TWI838990B - Intestinal immune activators, IgA production promoters and gene expression promoters - Google Patents

Abstract

The subject of the present invention is to clarify the novel properties of lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus and to provide new uses based on the properties. The intestinal immune activator, IgA production promoter and gene expression promoter of the present invention contain lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus as an active ingredient.

Description

Intestinal immune activators, IgA production promoters and gene expression promoters

The present invention relates to an intestinal immune activator, an IgA production promoter and a gene expression promoter, which contain lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus as an effective ingredient.

It has been known that lactic acid bacteria or their fermentation products have various physiological functions. For example, Patent Document 1 states that lactic acid bacteria belonging to Lactobacillus kunsii have a high IgA production promoting effect.

In addition, the applicant of this case discovered that the enzyme lactobacillus 10H strain, which is a lactic acid bacterium isolated from vegetable brown sugar fermentation liquid, is a fructose-loving lactic acid bacterium with a novel genome structure different from other lactic acid bacteria, and has excellent IgA production promotion effect (and immune activation effect) (refer to patent document 2; furthermore, "Lactobacillus kosoi" in patent document 2 refers to the same as "enzyme lactobacillus" in this specification). [Prior art document] [Patent document]

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

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

As mentioned above, it is known that certain species of lactic acid bacteria belonging to the genus Lactobacillus apis have an IgA production promoting effect (and an immunostimulating effect), but the cause is unknown.

The present invention is completed in view of the above situation, and its subject is to clarify the novel properties of substances derived from lactic acid bacteria of the genus Lactobacillus apis and to provide new uses based on the properties.

[Technical means to solve the problem]

In order to solve the above problems, the inventors of the present invention focused on lipoteichoic acid in the substances contained in lactic acid bacteria of the genus Lactobacillus and found that the structure of lipoteichoic acid in lactic acid bacteria of the genus Lactobacillus is different from the typical structure of lipoteichoic acid in lactic acid bacteria of the genus Lactobacillus and has excellent IgA production promotion and immune activation effects, thereby completing the present invention. That is, the present invention includes the following embodiments.

(1) A gastrointestinal immune activator comprising lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus as an active ingredient. (2) The gastrointestinal immune activator as described in (1), wherein the lactic acid bacteria of the genus Lactobacillus are lactic acid bacteria belonging to the genus Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum. (3) The intestinal immune activator as described in (2), wherein the lactic acid bacteria of the genus Lactobacillus are enzyme Lactobacillus kosoi 10H strain, Lactobacillus kunkeei JCM16173 strain or Lactobacillus apinorum JCM30765 strain. (4) The intestinal immune activator as described in any one of (1) to (3), which is in the form of a food, a medicine, a feed or a combination of active ingredients formulated therein. (5) An IgA production promoter, which contains lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus as an active ingredient. (6) The IgA production promoter as described in (5), wherein the lactic acid bacteria of the genus Lactobacillus are lactic acid bacteria belonging to the enzyme Lactobacillus kosoi, Lactobacillus kunkeei or Lactobacillus apinorum. (7) The IgA production promoter as described in (6), wherein the lactic acid bacteria of the genus Lactobacillus are Lactobacillus kosoi 10H strain, Lactobacillus kunkeei JCM16173 strain or Lactobacillus apinorum JCM30765 strain. (8) An IgA production promoter as described in any one of (5) to (7), which is in the form of a food, a medicine, a feed, or a combination of active ingredients formulated therein. (9) A gene expression promoter, which contains lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus as an active ingredient, and promotes the expression of at least one factor among IL-6, IL-10 and retinal aldehyde dehydrogenase 2 (RALDH2) in dendrite cells. (10) A gene expression promoter as described in (9), wherein the lactic acid bacteria of the genus Lactobacillus are lactic acid bacteria belonging to the enzyme Lactobacillus kosoi. (11) A gene expression enhancer as described in (10), wherein the lactic acid bacteria of the genus Lactobacillus kosoi is the enzyme Lactobacillus kosoi 10H strain. (12) A gene expression enhancer as described in any one of claims 9 to 11, which is in the form of a food, a medicine, a feed, or a combination of active ingredients formulated therein. (13) A use of lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus kosoi, which is used to produce a medicine, a food, a feed, or a combination of active ingredients formulated therein for intestinal immunity enhancement or IgA production promotion in humans or non-human animals. (14) A use of lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus apis for the manufacture of a gene expression-promoting pharmaceutical, food, feed, or an active ingredient composition formulated therein that promotes the expression of at least one factor among IL-6, IL-10, and retinal aldehyde dehydrogenase 2 (RALDH2) in dendrite cells in humans or non-human animals. [Effect of the invention]

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

The following describes various embodiments of the present invention with reference to the attached drawings. Furthermore, the embodiments described below do not limit the scope of the invention to be applied for, and the elements and their combinations described in the embodiments may not all be necessary for the solution of the present invention.

