome size and lower GC content than other FLAB and lactic acid bacteria (see Figure 3 of Filannino et al.).

Lactobacillus kunstii is a lactic acid bacterium isolated from slow-fermenting wine and is typically associated with bees or flowers. Lactobacillus kunstii strain JCM16173 is the model strain of Lactobacillus kunstii strain, which is the same as YH-15 strain, ATCC700308 strain and DSM12361 strain. Since this lactic acid bacterium has been well known from the past, detailed description of its properties will be omitted.

Colonial Lactobacillus apis is a lactic acid bacterium isolated from the honey stomach of honey bees. The Lactobacillus swarming strain JCM30765 is the type strain of Lactobacillus swarming strain, which is the same as the Fhon13N strain, the DSM26257 strain and the CCUG63287 strain. Since this lactic acid bacterium has been well known for a long time, detailed description of its properties will be omitted.

Lipoteichoic acid (LTA) is a component of the cell membrane of Gram-positive bacteria. Generally, lipoteichoic acid consists of a structure in which a main chain (glycerol phosphate chain) with glycerol phosphate as a repeating unit is combined with an anchoring glycolipid containing several sugars and fatty acids with several residues. 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 made any progress, and its detailed structure or physiological activity has not yet been clarified.

(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, it can be 0.01 to 1 per time for adults 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) Gene expression promoter In this specification, "gene expression promoter" refers to one that promotes the expression of specific factors (genes) from specific cells. As described in detail below, the gene expression promoter of the present invention includes the form of food, drink, medicine, feed, or active ingredient composition, etc. 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. In addition, retinal dehydrogenase 2 is an enzyme that metabolizes retinal into retinoic acid. The synthesis of IL-6, retinoic acid and IL-10 in dendritic cells in the intestine acts on Foxp3 ⁺ T cells, causing them to differentiate into follicular helper T cells (Tfh cells) that interact with B cells. In addition, the synthesis of retinoic acid and IL-10 in dendritic cells in the intestine promotes IgA type switching recombination and IgA production in B cells in the germinal center of Peyer's lymph nodes. In addition, dendritic cells expressing IL-6 enhance IgA production from B cells through IL-6R signaling. Furthermore, retinoic acid is necessary for the homing of IgA-producing B cells to the intestine. As mentioned above, the expression of IL-6, IL-10 and retinal dehydrogenase 2 in dendritic cells is closely related to the promotion of IgA production.

(IV) Food, drink, medicine, feed, or active ingredient compositions formulated therein (active ingredient composition) The intestinal immune stimulating agent, IgA production promoter, and gene expression 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 pure lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus apii as an active ingredient separated from lactic acid bacteria can be used directly, but also crudely purified lipoteichoic acid containing lipoteichoic acid can be used. or purified products, freeze-dried products, or cell wall differentiated parts obtained by treating bacterial cells with enzymes or physical methods.

The active ingredient composition of this embodiment is preferably prepared in the form of food, drink, medicine, 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, IgA production promoter and gene expression promoter of the present embodiment are taken into the form of pharmaceuticals, it is pharmaceutically acceptable to use them together with lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api. Appropriate preparation carriers are prepared into the form of pharmaceutical compositions for actual use. 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 the 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, maintaining/improving health, etc.

When the intestinal immune stimulating agent, IgA production promoter, and gene expression promoter of the present embodiment are used as food or drink, examples thereof include: fermented milk, lactic acid bacteria beverage, fermented vegetable beverage, fermented fruit beverage, fermented fruit beverage, Fermented soy milk drinks, etc. "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, gummies, 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 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, promoting IgA production, or promoting gene expression, for example, relative to the total mass of each food and drink, it is preferably 0.001 mass % or more, more preferably 0.01 mass % or more, and still more preferably 0.1 Quality% or more. 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, IgA production promoter and gene expression promoter of the present embodiment are in the form of feed, they can be used to treat infections during the non-antibiotic administration period of chickens or the weaning period of pigs, cows, etc. For disease prevention, it can be prepared 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 cultivation of lactic acid bacteria In order to obtain lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api, Lactobacillus apiensis strain JCM30765, Lactobacillus apiensis 10H strain and Lactobacillus kunstii JCM16173 strain were prepared. Furthermore, for comparison, lactic acid bacteria other than Lactobacillus api, Lactiplantibacillus plantarum subsp. plantarum (hereinafter, also referred to as "Lactobacillus plantarum") JCM1149 strain and Lactobacillus rhamnosus were also prepared. (Lacticaseibacillus rhamnosus) GG strain (ATCC53103). Furthermore, Lactobacillus plantarum subsp. plantarum strain JCM1149 is a model strain.

