JP2022023806A - Regulatory t-cell regulator and applications thereof - Google Patents

Abstract

To provide a technique to control the proportion of regulatory T-cells in helper T-cells within an optimal range.SOLUTION: A regulatory T-cell regulator contains lactic acid bacteria as an active ingredient.SELECTED DRAWING: Figure 1

Description

There is an application for exception of loss of novelty

The present invention relates to a regulatory T cell regulator containing lactic acid bacteria as an active ingredient and its use.

Regulatory T cells play a role in suppressing the excessive immune response that causes the disease of autoimmune disorders. Therefore, regulatory T cells are reduced in patients with the above diseases.

Therefore, regulating the amount of regulatory T cells is an important technique for the prevention and treatment of the above-mentioned diseases. So far, techniques for regulating the amount of various regulatory T cells have been proposed, and for example, a regulatory T cell enhancer containing trehalose as an active ingredient has been reported (Patent Document 1).

However, this regulatory T cell enhancer has shown that trehalose proliferates regulatory T cells through an increase in enteric bacteria of the phylum Bacillota, where the bacteria of the phylum Bacillota have safety issues. Because of this, it may be unfavorable.

Japanese Unexamined Patent Publication No. 2018-188408

Therefore, an object of the present invention is to provide a technique for adjusting the ratio of regulatory T cells to helper T cells to an optimum range.

As a result of diligent research to solve the above problems, the present inventors have found that lactic acid bacteria optimize the ratio of regulatory T cells to helper T cells, and have completed the present invention.

That is, the present invention is a regulatory T cell regulator containing lactic acid bacteria as an active ingredient.

The present invention is also an autoimmune disease prophylactic and / or therapeutic agent containing the regulatory T cell regulator.

The regulatory T cell regulator of the present invention contains lactic acid bacteria that have previously been eaten as an active ingredient and is safe.

In addition, the regulatory T cell regulator of the present invention is useful for the prevention and / or treatment of autoimmune diseases because it regulates the ratio of regulatory T cells in helper T cells to an optimum range.

It is a figure which shows the ratio of regulatory T cells (Treg) in blood (in the figure, 1 is before the start of drinking, 2 is the period of drinking, 3 is after drinking. The ratio (%) of regulatory T cells is Calculated by measuring the ratio of CD25 + Foxp3 + cells contained in CD4 + T cells). It is a figure which shows the ratio of regulatory T cells (Treg) in the blood for a healthy person and a mild person (in the figure, 1 is before the start of drinking, 2 is the period of drinking, and 3 is after drinking. The proportion (%) of T cells was calculated by measuring the proportion of CD25 + Foxp3 + cells contained in CD4 + T cells).

The regulatory T cell regulator of the present invention contains lactic acid bacteria as an active ingredient. The lactic acid bacteria are not particularly limited, and are, for example, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus helveticus, and Lactobacillus helveticus. (Lactobacillus salivarius), Lactobacillus fermentum, Lactobacillus del Brucchii Subspecies. Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus delbrueckii subspice. Lactobacillus delbrueckii subsp. Delbrueckii, Lactobacillus johnsonii, Lactobacillus mali, Lactobacillus plantarum, etc. , Lactobacillus subspecies, Lactobacillus subspecies, such as Streptococcus thermophilus. Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Lactococcus lactis subsp. Cremoris, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus cremoris, Lactococcus cremoris, etc. ), Enterococcus faecium and other Lactococcus spp., And one or more of these lactic acid bacteria can be used.

In recent years, lactic acid bacteria of the genus Lactobacillus have been reclassified (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. . J. Syst. Evol. Microbiol. 2020 Apr; 70 (4): 2782-2858 DOI 10.1099 / ijsem.0.004107). The classification of lactic acid bacteria of the genus Lactobacillus is as follows when reclassified based on the above literature.