(I) Lipoteichoic acid as an active ingredient In one embodiment of the present invention, the active ingredient is lipoteichoic acid derived from lactic acid bacteria of the genus Apilactobacillus. Preferably, the lactic acid bacteria of the genus Apilactobacillus are lactic acid bacteria belonging to the enzyme Apilactobacillus kosoi, Apilactobacillus kunkeei, or Apilactobacillus apinorum. Furthermore, it is further preferred that the lactic acid bacteria of the genus Apilactobacillus are the enzyme Apilactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain, or Apilactobacillus apinorum JCM30765 strain.

As lactic acid bacteria of the genus Lactobacillus, in addition to Lactobacillus enzyme, Lactobacillus kunsii, and Lactobacillus swarmii, there are known Lactobacillus micheneri, Lactobacillus ozensis, Lactobacillus quenuiae, and Lactobacillus timberlakei.

Lactobacillus apis is a group of lactic acid bacteria in the Lactobacillus genus called Lactobacillus kunsii. Lactobacillus apis is Gram-positive, rod-shaped, and heterofermentative. It usually grows in the range of 15-37°C and most of them grow in acidic conditions with a pH value of less than 3.0. The genome size of Lactobacillus apis is relatively small, about 1.42-1.58 Mbp. The G+C content in the DNA is in the range of 30.5-36.4. Lactobacillus apis converts fructose into mannitol. In addition, although it can 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 apis is a recently discovered fructophilic lactic acid bacteria (FLAB), which is believed to be a bacterium that evolved from the 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 lives in fructose-rich environments such as flowers or fruits, fermented foods, and the digestive tract of insects that feed on fructose. FLAB is believed to be a heterofermentative lactic acid bacterium that prefers fructose rather than glucose as a carbon source, but promotes growth in the presence of glucose by adding electron acceptor substrates such as oxygen. Compared with other FLAB and lactic acid bacteria, the enzyme Lactobacillus apiosus 10H strain has a relatively small genome size and a lower GC content (see Figure 3 of Filannino et al.).

Lactobacillus kunsii is a lactic acid bacterium isolated from slow-fermenting wine, and is typically associated with bees or flowers. Lactobacillus kunsii JCM16173 is the type strain of Lactobacillus kunsii, and is the same as YH-15, ATCC700308, and DSM12361. This lactic acid bacterium has been widely known for a long time, so the detailed description of its properties is omitted.

Lactobacillus mellifera is a lactic acid bacterium isolated from the honey stomach of honey bees. Lactobacillus mellifera JCM30765 is a type strain of Lactobacillus mellifera, which is the same as Fhon13N, DSM26257 and CCUG63287. This lactic acid bacterium has been widely known for a long time, so the detailed description of its properties is omitted.

Lipoteichoic acid (LTA) is a constituent of the cell membrane of Gram-positive bacteria. General lipoteichoic acid consists of a structure consisting of a main chain (glycerophosphate chain) with glycerophosphate as a repeating unit and an anchoring sugar lipid containing several sugars and several residual fatty acids. The structure of lipoteichoic acid varies depending on the bacteria from which it originates. Compared with lipopolysaccharide (LPS) of Gram-negative bacteria, research on lipoteichoic acid has not made much progress, and its detailed structure or physiological activity has not been much determined.

(II) IgA production promoter and intestinal immune activator In this specification, "IgA production promoter" refers to a substance with the ability to induce IgA production, which, when added to a culture medium containing Peyer's lymph node cells containing a large number of IgA-producing cells and cultured for a specific period of time, secretory IgA secreted in the culture medium after culture increases compared to the case where no IgA is added. The IgA production promoter of the present invention is described in detail below, including the form of food, medicine, feed or active ingredient combination. For example, IgA production promoters can be administered together with vaccines to enhance the production of antibodies corresponding to the antigens contained in the vaccine, enhance the effect of the vaccine, and have a higher possibility of inhibiting the side effects of the vaccine. In other words, the production of antibodies against the antigens contained in the vaccine is enhanced, so that the induction of defensive immunity becomes better and the effect of the vaccine is enhanced.

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 is administered before or after the vaccine to enhance the effect. The dosage of the IgA production promoter varies depending on the type and quality of the vaccine used, or the age and symptoms of the vaccine recipient. For example, when used for prevention, an example is that an adult can take about 0.01 to 1 g each time in terms of solid content, and it is ideal to take it three times a day about 30 minutes before meals.