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.

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. The strain of lactic acid bacteria used for comparison was cultured using MRS culture medium for lactobacilli (Difco Laboratories). Each strain was precultured at 30°C for one night, followed by formal culture at 30°C for one day.

[Example 2] Purification of lipoteichoic acid Purification of lipoteichoic acid was carried out according 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, centrifuge the officially cultured lactic acid bacteria cells to collect them, add 0.1 M citric acid buffer (pH 4.7) and suspend them. Next, Multi-beads Shoker (registered trademark) (manufactured by Yasui Instruments Co., Ltd.) was used to crush the beads on ice using 0.3 mm zirconia beads. Set the crushing time to 1 minute and repeat it 6 times. After temporarily freezing the broken lactic acid bacteria cells at -80°C, an equal amount of butanol was added and stirred for 2 hours to remove lipophilic cell molecules. It is centrifuged and the aqueous layer is recovered and freeze-dried. The freeze-dried sample was dissolved in column equilibration buffer (0.1 M sodium acetate buffer containing 15% n-propanol, pH 4.7), and the solid content was removed by centrifugation for 30 minutes, and then loaded into a column Hydrophobic chromatography was performed using a Sepharose 4 fast flow column (manufactured by GE Healthcare). Lipoteichoic acid was dissolved using a linear gradient from 15% to 60% n-propanol in 0.1 M sodium acetate buffer (pH 4.7). Fractions containing lipoteichoic acid are identified by measuring the phosphate and sugar content. The phosphate content is determined by the phosphorus molybdenum test. The sugar content was determined by the phenol sulfuric acid method using glucose as the standard. Furthermore, it was confirmed that the recovered fraction did not contain nucleic acid and protein by measuring UV absorption at 260 nm and 280 nm respectively. The fraction recovered as described above was temporarily freeze-dried, suspended in 10 ml of Milli-Q water, dialyzed with Milli-Q water, and freeze-dried again. The purity of lipoteichoic acid was determined by measuring the endotoxin content using LAL reagent (<0.0001%) (manufactured by Biochemical Industry Co., Ltd.).

[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 lipoteichoic acid obtained in Example 2 was added to the suspension of Peyer's lymph node cells at a final concentration of 50 μg/ml, and placed in a 96-well T cell activation plate (Becton Dickinson). Co-culture was performed 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).

The results are shown in Figure 1 . In addition, in Figure 1, the results of saline as a negative control are arranged at the top. In addition, the marks "a", "b" and "c" in Figure 1 indicate that there are significant differences (P<0.05) between groups marked with different letters. The results of the experiment showed that lipoteichoic acid derived from Lactobacillus apiensis lactic acid bacteria, Lactobacillus apiensis 10H strain, Lactobacillus kunstii JCM16173 strain, and Lactobacillus apiensis JCM30765 strain was found to be similar to lipoteichoic acid derived from other lactic acid bacteria. Lipoteichoic acid has a clearly higher IgA production inducing ability than lipoteichoic acid.

Based on the above results, it is expected that lipoteichoic acid derived from lactobacilli of the genus Lactobacillus, particularly those derived from enzymes Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus lipoteichoic acid of lactobacillus apinorum), furthermore, lipids derived from enzyme Lactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum JCM30765 strain Teichoic acid acts as an immune activator with significant effects.

[Example 4] Analysis of gene expression in dendritic cells (generation of bone marrow-derived dendritic cells from mouse bone marrow cells) A bone marrow-derived dendritic cell line was used from 4-week-old female BALBc/A mice (from CREA Purchased from Japan), the bone marrow cells of the femur and tibia are produced. Bone marrow cells collected from mice were washed, and the number of cells was adjusted to 1×10 ⁶ cells/ml, and then suspended in granulocyte macrophage colony-stimulating factor (manufactured by PeproTech) to a concentration of 20 ng/mL. method was added to RPMI 1640 medium (PSMF), and cultured at 37°C and 5% _CO2 . On the 3rd and 5th day of culture, half of the culture medium was replaced with new culture medium. On the 6th day of culture, cells containing dendritic cells were collected, magnetically labeled with anti-CD11c beads (manufactured by Miltenyi Biotec), and dendritic cells were isolated using AutoMACS (manufactured by Miltenyi Biotec) according to conventional methods.