Lactobacillus lactic acid bacteria according to the new classification include Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus acidophilus, Lactobacillus acidophilus, and Lactobacillus acidophilus. , Lactobacillus del Brucchii Subspecies. Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus delbrueckii subspice. Lactobacillus delbrueckii subsp. Delbrueckii, Lactobacillus johnsonii, etc., Lactobacillus salivarius, etc., Lactobacillus salivarius, etc. Lactobacillus genus bacteria such as fermentum (Limosilactobacillus fermentum), Lactobacillus genus bacteria such as Liquorilactobacillus mali, Lactiplantibacillus plantarum and the like. Etc., Lactobacillus subspecies, Lactobacillus subspecies, such as Streptococcus thermophilus. Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Lactococcus lactis subsp. Cremoris, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus cremoris, Lactococcus cremoris, Lactococcus cremoris, etc. , Enterococcus faecium and other Lactococcus spp., And one or more of these lactic acid bacteria can be used.

In the present invention, lactic acid bacteria of the genus Lactobacillus are included as lactic acid bacteria of the genus Lactobacillus in the old classification.

Among these, Lactiplanti Bacillus plantarum is preferable, and Lactiplanti Bacillus plantarum YIT 0132 (FERM BP-11349) is particularly preferable.

Lactiplantibacillus plantarum YIT 0132 was named Lactiplantibacillus plantarum YIT 0132 (belonging to Lactobacillus plantarum YIT 0132 in the old classification) and was deposited with the deposit number FERM BP-11349 on February 24, 2010. It has been internationally deposited at the Patent Organism Depositary Center (Central 6th, 1-1-1, Higashi, Tsukuba City, Ibaraki Prefecture, 305-8566), Incorporated Administrative Agency, Industrial Technology Research Institute.

The form of the lactic acid bacterium is not particularly limited, and may be a live bacterium or a dead bacterium, and may be a wet bacterium or a dried bacterium. In addition, lactic acid bacteria may be used as a culture, a concentrate thereof, a dried product, or the like, or may be a cell-treated product such as a crushed product or an extract of the cell.

The regulatory T cell regulator of the present invention can be formulated with the above-mentioned lactic acid bacterium in combination with a pharmaceutically acceptable carrier, if necessary.

Pharmaceutically acceptable carriers include, for example, glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acids, gelatin, albumin. , Water, physiological saline and the like. Further, if necessary, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents, and excipients can be appropriately added.

The dosage form of the regulatory T cell regulator of the present invention is not particularly limited, and examples thereof include liquids, powders, granules, capsules, tablets, suppositories, and external preparations, which can be prepared according to a conventional method. ..

The content of lactic acid bacteria in the regulatory T cell regulator of the present invention is not particularly limited, but since the regulatory T cell regulatory effect is more effectively exerted, the total amount contained in the regulatory T cell regulator is included. The number of bacteria is preferably 10 ⁸ cfu / g or more, and particularly preferably 10 ⁸ cfu / g to 10 ¹⁰ cfu / g. The administration method may be either oral administration or parenteral administration, but oral administration is preferable because administration is easy and suitable for long-term administration. The dose thereof is not particularly limited, but the total number of lactic acid bacteria is preferably 108 cfu or more, more preferably ^{10 10 cfu} or more, and further preferably 10 ¹¹ cfu or more per day. , Particularly preferably 10 ¹¹ to 10 ¹² cfu.

The regulatory T cell regulator of the present invention may be in the form of food or drink, and can be prepared according to a conventional method by blending the above-mentioned lactic acid bacteria with known food additives and / or food materials. The form is not particularly limited, and may be, for example, liquid, tablets, capsules, pastes, granules and the like. Food and drink includes fermented milk food and drink, fermented soy milk, fermented fruit juice, fermented vegetable juice and other fermented food and drink, bread, biscuits, hot cakes, noodles, tablet confectionery and other starch-based foods, gum, candy, and Japanese confectionery. Confectionery such as ham, sausage and other livestock foods, chikuwa, kamaboko and other fish meat foods, seafood foods, dressings, soy sauce, jam, sprinkles and other seasonings, tea, juice, soft drinks, liquor and other beverages Can be mentioned. Among these, fermented foods and drinks are preferable, fermented fruit juice is more preferable, and lactic acid bacteria fermented fruit juice is further preferable.