In addition, in this specification, "intestinal immune activator" means an agent that promotes the secretion of IgA in the mucosal epithelium of the intestine and activates the host's immune mechanism. The intestinal immune activator of the present invention is described in detail below, including the form of food, medicine, feed or active ingredient combination. Moreover, among these, health food is preferred, and in particular, a food composition for maintaining and enhancing the health of a subject with reduced immunity is 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 a promoter that promotes the expression of a specific factor (gene) from a specific cell. The gene expression promoter of the present invention includes the form of a food, a pharmaceutical, a feed, or an active ingredient combination, as described in detail below. 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 aldehyde dehydrogenase 2 (RALDH2) in dendritic cells. IL (interleukin) -6 and IL-10 are cytokines. In addition, retinal aldehyde dehydrogenase 2 is an enzyme that metabolizes retinal aldehyde into retinoic acid. The synthesis of IL-6, retinoic acid, and IL-10 in dendrite cells in the intestine acts on Foxp3 ⁺ T cells 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 dendrite cells in the intestine promotes IgA type switch recombination and IgA production in B cells in the germinal center of the Peyer's lymph nodes. In addition, dendrite cells expressing IL-6 enhance the production of IgA 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 aldehyde dehydrogenase 2 in dendrite cells is closely related to the promotion of IgA production.

(IV) Foods, medicines, feeds, or active ingredient compositions formulated therein (Active ingredient compositions) The intestinal immune activator, IgA production promoter, and gene expression promoter of the present embodiment can be used in the form of foods, medicines, feeds, or active ingredient compositions formulated therein. When used in the form of active ingredient compositions, not only pure lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus separated from lactic acid bacteria can be used directly as an active ingredient, but also crude or purified products containing lipoteichoic acid, freeze-dried products thereof, or cell wall fractions obtained by treating bacteria using enzymes or physical methods can be used.

The active ingredient composition of this embodiment is preferably prepared into the form of a food, medicine, etc. as described below by appropriately mixing with a suitable edible carrier (food material), a pharmaceutically acceptable carrier, etc.

(Pharmaceuticals) When the intestinal immune activator, IgA production promoter, and gene expression promoter of the present embodiment are in the form of pharmaceuticals, they are prepared in the form of pharmaceutical compositions using a suitable pharmaceutical carrier permitted in pharmaceutical preparation together with lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus bee. Examples of the pharmaceutical carrier include diluents or excipients such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, lubricants, etc. that are commonly used in the field.

As the dosage unit form of the pharmaceutical product, various forms can be selected, and oral dosage forms are preferred. Representative oral dosage forms include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, and the like.

When the tablet is formed, as a preparation carrier, for example, lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate and the like can be used as a shaping agent; water, ethanol, propanol, monosaccharide slurry, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, polyvinyl pyrrolidone and the like can be used as a binder; sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, dry starch, Disintegrants such as sodium alginate, agar powder, kelp powder, sodium bicarbonate, calcium carbonate; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid; disintegration inhibitors such as white sugar, stearin, cocoa butter, hydrogenated oil; absorption promoters such as quaternary ammonium salt, sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid; lubricants such as refined talc, stearate, boric acid powder, polyethylene glycol, etc. Tablets may be provided with a usual coating as required, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, or double-layer tablets or multi-layer tablets.

When forming into a pill form, as a preparation carrier, for example, there can be used: a shaping agent such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, etc.; a binder such as gum arabic powder, astragalus powder, gelatin, ethanol, etc.; a disintegrant such as kelp, agar, etc., etc.

Furthermore, the pharmaceutical product may contain colorants, preservatives, spices, flavoring agents, sweeteners, or other pharmaceutical products as needed.

The method of administration of the pharmaceutical product of this embodiment is not particularly limited and is determined according to the form of the preparation, the patient's age, gender, and other conditions, the severity of the disease, etc. In addition, the dosage is appropriately determined according to the usage, the patient's age, gender, and other conditions, the severity of the disease, etc. Generally, the above-mentioned active ingredient composition can be set to about 0.5 to 100 mg per day relative to 1 kg of body weight. The pharmaceutical product can be administered to humans 1 to 4 times a day.

(Food and Beverage) The term "food and beverage" in this manual includes all forms of food and beverage that are specifically taken orally for consumption (including drinks, for example). Even if the form is tablets, as long as they are specifically used for consumption, they are also included in the food and beverage in this manual. For example, health foods, health supplementary foods, foods for patients, nutritional supplementary foods, or health functional foods (specific health foods, nutritional functional foods) that are designed to prevent infection or diarrhea and whose contents are displayed as needed are also included in the food and beverage in this manual. Health foods refer to foods that are used for health care, maintenance/improvement of health, etc. in a more positive sense than ordinary foods.

