KR20240132831A - Novel Lactobacillus curvatus NE416 strain and use thereof - Google Patents

Abstract

The present invention relates to a novel Lactobacillus curvatus NE416 strain and uses thereof, and more particularly, the present invention relates to Lactobacillus curvatus NE-416 strain (Accession No.: KCTC15309BP), a pharmaceutical composition containing the strain as an active ingredient for preventing or treating Graves' disease, and a health functional food for preventing or improving Graves' disease. The novel Lactobacillus curvatus NE416 strain according to the present invention has effects of preventing, improving, and treating Graves' disease, such as reducing the concentration of thyroid hormones increased in the blood due to Graves' disease and reducing the size of the thyroid gland, and thus can be usefully used in the manufacture of a new therapeutic agent and health functional food for Graves' disease.

Description

{Novel Lactobacillus curvatus NE416 strain and use thereof}

The present invention relates to a novel Lactobacillus curbatus NE-416 strain and its use.

Graves' disease (GD) is an autoimmune disease that affects the thyroid gland. The patient's own immune cells produce antibodies that can bind to thyroid cells. These antibodies stimulate the thyroid gland to produce excessive amounts of thyroid-stimulating hormone. These antibodies also react to the tissue behind the eye, causing eye symptoms such as bulging eyes.

Meanwhile, the exact cause of Graves' disease has not been identified, but it is predicted that genetic and environmental factors may interact in combination.

Treatment methods for Graves' disease include antithyroid drugs that reduce thyroid hormone production, radioactive iodine therapy, and thyroidectomy (surgical removal). In fact, it is dangerous to perform surgery directly on patients with hyperthyroidism, so antithyroid drugs are administered before thyroidectomy to induce hypothyroidism before surgery.

Antithyroid drug treatment requires 6 months to 2 years to be effective, and hyperthyroidism can recur when the drug is discontinued. The risk of recurrence is approximately 40-50%, and lifelong antithyroid drug treatment can cause side effects such as agranulocytosis and liver disease. In addition, there is the problem of a potentially fatal decrease in white blood cell count as a side effect of antithyroid drugs. Radioactive iodine therapy is the most common treatment used in the United States, but it has the disadvantage of requiring daily thyroid hormone replacement, is used slowly (over months to years) to destroy the thyroid, and may not be effective for all patients.

Therefore, there is a need to develop a new treatment for Graves' disease that has excellent therapeutic effects without causing side effects in the body.

Republic of Korea Publication Patent No. 10-2021-0089214

Accordingly, the inventors of the present invention completed the present invention by confirming that a novel Lactobacillus curbatus NE416 strain isolated from kimchi has excellent preventive, ameliorating, and therapeutic effects on Graves' disease while conducting research to discover a new therapeutic agent that is safe for the body and targets lactic acid bacteria, while solving the problems of conventional Graves' disease treatment methods.

Therefore, the purpose of the present invention is to provide Lactobacillus curvatus NE-416 strain (Accession Number: KCTC15309BP).

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating Graves' disease, comprising Lactobacillus curvatus NE-416 strain (Accession No.: KCTC15309BP) as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving Graves' disease, comprising Lactobacillus curvatus NE-416 strain (Accession No.: KCTC15309BP) as an effective ingredient.

In order to achieve the above-described purpose of the present invention, the present invention provides Lactobacillus curvatus NE-416 strain (Accession Number: KCTC15309BP).

In one embodiment of the present invention, the strain may be isolated and identified from kimchi.

In addition, the present invention provides a pharmaceutical composition for preventing or treating Graves' disease, comprising Lactobacillus curvatus NE-416 strain (Accession No.: KCTC15309BP) as an active ingredient.

In one embodiment of the present invention, the strain may be selected from among a cell of the strain, a fragment of the cell, a culture of the strain, a culture solution from which the cell is removed from a culture of the strain, an extract of the cell of the strain, an extract of the culture of the strain, or an extract of the culture solution from which the cell is removed from a culture of the strain.

In one embodiment of the present invention, the strain may be isolated and identified from kimchi.

In one embodiment of the present invention, the composition may have an effect of reducing the concentration of thyroid hormone in the blood increased by Graves' disease and reducing the size of the thyroid gland.

