JP2021101711A - LACTIC ACID BACTERIUM HAVING HIGH AhR ACTIVATING ABILITY - Google Patents

Abstract

To provide a lactic acid bacterium having high activating ability of aromatic hydrocarbon receptor (AhR).SOLUTION: Provided is a lactic acid bacterium being Lactobacillus johnsonii (NITE BP-02003) that increases secretion of secretion-type alkali phosphatase being an activation indicator of aromatic hydrocarbon receptor in a transformed HeXS34 cell to 14 times or more comparing with a PBS group, and a composition for oral ingestion containing a cultured product, a secretion product, a dead bacterial cell of the lactic acid bacterium or a pulverized product thereof.SELECTED DRAWING: Figure 1

Description

The present invention relates to a lactic acid bacterium that exerts an anti-inflammatory effect in the intestine by activating an aromatic hydrocarbon receptor (AhR).

The aromatic hydrocarbon receptor (AhR) is a transcription factor belonging to the bHLH-PAS family, which is expressed in most cells and tissues, and causes transcriptional activation of various genes by binding of a ligand. It has been known. Compounds such as dioxin and polychlorinated biphenyl are known as ligands for AhR.

Immunological findings indicate that activation of AhR improves autoimmune diseases such as type I diabetes, autoimmune vasculitis, and polysclerosis through induction of regulatory T cells and suppression of Th17 cell differentiation. It is known to contribute to the improvement of food allergies such as peanut allergies. From this, it is considered that substances and materials having a high ability to activate AhR may be useful substances capable of suppressing an excessive immune response such as autoimmune diseases and allergies.

So far, various explorations and studies have been conducted on substances and materials having a high ability to activate AhR. For example, in Japanese Patent No. 5919193 (Patent Document 1), as a lactic acid bacterium having an ability to activate AhR, Lactobacillus delbruecchii subsp. bulgaricus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. Cremoris, Streptococcus thermophilus are described. Japanese Unexamined Patent Publication No. 2011-162482 (Patent Document 2) describes an enteric cancer inhibitor containing a substance that activates an aromatic hydrocarbon receptor as an active ingredient, and indole is a specific active ingredient. Derivatives and flavonoids are listed. However, there is a demand for substances and materials that have a higher ability to activate AhR, are in a form that is easy to ingest, are safe, and have no side effects.

Japanese Patent No. 5919193 Japanese Unexamined Patent Publication No. 2011-162482

Against this background, the present invention focused on lactic acid bacteria as a safe and side-effect-free material that is easy to ingest. An object of the present invention is to provide a lactic acid bacterium having an unprecedented high ability to activate AhR.

In order to solve the above problems, the present inventors focused on a predetermined lactic acid bacterium as a material that can be easily ingested in a familiar manner, and examined in detail the ability to activate AhR. As a result, they have found that among lactic acid bacteria, Lactobacillus jhonsiini and Lactobacillus crispatus, which satisfy certain conditions, have a remarkable ability to activate AhR, and have completed the present invention.

That is, the present invention is as follows.
[1] A composition for oral ingestion for activating an aromatic hydrocarbon receptor (AhR), which contains Lactobacillus jhonsiii.
[2] By adding cell treatment (weight concentration of lyophilized cells is 50 mg / mL) to HeXS34 cells at 37 ° C. for 24 hours, Lactobacillus jonsonii having 14 times or more SEAP activity as compared with the PBS group was obtained. The composition for oral ingestion according to [1], which is contained.
[3] The composition for oral ingestion according to [1] or [2], which comprises the culture, secretions, killed cells, or crushed product thereof as Lactobacillus jhonsiii.

According to the present invention, it is possible to provide a lactic acid bacterium having an unprecedented high ability to activate AhR. By ingesting the lactic acid bacterium of the present invention or, for example, fermented milk prepared by using the lactic acid bacterium of the present invention, an anti-inflammatory effect by activating AhR can be obtained. In particular, lactic acid bacteria are foods with abundant eating experience and can be easily and safely ingested in a familiar manner, and thus can greatly contribute to the suppression of excessive immune responses such as autoimmune diseases and allergies.