When the intestinal immune activator, IgA production promoter and gene expression promoter of the present embodiment are set as beverages, for example, fermented milk, lactic acid bacteria beverages, fermented vegetable beverages, fermented fruit beverages, fermented soy milk beverages, etc. "Fermented milk" refers to a paste or liquid obtained by fermenting milk or dairy products using lactic acid bacteria or yeast. Therefore, the fermented milk includes beverage form and yogurt form. In addition, "lactic acid bacteria beverage" refers to a beverage obtained by diluting a paste or liquid obtained by fermenting milk or dairy products using lactic acid bacteria or yeast as the main ingredient with water.

Examples of other food and beverage forms include fermented foods such as pickles, bean paste, fermented tea, bread, weaning foods, milk powder, baby foods, and other infant foods, foaming preparations, chewing gum, soft candy, pudding and other snacks, noodles, capsules, granules, powders, tablets and other nutritional supplements, and dairy products other than the above-mentioned fermented milk and lactic acid bacteria beverages.

The content of the active ingredient composition in the food of this embodiment is not particularly limited and can be appropriately determined. From the perspective of exerting the effects of intestinal immune activation, IgA production promotion or gene expression promotion, relative to the total mass of each food, 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. On the other hand, the upper limit of the content of the active ingredient composition in the food is not particularly limited and can usually be appropriately adjusted according to the form of the food.

(Feed) When the enteral immunoactivator, IgA production promoter and gene expression promoter of this embodiment are provided in the form of feed, for example, they can be used as a non-antibiotic administration period for chickens or for the prevention of infectious diseases during the weaning period of pigs and cattle, and can be prepared into a preparation form for oral administration (aqueous solution, emulsion, granules, powder, capsule, tablet, etc.).

[Examples] The present invention is further described in detail below with reference to examples, but the present invention is not limited by these examples. In addition, in the following examples, the unit % of the numerical value representing the addition amount of various components, etc., means mass % unless otherwise specified.

[Example 1] Acquisition and cultivation of lactic acid bacteria In order to obtain lipoteichoic acid from lactic acid bacteria of the genus Lactobacillus apis, Lactobacillus apis JCM30765 strain, Lactobacillus apis enzyme 10H strain, and Lactobacillus apis kunsii JCM16173 strain were prepared. In addition, for comparison, lactic acid bacteria other than the genus Lactobacillus apis, Lactiplantibacillus plantarum subsp. plantarum (hereinafter, also referred to as "Lactiplantibacillus plantarum") JCM1149 strain, and Lactobacillus rhamnosus GG strain (ATCC53103) were also prepared. In addition, Lactiplantibacillus plantarum subsp. plantarum JCM1149 strain was used as a model strain.

Among the above lactic acid bacteria, the enzyme Lactobacillus mellifera 10H strain was a strain stored in the Matsuzaki Laboratory of Ishikawa Prefectural University, the rhamnosus casei GG strain (ATCC53103) was obtained from the American Type Culture Collection (ATCC), and the others were obtained from the Microbial Material Development Laboratory (JCM), RIKEN Bioresource Research Center, Japan.

Lactobacillus apiosporium JCM30765 and Lactobacillus apiosporium enzyme 10H were cultured in MRS medium for lactobacillus supplemented with 10% fructose. Lactobacillus kunsii JCM16173 was cultured in MRS medium for lactobacillus supplemented with 10% tomato juice and 0.05% L-cysteine hydrochloride. The lactic acid bacteria strains used for comparison were cultured in MRS medium for lactobacillus (Difco Laboratories). Each strain was pre-cultured at 30°C overnight and then cultured at 30°C for one day.