(Gene expression analysis) The bone marrow-derived dendritic cell line obtained as described above was cultured at 1.0×10 ⁹ cells/well (3 ml) using RPMI 1640 medium (PSMF) for 6 hours. This culture system was performed on both those containing lipoteichoic acid derived from the enzyme Lactobacillus apiensis 10H strain at a concentration of 50 μg/ml and those without added lipoteichoic acid (control). Thereafter, total RNA was isolated from bone marrow-derived dendritic cells using the QuickPrep total RNA extraction kit (manufactured by GE Healthcare), and synthesized from the total RNA using the SuperScript (registered trademark) III reverse transcription kit (manufactured by Invitrogen). cDNA. The real-time PCR method was implemented using StepOne real-time PCR system (manufactured by Applied Biosystems) and Power SYBR (registered trademark) Green Master Mix (manufactured by Applied Biosystems). The following primers were used to amplify DNA.

(Primers for IL-6 amplification: bone marrow-derived dendritic cell IL-6 PCR primer) Forward: 5'-AATAGTCCTTCCTACCCCAATTTC-3' (Sequence Number 1) Reverse: 5'-ATTTCAAGATGAATTGGATGGTCT-3' (Sequence Number 2 ) (IL-10 amplification primer: bone marrow-derived dendritic cell IL-10 PCR primer) Forward: 5'-ATGCAGGACTTTAAGGGTTACTTG-3' (Sequence Number 3) Reverse: 5'-GAATTCAAATGCTCCTTGATTTCT-3' (Sequence Number 4) (Primers for RALDH2 amplification: bone marrow-derived dendritic cells RALDH2 PCR primer) Forward: 5'-GACTTGTAGCAGCTGTCTTCACT-3' (Sequence Number 5) Reverse: 5'-TCACCCATTTTCCTCCCATTTCC-3' (Sequence Number 6)

As an endogenous control, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used. The following primers were used for amplification.

(Primers for GAPDH amplification: bone marrow-derived dendritic cells GAPDH PCR primers) Forward: 5'-CTACACTGAGGACCAGGTTGTCT-3' (sequence number 7) Reverse: 5'-ATTGTCATACCAGGAAATGAGCTT-3' (sequence number 8)

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. ***p<0.001

The results are shown in Figure 2 . The degree of gene expression in Figure 2 is expressed as the ratio of the results of the control to the sample containing lipoteichoic acid. The results of the experiment confirmed that lipoteichoic acid derived from Lactobacillus apiensis strain 10H has the effect of promoting the gene expression of IL-6, IL-10 and retinal dehydrogenase 2 (RALDH2) in bone marrow-derived dendritic cells. .

Based on the above results, it can be expected that lipoteichoic acid derived from lactobacilli of the genus Lactobacillus kosoi, particularly lipoteichoic acid derived from lactic acid bacteria belonging to the genus Apilactobacillus kosoi, and furthermore, lipoteichoic acid derived from lactic acid bacteria belonging to the genus Apilactobacillus Kosoi)10H strain lipoteichoic acid acts as a gene expression promoter that promotes the expression of IL-6, IL-10 and retinal dehydrogenase 2 (RALDH2) in dendritic cells.

[Example 5] Analysis of the glycerol-phosphate chain of lipoteichoic acid The analysis of the glycerol-phosphate chain (repeating structure, polymer site) of lipoteichoic acid was performed by acquiring ^{a 1} H-NMR spectrum. First, the lipoteichoic acid obtained in Example 2 was dissolved in 0.6 ml of 99.8% D ₂ O (purchased from Fujifilm Wako Pure Chemical Industries, Ltd.). ¹ H-NMR spectrum was obtained using a 500 MHz Varian Unity Inova 500 spectrometer (manufactured by Agilent Technologies) at 25°C. As a reference material for chemical shifts, sodium 3-(trimethylsilyl)propionate-2,2,3,3-d ₄ (purchased from Fujifilm Wako Pure Chemical Industries, Ltd.) was used.