In addition, the present invention provides a health functional food for preventing or improving Graves' disease, comprising Lactobacillus curvatus NE-416 strain (Accession No.: KCTC15309BP) as an effective ingredient.

In one embodiment of the present invention, the strain may be selected from among a cell of the strain, a fragment of the cell, a culture of the strain, a culture solution from which the cell is removed from a culture of the strain, an extract of the cell of the strain, an extract of the culture of the strain, or an extract of the culture solution from which the cell is removed from a culture of the strain.

In one embodiment of the present invention, the strain may be isolated and identified from kimchi.

In one embodiment of the present invention, the composition may have an effect of reducing the concentration of thyroid hormone in the blood increased by Graves' disease and reducing the size of the thyroid gland.

The novel Lactobacillus curbatus NE416 strain according to the present invention has the effect of preventing, improving and treating Graves' disease by reducing the concentration of thyroid hormones increased in the blood due to Graves' disease and reducing the size of the thyroid gland, and thus can be usefully used in the manufacture of a new therapeutic agent for Graves' disease and a health functional food.

Figure 1 shows an experimental schedule for establishing a Graves' disease mouse model and administering lactic acid bacteria isolated from kimchi in one embodiment of the present invention.
Figure 2 shows the results of analyzing the concentration of thyroid hormone (T4) in the blood of each experimental mouse group to confirm the efficacy of lactic acid bacteria isolated from kimchi in treating Graves' disease in one embodiment of the present invention.
Figure 3 is a photograph of the morphology of the new strain Lactobacillus curvatus NE-416 isolated and identified in the present invention observed by an electron microscope.
Figure 4 is a photograph comparing the size and color of thyroid glands isolated from a group of Graves' disease mice (Ad-TSHR) administered with an adenovirus that induces TSHR expression (Ad-TSHR), a group of mice administered with Ad-TSHR and the novel Lactobacillus curbatus NE-416 strain of the present invention, and a control group (PBS, EGFP).
Figure 5 shows a photograph of thyroid cells isolated from each mouse group of Figure 4 observed under a microscope.
Figure 6 shows the results of measuring the concentration of thyroid hormone (T4) in the blood of a group of mice for Graves' disease administered an adenovirus (Ad-TSHR) that induces TSHR expression, a group of mice administered Ad-TSHR and the new strain of Lactobacillus curbatus NE-416 of the present invention together, a control group (PBS, EGFP) mice, and a group of mice administered PTU, a Graves' disease treatment drug, as a positive control group.

The present invention is characterized by the fact that it has been found that a novel strain of Lactobacillus curbatus NE416 can be used as a new therapeutic agent for preventing or treating Graves' disease (GD).

The present inventors, while conducting research to develop a microbial therapeutic agent rather than the conventional synthetic compounds as a new treatment for Graves' disease, isolated a number of lactic acid bacteria from kimchi, and selected the lactic acid bacteria that can most effectively suppress the concentration of thyroid hormone (thyroxine (T4)) in the blood that appears when Graves' disease occurs among the isolated lactic acid bacteria, and as a result, identified a novel, previously unknown Lactobacillus curbatus strain, named it 'Lactobacillus curbatus NE416', and deposited it with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on February 1, 2023, and was assigned the deposit number KCTC15309BP.

Therefore, the present invention can provide a novel Lactobacillus curvatus NE-416 strain (Accession Number: KCTC15309BP).

In addition, the present invention can provide a pharmaceutical composition for preventing or treating Graves' disease, comprising Lactobacillus curvatus NE-416 strain (Accession No.: KCTC15309BP) as an active ingredient.

In the present invention, the strain that can be included as an effective ingredient in the composition is not limited thereto, but may include all of a cell of the strain, a fragment of the cell, a culture of the strain, a culture solution from which the cell is removed from a culture of the strain, an extract of the cell of the strain, an extract of a culture of the strain, or an extract of a culture solution from which the cell is removed from a culture of the strain.

In addition, the novel Lactobacillus curbatus NE-416 strain of the present invention has the characteristic of being able to prevent and treat Graves' disease.