[Example 2] Purification of lipoteichoic acid The purification of lipoteichoic acid was carried out with reference to the known method (Morath et al., J. Exp. Med., 193: 393-397, 2001 and Claes et al., Microbial Cell Factories, 11: 161-168, 2012). First, the lactic acid bacteria cells after formal culture were collected by centrifugation and suspended by adding 0.1 M citric acid buffer (pH 4.7). Secondly, Multi-beads Shoker (registered trademark) (manufactured by Anjing Instrument Co., Ltd.) was used to crush the cells on ice using 0.3 mm zirconium oxide beads. The crushing time was set to 1 minute and repeated 6 times. The disrupted lactic acid bacteria cells were temporarily frozen at -80°C, and then an equal amount of butanol was added and stirred for 2 hours to remove lipophilic cell molecules. The mixture was centrifuged, and the aqueous layer was 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 components were removed by centrifugation for 30 minutes. The column was loaded onto an Octyl-Ag-Se 4 Fast Flow column (manufactured by GE Healthcare) and subjected to hydrophobic chromatography. Lipoteichoic acid was eluted using a linear gradient of n-propanol from 15% to 60% in 0.1 M sodium acetate buffer (pH 4.7). The fraction containing lipoteichoic acid was identified by measuring the phosphate and sugar contents. The phosphate content was determined by the molybdenum phosphate test. The sugar content was determined by the phenol-sulfuric acid method with glucose as the standard. In addition, the UV absorption at 260 nm and 280 nm was measured to confirm that the recovered fraction did not contain nucleic acids and proteins. 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 Biochem Co., Ltd.).

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

The mice were operated according to the guidelines for appropriate conduct of animal experiments issued by the Japan Academic Conference in 2006. After 1 week of feeding, the mice were euthanized by carbon dioxide, and the Peyer's lymph nodes of the small intestine were removed by laparotomy.

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

(IgA determination) The lipoteichoic acid obtained in Example 2 was added to a suspension of Peyer's lymph node cells at a final concentration of 50 μg/ml, and co-cultured in a 96-well T cell activation plate (Becton Dickinson) for 5 days at 37°C and 5% CO _2. The amount of IgA in the obtained culture supernatant was then determined using a mouse IgA ELISA kit (Bethyl Laboratories).

The results are shown in FIG1 . In FIG1 , the results of physiological saline as a negative control are arranged at the top. In FIG1 , "a", "b" and "c" indicate that there is a significant difference (P < 0.05) between the groups labeled with different letters. The results of the experiment showed that the lipoteichoic acid derived from the lactic acid bacteria of the genus Lactobacillus, namely, the enzyme Lactobacillus 10H strain, the Lactobacillus kunsii strain JCM16173 strain and the Lactobacillus swarming strain JCM30765 strain, had a significantly higher IgA production inducing ability than the lipoteichoic acid derived from other lactic acid bacteria.

Based on the above results, it can be expected that lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus, especially lipoteichoic acid derived from lactic acid bacteria belonging to the enzyme Lactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum, and further lipoteichoic acid derived from the enzyme Lactobacillus kosoi 10H strain, Apilactobacillus kunkeei JCM16173 strain or Apilactobacillus apinorum JCM30765 strain, can be used as an immunostimulant with significant effects.

[Example 4] Analysis of gene expression in dendritic cells (generation of bone marrow-derived dendritic cells from mouse bone marrow cells) Bone marrow-derived dendritic cells were generated using bone marrow cells from the femur and tibia of 4-week-old female BALBc/A mice (purchased from CREA Japan). Bone marrow cells collected from mice were washed, and the cell number was set to 1×10 ⁶ cells/ml, and suspended in RPMI 1640 medium (PSMF) to which granulocyte macrophage colony stimulating factor (manufactured by PeproTech) was added at a concentration of 20 ng/mL, and cultured at 37°C and 5% CO ₂ . On the 3rd and 5th day of culture, half of the culture medium was replaced with a new medium. On the 6th day of culture, cells including dendrites were collected, magnetically labeled with anti-CD11c microbeads (Miltenyi Biotec), and dendrites were separated using AutoMACS (Miltenyi Biotec) according to conventional methods.

(Gene expression analysis) The bone marrow-derived dendritic cells obtained as described above were cultured at 1.0×10 ⁹ cells/well (3 ml) in RPMI 1640 medium (PSMF) for 6 hours. The culture was performed for both the cells containing lipoteichoic acid derived from the enzyme Lactobacillus mellifera 10H strain at a concentration of 50 μg/ml and the cells without lipoteichoic acid (control). Thereafter, total RNA was isolated from the bone marrow-derived dendritic cells using the QuickPrep Total RNA Extraction Kit (manufactured by GE Healthcare), and cDNA was synthesized from the total RNA using the SuperScript (registered trademark) III Reverse Transcription Kit (manufactured by Invitrogen). The real-time PCR method was carried out using the 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 for amplifying DNA.