The above-mentioned lactic acid bacterium fermented fruit juice is obtained by fermenting fruit juice with lactic acid bacteria and contains lactic acid bacteria.

Here, the fruit juice is not particularly limited, and examples thereof include fruit juices of bananas, apples, grapes, and citrus fruits of the genus Citrus such as oranges and Satsuma mandarin. Among these, citrus juice is preferable, and citrus juice of the citrus genus is more preferable. The Brix of the fruit juice is not particularly limited, but is, for example, 1 to 10, preferably 2 to 8.

The conditions for fermenting the fruit juice with lactic acid bacteria are not particularly limited, but for example, the fruit juice is inoculated with lactic acid bacteria in an amount of 0.1 to 3.0% by mass (hereinafter, simply referred to as "%"), preferably 0.2 to 1.0%. Then, it may be cultured at 30 to 37 ° C., preferably 33 to 37 ° C. for 24 to 48 hours, preferably 36 to 48 hours.

In addition, at the time of culturing, vegetable juice such as lettuce, cabbage, carrot, and kale, and milk components such as milk, fresh cream, skim milk powder, and whole milk powder may be added to the fruit juice.

After culturing, a sweetener such as fructose, sucrose, glucose, maltose, sucralose, and stevia, a pH adjuster, and the like may be added to adjust the flavor.

The lactic acid bacterium fermented fruit juice may be concentrated, powdered, frozen or the like in the same manner as the conventionally known fruit juice.

Further, since the lactic acid bacterium fermented fruit juice can be stored at room temperature, it is preferable to sterilize it by heating to make the lactic acid bacterium dead. The conditions for heat sterilization are not particularly limited, but are, for example, 5 to 60 seconds at 95 to 100 ° C.

As a preferred embodiment of the lactic acid bacterium fermented fruit juice, the fruit juice of Satsuma mandarin (Brix 1 to 10) is inoculated with Lactiplantibacillus plantarum YIT 0132 to 0.4%, fermented under aerobic conditions, and finally sterilized by heating. It was done. This lactic acid bacterium fermented fruit juice contains dead bacillus lactiplantibacillus plantarum at 5.0 × ¹⁰⁸ cfu / ml.

The regulatory T cell regulator of the present invention thus obtained can regulate the ratio of regulatory T cells to helper T cells, for example, CD4 + CD25 + Foxp3 + cells (+ indicates positive) in an optimum range. .. That is, it is possible to increase or decrease the ratio of regulatory T cells to helper T cells, and it is also possible to suppress the decrease in the ratio of regulatory T cells to helper T cells for some reason. , It is particularly preferable to suppress the decrease. Therefore, it is particularly preferable that the regulatory T cell regulator of the present invention prevents and / or treats autoimmune diseases such as rheumatism in which the proportion of regulatory T cells in helper T cells decreases at the time of illness.

The subject of administration of the regulatory T cell regulator of the present invention is not particularly limited as long as it is an animal, and examples thereof include mammals such as humans, dogs, cats, monkeys, chimpanzees, cows, horses, and sheep. Since the QOL improving agent of the present invention improves the QOL of animals suffering from pollinosis, mammals such as humans, monkeys, chimpanzees, dogs and cats suffering from pollinosis are preferable among these animals. Humans are particularly preferred.