The results are shown in Figures 3 and 4. First, the structure of the glycerol-phosphate chain in lipoteichoic acid derived from lactic acid bacteria other than Lactobacillus apii will be explained.

Regarding lipoteichoic acid derived from Lactobacillus plantarum strain L - 137, based on past papers (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) as a reference to assign each peak (see Figure 3(b)). Lipoteichoic acid derived from Lactobacillus plantarum JCM1149 strain is considered to have a glycerol phosphate chain composed of a GroP unit, an AlaGroP unit and a GlcGroP unit.

Regarding lipoteichoic acid derived from Lactobacillus rhamnosus GG strain, based on past papers (Claes et al. Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG. Microbial Cell Factories, 11: 161 - 168, 2012 ) as a reference to assign each wave peak (refer to Figure 3(c)). Lactobacillus rhamnosus GG strain is considered to have a glycerol phosphate chain composed of a GroP unit and an AlaGroP unit.

The structure of the glycerol phosphate chain in the lipoteichoic acid derived from Lactobacillus plantarum JCM1149 strain and Lactobacillus rhamnosus GG strain is a structure commonly seen in lipoteichoic acid in lactic acid bacteria of the genus Lactobacillus genus.

On the other hand, the ¹ H-NMR spectrum of lipoteichoic acid derived ^from Lactobacillus apiensis strain 10H is significantly different from the 1 H-NMR spectrum of lipoteichoic acid derived from Lactobacillus plantarum strain JCM1149 and Lactobacillus rhamnosus GG strain. Different (see Figure 3(a)). The attribution of each peak is currently in progress. If we also take into account past reports and the results of ¹³ C-NMR and 2D NMR (not shown), it is believed that the lipoteichoic acid derived from the enzyme Lactobacillus apiensis 10H strain may have a GlcGroP unit . However, since there are also many unknown peaks, we will continue to clarify them through structural analysis and other methods in the future. At least it is believed that the glycerol phosphate chain in lipoteichoic acid derived from the enzyme Lactobacillus apiensis 10H strain has a unique structure that is not ordinary.

Furthermore, the ¹ H-NMR spectrum of lipoteichoic acid derived from Lactobacillus kunstii strain JCM16173 (refer to Figure 4(b)) and ^{the 1H} -NMR spectrum of lipoteichoic acid derived from Lactobacillus kunstii strain JCM30765 (refer to Figure 4(b)) Referring to Figure 4(c)), the ¹ H-NMR spectrum of lipoteichoic acid derived from Lactobacillus apiensis 10H strain is also shown (see Figure 4(a)); furthermore, Figure 4(a) and Figure 3(a) same) very similar results. Therefore, it is considered that the glycerol phosphate chain in lipoteichoic acid derived from lactobacilli of the genus Lactobacillus api and the usual lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus is considered to have a certain degree of similarity within the genus. common structure.

[Example 6] Analysis of anchored glycolipids of lipoteichoic acid The analysis of anchored glycolipids of lipoteichoic acid was performed by acquiring MALDI-TOF MS spectra.

(Isolation of anchored glycolipids) First, collect 100 μg of lipoteichoic acid into a tube made of PP. Next, add 0.1 ml of 48% (w/v) hydrofluoric acid and let stand at 4°C for 3 hours. After removing hydrofluoric acid by blowing nitrogen in the ventilation room, add 1 ml of chloroform, 1 ml of methanol, and 0.9 ml of water. After stirring thoroughly, centrifuge (20°C, 200×g, 30 seconds) to recover the organic matter in the lower layer. layer. By blowing nitrogen in the ventilation chamber to remove the organic solvent, the anchored glycolipid of lipoteichoic acid is obtained.

(Acquisition of MALDI-TOF MS spectrum) Dissolve the anchored glycolipid in 100 μl of chloroform/methanol (2:1, v/v), and then mix it with the same amount of matrix agent (10 mg/ml containing 0.1% trifluoroacetic acid (TFA)) on the target plate. ,5-dihydroxybenzoic acid (DHBA) water/methanol (7:3, v/v) solution) mixed. After the mixture was co-crystallized, MALDI-TOF mass spectra were acquired in positive ion mode and reflectron mode. As a mass spectrometer, a TOF/TOF 5800 system (manufactured by AB Sciex) was used.