(Primers for IL-6 expansion: bone marrow-derived dendritic cell IL-6 PCR primers) Forward: 5'-AATAGTCCTTCCTACCCCAATTTC-3' (SEQ ID NO. 1) Reverse: 5'-ATTTCAAGATGAATTGGATGGTCT-3' (SEQ ID NO. 2) (Primers for IL-10 expansion: bone marrow-derived dendritic cell IL-10 PCR primers) Forward: 5'-ATGCAGGACTTTAAGGGTTACTTG-3' (SEQ ID NO. 3) Reverse: 5'-GAATTCAAATGCTCCTTGATTTCT-3' (SEQ ID NO. 4) (Primers for RALDH2 expansion: bone marrow-derived dendritic cell RALDH2 PCR primers) Forward: 5'-GACTTGTAGCAGCTGTCTTCACT-3' (SEQ ID NO. 5) Reverse: 5'-TCACCCATTTCTCTCCCATTTCC-3' (SEQ ID NO: 6)

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

(GAPDH amplification primers: bone marrow-derived dendritic cell 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 Inc.). The results were analyzed using one-way ANOVA and Dunnett's post-hoc analysis. ***p<0.001

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

Based on the above results, it can be expected that lipoteichoic acid derived from lactic acid bacteria of the genus Lactobacillus , especially lipoteichoic acid derived from lactic acid bacteria belonging to the enzyme Lactobacillus kosoi, and further lipoteichoic acid derived from the enzyme Lactobacillus kosoi 10H strain, can be used as a gene expression promoter that promotes the expression of IL-6, IL-10 and retinal aldehyde dehydrogenase 2 (RALDH2) in dendrites.

[Example 5] Analysis of the glycerophosphate chain of lipoteichoic acid The analysis of the glycerophosphate chain of lipoteichoic acid (repeating structure, polymer part) was carried out by obtaining 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 Fuji Film Wako Pure Chemical Industries, Ltd.). The ¹ H-NMR spectrum was obtained at 25°C using a 500 MHz Varian Unity Inova 500 spectrometer (manufactured by Agilent Technologies). As a reference substance for chemical shift, 3-(trimethylsilyl) sodium propionate-2,2,3,3-d ₄ (purchased from Fuji Film Wako Pure Chemical Industries, Ltd.) was used.

The results are shown in Figures 3 and 4. First, the structure of the glycerophosphate chain in lipoteichoic acid derived from lactic acid bacteria other than Lactobacillus melitensis will be described.

Regarding the lipoteichoic acid from Lactobacillus plantarum strain JCM1149, the peaks were assigned with reference to a previous paper (Hatano et al. Scavenger receptor for lipoteichoic acid is involved in the potent ability of Lactobacillus plantarum strain L - 137 to stimulate production of interleukin - 12p40. International Immunopharmacology, 25 : 321 - 331, 2015) (see Figure 3(b)). It is believed that the lipoteichoic acid from Lactobacillus plantarum strain JCM1149 has a glycerophosphate chain composed of GroP units, AlaGroP units, and GlcGroP units.

Regarding lipoteichoic acid from Lactobacillus rhamnosus GG, the peaks were assigned with reference to a previous paper (Claes et al. Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG. Microbial Cell Factories, 11: 161-168, 2012) (see Figure 3(c)). It is believed that Lactobacillus rhamnosus GG has a glycerophosphate chain composed of GroP units and AlaGroP units.

The above-mentioned structures of the glycerophosphate chain in the lipoteichoic acid from Lactobacillus plantarum strain JCM1149 and Lactobacillus rhamnosus strain GG are structures commonly seen in lipoteichoic acid in lactic acid bacteria of the genus Lactobacillus.

On the other hand, the ¹ H-NMR spectrum of lipoteichoic acid from enzyme Lactobacillus mellifera 10H strain is significantly different from the ¹ H-NMR spectrum of lipoteichoic acid from plant lactobacillus plantarum JCM1149 strain and rhamnosus casei GG strain (see Figure 3(a)). The attribution of each peak is currently underway. If past reports and the results of ¹³ C-NMR and two-dimensional NMR (not shown) are also taken into account, it is believed that lipoteichoic acid from enzyme Lactobacillus mellifera 10H strain may have a GlcGroP unit. However, since there are many unknown peaks in addition, they will continue to be clarified through structural analysis in the future. At least it is believed that the glycerophosphate chain in lipoteichoic acid from enzyme Lactobacillus mellifera 10H strain has a unique structure that is not a normal structure.

In addition, the ¹ H-NMR spectrum of lipoteichoic acid from Lactobacillus kunsii JCM16173 strain (see Figure 4(b)) and the ¹ H-NMR spectrum of lipoteichoic acid from Lactobacillus mellifera JCM30765 strain (see Figure 4(c)) also show very similar results to the ¹ H-NMR spectrum of lipoteichoic acid from Lactobacillus mellifera 10H strain (see Figure 4(a); Figure 4(a) is the same as Figure 3(a)). Therefore, it is believed that the glycerophosphate chain in lipoteichoic acid from lactic acid bacteria of the genus Lactobacillus is very different from the glycerophosphate chain in the usual lipoteichoic acid from lactic acid bacteria of the genus Lactobacillus, and has a certain degree of common structure within the genus.