It is a graph which shows the result of having compared the SEAP activity of the strains shown in Table 1 in an Example. The horizontal bar shows the standard deviation of the measured values of each cell.

The present invention relates to Lactobacillus crispatus and Lactobacillus jhonsiii having the ability to activate aromatic hydrocarbon receptors (AhR). In the present invention, the "AhR activating ability" refers to the ability to activate the signal transduction pathway initiated by AhR activation, and the activation mechanism may be any. Therefore, the bacterial cell itself does not necessarily have to be a ligand for AhR. For example, the secretory substance produced by the bacterial cell may have an AhR activating ability, or AhR is activated by a dead bacterial cell or a crushed product thereof. May be done. Therefore, when referring to a specific bacterium in the present invention, not only the live bacterium itself but also a dead bacterium or a crushed product thereof, and a culture or secretion of the bacterium are included.

In the present invention, the method for evaluating the AhR activation ability is not particularly limited, and the AhR activation ability can be measured and evaluated by a known method. Examples of the method for evaluating the AhR activation ability include a method using secretory alkaline phosphatase (SEAP) activity as an index and a method for measuring the expression level of CYP1A1 in vitro using a predetermined human cell. From the viewpoint of simplicity, a method of measuring and evaluating SEAP activity is preferable.

The Lactobacillus crispatus and Lactobacillus jhonsiini of the present invention have an AhR activating ability and can exert an anti-inflammatory effect. Therefore, Lactobacillus crispatus and Lactobacillus jhonsiii of the present invention can be used as active ingredients of anti-inflammatory agents. When Lactobacillus crispatus or Lactobacillus jonsonii of the present invention is used as an active ingredient of an anti-inflammatory agent, live bacteria, dead bacteria, cultures and processed products thereof can be used. The culture is the one in which the culture supernatant and the medium component after the completion of the culture of Lactobacillus crispatus and Lactobacillus jonsonii of the present invention are used as they are, and the processed product is not particularly limited as long as it is derived from the culture, but for example, Examples thereof include those obtained by processing such as concentration, pasting, spray drying, freeze drying, vacuum drying, drum drying, liquefaction, dilution, and crushing of the culture. A known method can be appropriately used for these processes. In the culture or the processed product, the Lactobacillus crispatus and Lactobacillus jhonsiii of the present invention may be live or dead. From the viewpoint of suppressing the growth of harmful intestinal bacteria and improving the intestinal environment by ingestion, it is preferably contained in the composition as a viable bacterium.

In addition, Lactobacillus crispatus and Lactobacillus jhonsiii of the present invention, their cultures, or processed products thereof, appropriately include, for example, medium components, additives suitable for oral ingestion, and solvents such as water, sugars, proteins, lipids, and the like. Optional ingredients such as vitamins, bioessential trace metals, fragrances, pharmaceutically acceptable carriers, and food additives can be added to prepare a composition for oral ingestion such as a pharmaceutical composition or a food composition.

As used herein, the term "composition for oral ingestion" means any composition that can be ingested orally. Therefore, the composition for oral intake includes, but is not limited to, for example, a beverage composition, a food composition, a feed composition, a pharmaceutical composition, and the like.

The beverage composition of the present invention typically comprises one or more selected from Lactobacillus crispatus and Lactobacillus jhonsiii having AhR activating ability, a culture thereof and a processed product thereof. The beverage composition of the present invention further contains sugars, proteins, lipids, vitamins, bioessential trace metals (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.) as long as they do not interfere with the growth of Lactobacillus crispatus and Lactobacillus jhonsonii. ), Perfume and other formulations can be included. Such a beverage composition improves the intestinal environment of the ingested individual and exerts an ameliorating and / or preventive effect on inflammatory diseases.

The food composition of the present invention typically comprises one or more selected from Lactobacillus crispatus and Lactobacillus jhonsiii having AhR activating ability, cultures thereof and processed products thereof. The food composition of the present invention further includes sugars, proteins, lipids, vitamins, and bioessential trace metals (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.) as long as they do not interfere with the growth of Lactobacillus crispatus and Lactobacillus jhonsonii. , Fragrances and other formulations can be included. Such a food composition improves the intestinal environment of the ingested individual and exerts an ameliorating and / or preventive effect on inflammatory diseases.