The results are shown in Figures 5 and 6 . First, the structure of the anchor glycolipid in lipoteichoic acid derived from lactic acid bacteria other than Lactobacillus apiensis will be explained.

It is believed that the main structure of the anchored glycolipid in lipoteichoic acid derived from Lactobacillus plantarum JCM1149 strain is Hex ₃ DAG, that is, a structure in which diacylglycerol is bonded to a trisaccharide (see Figure 5(b) ). In addition, there are also peaks thought to be generated by AcylHex ₃ DAG, but the intensity of the peaks is weak and hidden in the background. Furthermore, there is also a peak (956) thought to be generated by Hex ₂ DAG, that is, a structure in which diacylglycerol is bonded to a disaccharide, but the intensity of the peak is also weak and it is not considered to definitely exist.

It is considered that the main structure of the anchoring glycolipid in lipoteichoic acid derived from Lactobacillus rhamnosus GG strain is Hex ₃ DAG (see Figure 5(c) ). Regarding the peak thought to be generated by AcylHex ₃ DAG, only one peak (1368) is visible, and the peak is also weak. Therefore, the anchored glycolipid in the lipoteichoic acid derived from Lactobacillus rhamnosus GG strain may not have AcylHex ₃ DAG. . The peak thought to be generated by Hex ₂ DAG exists similarly to the case of Lactobacillus plantarum JCM1149 strain (942, 956), but the intensity of the peak is still weak.

In summary, it is believed that the structure of the anchoring glycolipid in the lipoteichoic acid derived from Lactobacillus plantarum JCM1149 strain and Lactobacillus rhamnosus GG strain is Hex ₃ DAG. The trisaccharide-anchored glycolipid is a common structure for lipoteichoic acid in lactic acid bacteria of the genus Lactobacillus genus.

On the other hand, it is thought that the main structure of the anchor glycolipid in the lipoteichoic acid derived from the enzyme Lactobacillus apiensis 10H strain is Hex ₂ DAG (942, 956, 982) (see Figure 5(a) ). The anchoring glycolipid of the disaccharide is derived from the common structure of lipoteichoic acid in lactic acid bacteria of the genus Enterococcus, Lactococcus and Leuconostock. On the other hand, lipoteichoic acids derived from lactic acid bacteria of the genus Lactobacillus genus are commonly found in tri- or tetrasaccharide-anchored glycolipids. However, although Lactobacillus apiensis lactic acid bacteria belong to the genus Lactobacillus genus, it can be confirmed that the anchoring glycolipid in lipoteichoic acid is mainly a disaccharide. Furthermore, the anchoring glycolipid in the lipoteichoic acid derived from the enzyme Lactobacillus apiensis 10H strain does not have 3 residues in the bond commonly seen in the lipoteichoic acid derived from various Lactobacillus genus lactic acid bacteria or Lactococcus genus lactic acid bacteria. Anchor glycolipids based on fatty acids. Therefore, the structure of the anchored glycolipid in lipoteichoic acid derived from the enzyme Lactobacillus apiensis 10H strain shows characteristics that are quite different from the structure of the anchored glycolipid common to previously identified representative lactic acid bacteria.

Furthermore, the MALDI-TOF MS spectrum of anchored glycolipids in lipoteichoic acid derived from Lactobacillus kunstii strain JCM16173 (refer to Figure 6(b)) and lipoteichoic acid derived from Lactobacillus kunstii strain JCM30765 The MALDI-TOF MS pattern of anchored glycolipids (refer to Figure 6(c)) is also consistent with the MALDI-TOF MS pattern of anchored glycolipids in lipoteichoic acid derived from the enzyme Lactobacillus apiensis 10H strain (refer to Figure 6(c)). a); Furthermore, Figure 6(a) is the same as Figure 5(a)) showing similar results in terms of using Hex ₂ DAG as the main structure. Therefore, it is considered that the anchoring glycolipids in the lipoteichoic acid derived from the Lactobacillus api lactic acid bacterium are also very different from the anchored glycolipids in the usual lipoteichoic acid derived from the Lactobacillus genus lactobacillus, and have unique characteristics within the genus A somewhat common structure.