[Example 6] Analysis of lipoteichoic acid-anchored sugar lipids Analysis of lipoteichoic acid-anchored sugar lipids was performed by obtaining MALDI-TOF MS spectra.

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

(Acquisition of MALDI-TOF MS spectrum) The anchored glycolipid was dissolved in 100 μl of chloroform/methanol (2:1, v/v), and then mixed with the same amount of matrix agent (10 mg/ml 2,5-dihydroxybenzoic acid (DHBA) in water/methanol (7:3, v/v) containing 0.1% trifluoroacetic acid (TFA)) on a target plate. After the mixture was co-crystallized, MALDI-TOF mass spectra were acquired in cation mode and reflectron mode. As a mass analyzer, 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 anchoring sugar lipid in lipoteichoic acid derived from lactic acid bacteria other than Lactobacillus melitensis will be described.

The main structure of the anchored sugar lipid in the lipoteichoic acid from the plant milk bacterium Plantarum JCM1149 strain is considered to be Hex ₃ DAG, that is, a structure with diacylglycerol bonded to a trisaccharide (see Figure 5(b)). In addition, there is also a peak that is considered to be produced by AcylHex ₃ DAG, but the peak intensity is weak and hidden in the background. Furthermore, there is also a peak (956) that is considered to be produced by Hex ₂ DAG, that is, a structure with diacylglycerol bonded to a disaccharide, but its peak intensity is also weak and it is considered that it cannot be said to exist for sure.

The main structure of the sugar-anchored lipid in the lipoteichoic acid of Lactobacillus rhamnosus GG strain is Hex ₃ DAG (see Figure 5(c)). Regarding the peak that is believed to be produced by AcylHex ₃ DAG, only one peak (1368) is visible, and the peak is also weak. Therefore, the sugar-anchored lipid in the lipoteichoic acid of Lactobacillus rhamnosus GG strain may not have AcylHex ₃ DAG. Regarding the peak that is believed to be produced by Hex ₂ DAG, although it exists in the same way as in the case of Plantarum plantarum JCM1149 strain (942, 956), the peak intensity is still weak.

In conclusion, the structure of the glycolipid anchored in the lipoteichoic acid from the plant lactobacillus plantarum strain JCM1149 and the rhamnosus casei strain GG is considered to be Hex ₃ DAG. The trisaccharide-anchored glycolipid is a common structure of lipoteichoic acid in lactic acid bacteria of the genus Lactobacillus.

On the other hand, the main structure of the sugar-anchored lipid in the lipoteichoic acid derived from the enzyme Lactobacillus mellifera 10H strain is considered to be Hex ₂ DAG (942, 956, 982) (see Figure 5 (a)). Disaccharide-anchored lipids are common structures in the lipoteichoic acid derived from the lactic acid bacteria of the Enterococcus genus, the Lactococcus genus, and the Leuconostoc genus. On the other hand, the most common lipoteichoic acid derived from the lactic acid bacteria of the Lactobacillus genus is a trisaccharide or tetrasaccharide-anchored lipid. However, although the lactic acid bacteria of the Lactobacillus mellifera genus belong to the Lactobacillus genus, it can be confirmed that the sugar-anchored lipid in the lipoteichoic acid is mainly a disaccharide. Furthermore, the anchoring sugar lipids in the lipoteichoic acid derived from the enzyme Lactobacillus apis strain 10H do not have the anchoring sugar lipids with tri-residue fatty acids that are commonly found in the lipoteichoic acid derived from various lactic acid bacteria of the genus Lactobacillus or Lactococcus. Therefore, the structure of the anchoring sugar lipids in the lipoteichoic acid derived from the enzyme Lactobacillus apis strain 10H is very different from the structure of the anchoring sugar lipids common to the representative lactic acid bacteria identified previously.

In addition, the MALDI-TOF MS spectra of the sugar-anchored lipids in lipoteichoic acid from Lactobacillus kunsii strain JCM16173 (see Figure 6(b)) and the MALDI-TOF MS spectra of the sugar-anchored lipids in lipoteichoic acid from Lactobacillus kunsii strain JCM30765 (see Figure 6(c)) also showed similar results to the MALDI-TOF MS spectra of the sugar-anchored lipids in lipoteichoic acid from Lactobacillus kunsii strain 10H (see Figure 6(a); furthermore, Figure 6(a) is the same as Figure 5(a)) in terms of Hex ₂ DAG being the main structure. Therefore, it is believed that the glycosyl lipids in the lipoteichoic acid derived from the lactic acid bacteria of the genus Lactobacillus are also very different from the glycosyl lipids in the common lipoteichoic acid derived from the lactic acid bacteria of the genus Lactobacillus, and have a certain degree of common structure within the genus.