Examples of carbohydrates include sugars, modified starches (in addition to dextrin, soluble starches, oxidized starches, starch esters, starch ethers, etc.), dietary fiber and the like. Examples of proteins include total fat powder, defatted milk powder, partially defatted milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin. , Α-lactoalbumin, lactoferrin, soybean protein, chicken egg protein, meat protein and other animal and vegetable proteins, their hydrolyzates; butter, milky minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, lactose, etc. Examples of various milk-derived components of.

Examples of lipids include animal fats and oils such as lard, fish oil, and other fractionated oils, hydrogenated oils, and ester exchange oils; palm oil, safflower oil, corn oil, rapeseed oil, palm oil, and these fractionated oils. Examples thereof include vegetable oils and fats such as hydrogenated oils and ester exchange oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, and choline. , Folic acid and the like, and examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium and the like.

The categories and types of the beverage composition and the food composition of the present invention are not limited, and may be functional foods, foods for specified health use, foods for specified use, foods with nutritional function, health foods, foods for nursing care, and confectionery. It may be a lactic acid bacterium beverage, a dairy product such as cheese or yogurt, a seasoning, or the like. There are no restrictions on the shape of foods and drinks, and any foods and drinks that can be normally distributed, such as solid, liquid, liquid foods, jelly, tablets, granules, and capsules, can be taken, and various foods (milk, May be added to soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, formula milk powder, liquid foods, sick foods, nutritional foods, frozen foods, processed foods and other commercial foods. .. The above-mentioned food and drink can be produced by a conventional method of those skilled in the art.

As described above, the Lactobacillus crispatus and Lactobacillus jhonsiii of the present invention, their cultures, or their processed products can be processed into general foods and drinks including dairy products and fermented milk, and the production of dairy products and fermented milk such as yogurt and cheese. It can also be used as a starter for. When it is used as a starter, other microorganisms may be mixed as long as it does not interfere with the survival and proliferation of the probiotics of the present invention and as long as it does not interfere with the production of dairy products. For example, Lactobacillus delbruecchii subsp., Which is a major bacterial species as a lactic acid bacterium for yogurt. It may be mixed with bulgaricus, Streptococcus thermophilus, Lactobacillus acidofilus, etc., or may be mixed with other bacterial species generally used for yogurt or cheese to obtain a starter. Dairy products and fermented milk can be produced by the above starter according to a conventional method. For example, plain yogurt can be produced by mixing the above starter with milk or dairy products cooled after heating, mixing, homogenization, and sterilization, and fermenting and cooling.

The feed composition of the present invention typically comprises one or more selected from Lactobacillus crispatus and Lactobacillus jhonsiii having AhR activating ability, a culture thereof and a processed product thereof. The feed composition of the present invention further contains sugars, proteins, lipids, vitamins, and bioessential trace metals (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.) as long as they do not interfere with the growth of Lactobacillus crispatus and Lactobacillus jhonsonii. , Fragrances and other formulations can be included. Such a feed composition improves the intestinal environment of the ingested individual and exerts an ameliorating and / or preventive effect on inflammatory diseases.

The pharmaceutical composition of the present invention typically comprises one or more selected from Lactobacillus crispatus and Lactobacillus jhonsiii having AhR activating ability, a culture thereof and a processed product thereof. Such pharmaceutical compositions improve the intestinal environment of ingested individuals and have therapeutic, ameliorating and / or prophylactic effects on inflammatory diseases. The administration route of such a pharmaceutical composition is not particularly limited, but includes oral or parenteral administration, and examples thereof include oral administration, tube administration, and enteral administration. Oral administration is preferable from the viewpoint of convenience and safety. The dosage form is not particularly limited, but can be appropriately selected depending on the administration route. For example, an aerosol agent, a liquid agent, an extract agent, an elixir agent, a capsule agent, a granule, a pill, an eye ointment agent, or a transdermal agent. Absorbent preparations, suspending agents, emulsions, suppositories, powders, alcoholic preparations, tablets, syrups, dipping agents, decoctions, injections, patches, tinctures, eye drops, troches, ointments, poultices, aromatic waters. Examples include agents, liniment agents, limonade agents, flow extracts, and lotions.