According to the above-mentioned Examples 5 and 6, it was found that lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus meli has an overall different structure from normal lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus genus. It is believed that the structural difference between lipoteichoic acid derived from lactobacilli of the genus Lactobacillus api and conventional lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus is related to the fact that lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api shows higher IgA production. Inducibility.

Figure 1 is a graph showing the IgA production inducing ability of lipoteichoic acid in Example 3. FIG. 2 is a graph showing the results of gene expression analysis of lipoteichoic acid derived from Lactobacillus apiensis 10H strain in Example 4. Figure 3 (a) to (c) are ¹ H-NMR spectra of lipoteichoic acid in Example 5. Figure 4 (a) to (c) are ¹ H-NMR spectra of lipoteichoic acid in Example 5. Figure 5 (a) to (c) are MALDI-TOF MS spectra of anchored glycolipids of lipoteichoic acid in Example 6. Figure 6 (a) to (c) are MALDI-TOF MS spectra of anchored glycolipids of lipoteichoic acid in Example 6.

TW202335675A_111145803_SEQL.xml

Claims (21)

An intestinal immune stimulating agent containing lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api as an active ingredient. As claimed in claim 1, the intestinal immune activating agent is a lactic acid bacterium belonging to the genus Lactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum. Such as the intestinal immune activating agent of claim 2, wherein the above-mentioned Lactobacillus api lactic acid bacteria are Apilactobacillus kosoi strain 10H, Apilactobacillus kunkeei JCM16173 strain or 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 promoter containing lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus meli as an active ingredient. An IgA production promoter according to claim 5, wherein the lactic acid bacteria of the genus Lactobacillus are lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum. Such as the IgA production accelerator of claim 6, wherein the above-mentioned Lactobacillus genus lactobacillus is Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or 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 gene expression promoter that contains lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api and promotes IL-6, IL-10 and retinal dehydrogenase 2 (RALDH2) in dendritic cells. The manifestation of at least one factor. The gene expression accelerator of claim 9, wherein the above-mentioned Lactobacillus api lactic acid bacterium is a lactic acid bacterium belonging to the enzyme Apilactobacillus kosoi. The gene expression accelerator of claim 10, wherein the lactic acid bacteria of the genus Lactobacillus apiensis are Lactobacillus apiensis (Apilactobacillus kosoi) 10H strain. The gene expression promoter according to any one of claims 9 to 11 is in the form of a food, drink, medicine, feed, or an active ingredient composition formulated therein. A use of lipoteichoic acid derived from lactobacilli of the genus Lactobacillus api and used to manufacture pharmaceuticals, food and beverages, feeds for intestinal immune stimulation of humans or non-human animals, or active ingredient compositions formulated therein. Such as the use of lipoteichoic acid in Claim 13, wherein the above-mentioned Lactobacillus api lactic acid bacteria are lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum . Such as the use of lipoteichoic acid in claim 14, wherein the above-mentioned Lactobacillus api lactobacillus is Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum )JCM30765 strain. A use of lipoteichoic acid derived from lactobacilli of the genus Lactobacillus api and used to produce pharmaceuticals, foods, and feeds for promoting IgA production in humans or non-human animals, or active ingredient compositions formulated therein. Such as the use of lipoteichoic acid in claim 16, wherein the above-mentioned Lactobacillus api lactic acid bacteria are lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum . Such as the use of lipoteichoic acid in claim 17, wherein the above-mentioned Lactobacillus api lactobacillus is Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum )JCM30765 strain. A use of lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus api and used to produce IL-6, IL-10 and retinal dehydrogenase 2 (RALDH2) in dendritic cells in humans or non-human animals. ) pharmaceuticals, foods, and feeds for promoting the expression of genes that express at least one factor among them, or active ingredient compositions formulated therein. Such as the use of lipoteichoic acid in claim 19, wherein the above-mentioned Lactobacillus api lactic acid bacteria are lactic acid bacteria belonging to the enzyme Apilactobacillus kosoi. Such as the use of lipoteichoic acid in claim 20, wherein the above-mentioned Lactobacillus api lactic acid bacteria is Apilactobacillus kosoi 10H strain.