In one embodiment of the present invention, an adenovirus having a thyroid stimulating hormone receptor (TSHR) gene introduced therein to induce Graves' disease was administered to mice together with the Lactobacillus curbatus NE-416 strain of the present invention, and a group of mice in which Graves' disease was induced by administering only the adenovirus having the TSHR gene introduced therein (Ad-TSHR) and a control group (PBS treatment group, group administered an adenovirus having an EGFR gene introduced instead of TSHR) were subjected to thyroid extraction, and the size, color, and cells of the thyroid were analyzed.

As a result, in the group of mice in which Graves' disease was induced by TSHR overexpression, the size of the thyroid gland was significantly increased compared to the control group, and its color also changed to dark brown.

On the other hand, in the case of the group treated with the Lactobacillus curbatus NE-416 strain of the present invention, the size and color of the thyroid gland were found to be almost similar to those of the control group, and in the case of the Graves' disease-induced group, the cell size was found to be larger, whereas the group treated with the new strain of the present invention was found to have a cell size similar to that of the control group.

Through these results, the inventors of the present invention were able to determine that the novel Lactobacillus curbatus NE-416 strain of the present invention has the ability to prevent the onset of Graves' disease.

In another embodiment of the present invention, it was confirmed whether the novel strain of Lactobacillus curbatus NE-416 can treat Graves' disease. First, Graves' disease was induced in mice, and then the novel strain of Lactobacillus curbatus NE-416 of the present invention was orally administered to the mice. As a result, the group treated with the novel strain of the present invention showed a significant decrease in the concentration of thyroid hormone (T4) in the blood compared to the group not treated with the novel strain. This effect was shown to be at a similar level to the group treated with PTU, which is known as a conventional Graves' disease treatment agent.

Therefore, through these results, the inventors of the present invention were able to find out that the new strain of Lactobacillus curbatus NE-416 of the present invention has not only a preventive effect but also a therapeutic effect on Graves' disease.

The pharmaceutical composition according to the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods, and may include appropriate carriers, excipients, or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or excipient or diluent may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating, it can be manufactured using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

Solid preparations for oral administration can be prepared by mixing the new strain NE-416 of the present invention with at least one excipient, such as starch, calcium borate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerol gelatin.

The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, weight, degree of disease, drug form, route of administration, and period of administration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, it can be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. The administration can be administered once a day or divided into several times. However, the scope of the present invention is not limited by the above dosage.

The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes. All modes of administration can be administered, for example, orally, rectally, or by intravenous, intramuscular, or subcutaneous injection.

In addition, the present invention can provide a health functional food for preventing or improving Graves' disease, which contains Lactobacillus curvatus NE-416 strain (Accession No.: KCTC15309BP) as an effective ingredient.

The strain is as described above, and the dosage of the new strain of the present invention may be 1Х10 ⁶ CFU/mL to 1Х10 ¹⁰ CFU/mL, preferably 1Х10 ⁸ CFU/mL to 1Х10 ¹⁰ CFU/mL, but is not limited thereto.

In the health functional food composition according to the present invention, when the strain (NE-416) is used as an additive to a health functional food, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the effective ingredients can be appropriately determined according to each purpose of use, such as prevention, health, or treatment.

The formulation of health functional foods can be in the form of powders, granules, pills, tablets, capsules, or in the form of general food or beverages.

There is no particular limitation on the type of the above food, and examples of foods to which the above substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense can be included.

In general, when manufacturing food or beverage, the new strain of the present invention can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

Among the health functional foods according to the present invention, the beverage may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame may be used. The proportion of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food composition according to the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. In addition, the health functional food composition according to the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in mixtures. The ratio of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the health functional food composition according to the present invention.

In addition, since the Lactobacillus curbatus NE-416 strain according to the present invention is isolated from kimchi, it can be used to manufacture a fermented food by adding a fermentation starter or a starter composition for fermented food.

The above fermented foods can be diverse, such as yogurt, kimchi, and cheese, and the above fermented foods can also have the effect of preventing or improving Graves' disease.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited to these examples.

<Example 1>

Isolation and identification of a novel Lactobacillus curbatus NE-416 strain from kimchi

The present inventors performed the following experiments to isolate and identify microorganisms having Graves' disease therapeutic efficacy from kimchi. The suspension of commercially available kimchi in sterilized water was spread on MRS agar medium and cultured, and the resulting single colonies were collected. Then, each colony was cultured and the 16s rRNA base sequences for each cultured strain were analyzed.