The carriers that can be used in the pharmaceutical composition of the present invention include surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders and disintegrants. , Lubricants, fluidity promoters, flavoring agents and the like are listed as pharmaceutically acceptable carriers, but other commonly used carriers can be appropriately used. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, Examples thereof include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts.

Hereinafter, the present invention will be described in more detail based on Examples. It should be noted that this example does not limit the present invention. Regarding the microorganisms used in the examples, the strain described as JCM in the strain name is a reference strain obtained from the Japan Collection of Microorganisms, BioResource Center, RIKEN. The strain described as NCIMB in the strain name is a reference strain obtained from the National Collection of Industrial and Marine Bacteria in the United Kingdom. The strain described as MEP in the strain name is a strain owned by Meiji Co., Ltd.

In addition, Lactobacillus jhonsiii OLL203565 was sent to the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture) on February 3, 2015, with a consignment number of NITE BP. It is a strain deposited as -0203.
[Example]
HeXS34 cells were prepared according to previously reported (Kasai et al., Toxicol Appl Pharmacol. 2006; 211 (1): 11-19). Briefly, pXRE-SEAP, in which the SEAP gene was introduced downstream of two copies of the XRE consensus sequence (tctccagcaactccg), was transferred to HEPA-1c1c7 cells (Muscle Hepatoma cell line, American Type Culture Collection (Manassas, VA), USA). HeXS34 cells were prepared by stable transformation.

The HeXS34 cells were maintained in MEMα medium (manufactured by Invitrogen) supplemented with 10% fetal bovine serum (FBS), and then ^{seeded in 96-well plates at 1 × 10 4} cells / 100 μL per well. , 18 hours culture. Next, 10% of each lactic acid bacterium cell shown in Table 1 which had been heat sterilized and prepared at 50 mg / mL was added to the cells (final concentration; 5 mg / mL). For negative control, PBS (phosphate buffered saline) was added instead of the cells. After the addition of the cells, the cells were cultured at 37 ° C. for 24 hours, and the secretory alkaline phosphatase (SEAP) activity in the culture medium supernatant was measured as an index of AhR activation ability. The SEAP activity was measured 3 times for each sample using the Great EscAPe SEAP detection kit (manufactured by Clontech), and the average value of the obtained luminescence intensity (LU) was used for evaluation.

The measurement results of the SEAP activity of each strain of the example are shown in FIG. In Lactobacillus crispatus and Lactobacillus jhonsonii, luminescence intensity of 5000 or more was observed in any of the strains, suggesting that they have high AhR activation ability. Among Lactobacillus crispatus and Lactobacillus jhonsonii, Lactobacillus crispatus MEP201602, which had the lowest SEAP activity, also showed 14.5 times as much SEAP activity as the negative control.

According to the present invention, it is possible to provide a lactic acid bacterium having an unprecedented high ability to activate AhR, and by ingesting the lactic acid bacterium and a composition containing the lactic acid bacterium, it is possible to obtain an anti-inflammatory effect by activating AhR. it can. In particular, lactic acid bacteria are foods with abundant eating experience and can be easily and safely ingested in a familiar manner, so that they can greatly contribute to the suppression of excessive immune responses such as autoimmune diseases and allergies.

Claims (3)

A composition for oral ingestion for activating aromatic hydrocarbon receptors (AhRs), which contains Lactobacillus jhonsiii. By adding cell treatment to HeXS34 cells at 37 ° C. for 24 hours (weight concentration of lyophilized cells is 50 mg / mL), Lactobacillus jonsonii having 14 times or more SEAP activity as compared with the PBS group is contained. The composition for oral ingestion according to claim 1. The composition for oral ingestion according to claim 1 or 2, which comprises the culture, secretions, killed cells, or crushed product thereof as Lactobacillus jhonsiii.

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