According to the above-mentioned Examples 5 and 6, it was found that the lipoteichoic acid derived from the lactic acid bacteria of the genus Lactobacillus has a structure that is completely different from the general lipoteichoic acid derived from the lactic acid bacteria of the genus Lactobacillus. It is believed that the difference in structure between the lipoteichoic acid derived from the lactic acid bacteria of the genus Lactobacillus and the general lipoteichoic acid derived from the lactic acid bacteria of the genus Lactobacillus is related to the fact that the lipoteichoic acid derived from the lactic acid bacteria of the genus Lactobacillus shows a higher IgA production inducing ability.

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

TWI838990B_111145803_SEQL.xml

Claims (15)

A gastrointestinal immune activator comprising lipoteichoic acid derived from lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum as an active ingredient. For example, the intestinal immune activator of claim 1, wherein the lactic acid bacteria are enzyme bee lactobacillus (Apilactobacillus kosoi) 10H strain, Kunkeei bee lactobacillus (Apilactobacillus kunkeei) JCM16173 strain or bee colony bee lactobacillus (Apilactobacillus apinorum) JCM30765 strain. For example, the intestinal immune activator in claim 1 or 2 is in the form of food, medicine, feed, or a combination of active ingredients formulated in any of the above. An IgA production promoter comprising lipoteichoic acid derived from lactic acid bacteria belonging to Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum as an active ingredient. As in claim 4, the IgA production promoter, wherein the lactic acid bacteria are enzyme bee lactobacillus (Apilactobacillus kosoi) 10H strain, Kunkeei bee lactobacillus (Apilactobacillus kunkeei) JCM16173 strain or bee colony bee lactobacillus (Apilactobacillus apinorum) JCM30765 strain. For example, the IgA production promoter in claim 4 or 5 is in the form of a food, medicine, feed, or a combination of active ingredients formulated in any of the above. A gene expression promoter, comprising lipoteichoic acid derived from lactic acid bacteria belonging to the enzyme bee lactobacillus (Apilactobacillus kosoi) as an active ingredient, and promoting the expression of at least one factor among IL-6, IL-10 and retinal aldehyde dehydrogenase 2 (RALDH2) in dendrite cells. As in claim 7, the gene expression promoter, wherein the lactic acid bacteria is the enzyme bee lactobacillus (Apilactobacillus kosoi) 10H strain. For example, the gene expression enhancer in claim 7 or 8 is in the form of food, medicine, feed, or a combination of active ingredients formulated therein. A use of lipoteichoic acid derived from lactic acid bacteria belonging to the enzyme Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum for the manufacture of medicines, beverages, feeds or active ingredient compositions formulated therein for intestinal immunity of humans or non-human animals. For example, the use of lipoteichoic acid in claim 10, wherein the lactic acid bacteria are enzyme bee lactobacillus (Apilactobacillus kosoi) 10H strain, Kunkeei bee lactobacillus (Apilactobacillus kunkeei) JCM16173 strain or bee colony bee lactobacillus (Apilactobacillus apinorum) JCM30765 strain. A use of lipoteichoic acid derived from lactic acid bacteria belonging to the enzyme Apilactobacillus kosoi, Apilactobacillus kunkeei or Apilactobacillus apinorum for the manufacture of a pharmaceutical, food, feed or an active ingredient composition formulated therein for promoting IgA production in humans or non-human animals. For example, the use of lipoteichoic acid in claim 12, wherein the lactic acid bacteria are enzyme bee lactobacillus (Apilactobacillus kosoi) 10H strain, Kunkeei bee lactobacillus (Apilactobacillus kunkeei) JCM16173 strain or bee colony bee lactobacillus (Apilactobacillus apinorum) JCM30765 strain. A use of lipoteichoic acid derived from lactic acid bacteria belonging to the enzyme bee lactobacillus (Apilactobacillus kosoi) for the preparation of a gene expression-promoting pharmaceutical, food, feed, or an active ingredient composition formulated therein for promoting the expression of at least one factor among IL-6, IL-10 and retinal aldehyde dehydrogenase 2 (RALDH2) in dendrite cells in humans or non-human animals. For example, the use of lipoteichoic acid in claim 14, wherein the lactic acid bacteria is the enzyme bee lactobacillus (Apilactobacillus kosoi) 10H strain.