A total of 32 colonies were isolated from various types of kimchi, and analysis of the 16s rRNA base sequence revealed that they were lactic acid bacteria, including Bacillus subtilis, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus curbatus, Leuconostoc mesenteroides, and Leuconostoc lactis.

To select lactic acid bacteria with the efficacy of treating Graves' disease among lactic acid bacteria isolated from kimchi, the following experiments were conducted using an animal model of Graves' disease.

In the present invention, the kimchi-derived lactic acid bacteria isolated and an adenovirus having a coding base sequence of a peptide including the 1st to 289th amino acids of the TSHR (thyroid stimulating hormone receptor) protein were administered to 6-week-old Balb/c female mice. The improvement of Graves' disease due to the administration of the lactic acid bacteria was confirmed by measuring the concentration of blood thyroid hormone T4. Here, the amino acid sequence of the TSHR protein is shown in SEQ ID NO: 2.

In addition, the administration of adenovirus containing the base sequence encoding the above TSHR protein was administered three times for three weeks at 10 ¹⁰ particles, and the lactic acid bacteria were administered daily at 10 ⁹ /kg for 9 weeks (Fig. 1). The experiment was conducted in a total of 9 groups (PBS, EGFP, Ad-TSHR, lactic acid bacteria treatment) including the control group, with 5 animals per group.

As shown in Fig. 2, the analysis results showed that, compared to the control group (PBS, EGFP treatment group) that did not induce Graves' disease, the T4 concentration in the blood of mice in which Graves' disease was induced with Ad-TSHR for 9 weeks increased by approximately 3 times compared to before induction.

These results indicate that a Graves' disease mouse model was well established through infection with an adenovirus introduced with the TSHR base sequence according to the present invention.

In addition, in the five groups of mice that were administered Ad-TSHR and lactic acid bacteria isolated from kimchi together, the increase in blood T4 was significantly reduced compared to the control group and the group administered Ad-TSHR alone. In particular, in the case of the Lactobacillus curvatus strain, it was confirmed that the increase in T4 was relatively lower compared to other strains.

Based on the results above, the strain with the best effect of inhibiting the increase in T4 concentration among the lactic acid bacteria isolated from kimchi was selected, and the 16s rRNA base sequence of the strain was analyzed. As a result, it was confirmed to be a novel strain that had 99% homology with the Lactobacillus curvatus strain and was not previously known.

Accordingly, the inventors of the present invention named the strain ‘Lactobacillus curvatus NE416’ and deposited it with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on February 1, 2023, and were assigned the deposit number KCTC 15309BP.

The 16s rRNA base sequence of the Lactobacillus curbatus NE416 strain identified in the present invention is shown in sequence number 1, and the results of observing the morphology of the strain using an electron microscope are shown in Fig. 3.

<Example 2>

Confirmation of Graves' disease prevention efficacy of the novel strain of Lactobacillus curbatus NE-416 of the present invention

The present inventors performed the following experiments to confirm whether the new strain of Lactobacillus curbatus NE-416 identified in Example 1 above has the effect of preventing or treating Graves' disease.

Thyroid glands were extracted from mice in the group administered only the adenovirus expressing TSHR of Example 1 (Ad-TSHR), the group administered together with Ad-TSH and the new strain of Lactobacillus curbatus NE-416, and the control group (PBS, EGFR), and the changes in size were analyzed.

As a result, as shown in Fig. 4, in the case of the control group (PBS, EGFR), the thyroid gland of the mice was found to be slightly smaller and brighter in color. On the other hand, the thyroid gland of the mice in which Graves' disease was induced by Ad-TSHR administration was found to be dark brown and larger in size. This result corresponds to one of the characteristics of Graves' disease.

When the novel Lactobacillus curbatus NE-416 strain of the present invention was administered together with Ad-TSHR, the thyroid size of the mice was found to be almost similar in size and color to the control group.

Through these results, the inventors of the present invention were able to find out that the novel Lactobacillus curbatus NE-416 strain identified in the present invention can improve the symptoms of Graves' disease and prevent Graves' disease.

Furthermore, the inventors of the present invention isolated cells from thyroid glands extracted from the experimental mouse group and observed the shape of the cells.

As a result, as shown in Fig. 5, in the case of the control group (PBS, EGFR), the cell size appeared to be small and constant, whereas the thyroid cells in the group in which Graves' disease was induced by Ad-TSHR administration appeared to be large and dark in color.

In contrast, when the NE-416 lactic acid bacteria of the present invention and Ad-TSHR were treated simultaneously, it was confirmed that the cell size and color were similar to those of the control group.

This result also means that the new strain of Lactobacillus curbatus NE-416 of the present invention can prevent Graves' disease.

<Example 3>

Confirmation of Graves' disease treatment efficacy of the novel strain of Lactobacillus curbatus NE-416 of the present invention

Based on the results of Example 2 above, the inventors of the present invention confirmed that the novel Lactobacillus curbatus NE-416 strain of the present invention can prevent Graves' disease, and therefore conducted an experiment to confirm whether the strain can treat Graves' disease.

To this end, Graves' disease was induced in mice by administering Ad-TSHR for 9 weeks, and then the novel strain of Lactobacillus curbatus NE-416 of the present invention was orally administered daily at a concentration of 10 ⁹ /kg for 9 weeks, with each experimental group consisting of 7 mice.

Blood was collected from each experimental group of mice and the amount of thyroid hormone T4 in the blood was confirmed.

As a result, mice treated with PBS as a control group and mice overexpressing EGFP showed low levels of T4 in their blood, whereas the group in which Graves' disease was induced by administering Ad-TSHR showed a 1.6-fold increase in the concentration of T4 in their blood compared to the control group, and the group treated with propylthiouracil (PTU), a Graves' disease treatment, showed a decrease in the concentration of T4 to the level of the control group.

In addition, it was shown that the concentration of T4 in the blood was effectively reduced in the group administered the new strain of Lactobacillus curbatus NE-416 of the present invention (Fig. 6).

Through the above results, the inventors of the present invention were able to find that the novel strain of Lactobacillus curbatus NE-416 isolated and identified from kimchi can be usefully used in medicines and health functional foods that can prevent, improve, or treat Graves' disease.

The present invention has been described with reference to preferred embodiments thereof. Those skilled in the art will appreciate that the present invention may be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC15309BP 20230201

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Claims (10)

Lactobacillus curvatus NE-416 strain (Accession number: KCTC15309BP). In the first paragraph,
The above strain is a Lactobacillus curbatus NE-416 strain, characterized in that it is isolated and identified from kimchi. A pharmaceutical composition for preventing or treating Graves' disease, comprising Lactobacillus curvatus NE-416 strain (Accession No.: ) as an active ingredient. In the third paragraph,
A pharmaceutical composition for preventing or treating Graves' disease, characterized in that the strain is selected from among cells of the strain, a fragment of cells, a culture of the strain, a culture solution from which cells have been removed from a culture of the strain, an extract of cells of the strain, an extract of a culture of the strain, or an extract of a culture solution from which cells have been removed from a culture of the strain. In the third paragraph,
A pharmaceutical composition for preventing or treating Graves' disease, characterized in that the strain is isolated and identified from kimchi. In the third paragraph,
A pharmaceutical composition for preventing or treating Graves' disease, characterized in that the composition has the effect of reducing the concentration of thyroid hormone increased in the blood due to Graves' disease and reducing the size of the thyroid gland. A health functional food for preventing or improving Graves' disease, containing Lactobacillus curvatus NE-416 strain (Accession No.: KCTC15309BP) as an active ingredient. In Article 7,
A health functional food for preventing or improving Graves' disease, characterized in that the strain is selected from among cells of the strain, a fragment of cells, a culture of the strain, a culture solution from which cells have been removed from a culture of the strain, an extract of cells of the strain, an extract of a culture of the strain, or an extract of a culture solution from which cells have been removed from a culture of the strain. In Article 7,
A health functional food for preventing or improving Graves' disease, characterized in that the above strain is isolated and identified from kimchi. In Article 7,
The composition is a health functional food for preventing or improving Graves' disease, characterized in that it has the effect of reducing the concentration of thyroid hormone increased in the blood due to Graves' disease and reducing the size of the thyroid gland.