The dose of the regulatory T cell regulator of the present invention is not particularly limited, but for example, when the regulatory T cell regulator contains lactic acid bacteria fermented fruit juice as an active ingredient, the daily intake is 125 ml. The number of lactic acid bacteria to be produced is 1.0 × 10 ⁶ cfu / ml to 1.0 × 10 ¹⁰ cfu / ml, preferably 5.0 × 10 ⁸ cfu / ml or more.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1
Preparation of test beverage:
Brix8 juice (manufactured by Nankai Fruit Factory Co., Ltd.) of Satsuma mandarin (scientific name: Citrus unshiu) was inoculated with Lactiplantibacillus plantarum YIT 0132 (FERM BP-11349) so as to be 0.4%. Then, it was cultured at 37 ° C. under aerobic conditions for 2 days to obtain a fermented product. A lactic acid bacterium containing a heat-killed cell of Lactiplantibacillus plantarum at 5.0 × ¹⁰⁸ cfu / mL by sterilizing this fermented product with fructose added to 3% by heating at 100 ° C. for 35 seconds. Fermented fruit juice was obtained. 125 mL of this was filled in a paper carton to prepare a test beverage. It was stored at room temperature until it was used.

Reference example 1
Placebo Beverage Preparation:
Brix8 juice (manufactured by Nankai Fruit Factory Co., Ltd.) of Satsuma mandarin (scientific name: Citrus unshiu) was sterilized by heating at 100 ° C. for 35 seconds with fructose added to 3% to obtain a placebo beverage. Each 125 mL of this was filled in a paper carton. It was stored at room temperature until it was used.

Example 2
Measurement of the proportion of regulatory T cells in the blood:
The test beverage of Example 1 and the placebo beverage of Reference Example 1 were given to 50 persons with pollinosis selected as follows, one bottle per day for 8 weeks (placebo-controlled randomized allocation 2). Heavy-blind parallel group-to-group comparative study). Blood was collected once before the start of drinking (-30 days to -15 days), during the drinking period (26-38 days), and after the end of drinking (59-72 days), and the proportion of regulatory T cells in the blood. Was measured. The results are shown in FIG.

<Selection of people with hay fever>
Those who showed hay fever symptoms during the previous year's hay fever season (February-April) and had a sneezing-specific IgE of class 2 or higher (positive), and the degree of hay fever symptoms was the symptom score of last season (questionnaire). Runny nose, sneezing, stuffy nose, nasal itching, itchy eyes, watery eyes / congestion are judged by a 0-4 index on a 5-point scale. Those who have an average of 2 or more

<Measurement method of the ratio of regulatory T cells>
The proportion of regulatory T cells in human peripheral blood was calculated by measuring the proportion of CD25 + Foxp3 + cells in CD4 + T cells (+ indicates positive). The number of CD4 + T cells and the number of CD4 + CD25 + Foxp3 + cells were measured by flow cytometry.

(result)
In the placebo group, the proportion of regulatory T cells decreased significantly with the scattering of pollen (before drinking (1) → during drinking (2), before drinking (1) → after drinking (3)). In the test beverage group, the decrease in the proportion of regulatory T cells was suppressed. In addition, during drinking (2), the proportion of regulatory T cells in the test beverage group was significantly higher than that in the placebo group. Regulatory T cells are known to have a function of suppressing an excessive immune response that causes autoimmune diseases, suggesting that the test beverage can be used for the prevention and / or treatment of autoimmune diseases.

Example 3
Comparison between different groups:
The same measurement was performed except for those with pollinosis who measured the ratio of regulatory T cells in the blood in Example 2 and who were constantly taking an antiallergic drug. That is, 16 healthy subjects (those who do not take anti-allergic drugs) and 28 mildly ill subjects (those who occasionally take anti-allergic drugs) in the test beverage group, a total of 44 people, and 32 healthy people and 17 mildly ill people in the placebo group. The names were 49, and the proportion of regulatory T cells in the blood was compared for these groups. The results are shown in FIG.

(result)
Similar to Example 2, in the case of healthy and mildly ill patients with hay fever, the pollen was scattered in the placebo group (before drinking (1) → during drinking (2), before drinking (1) → after drinking (3). ) Significantly decreased the proportion of regulatory T cells, whereas the test beverage group suppressed the decrease in the proportion of regulatory T cells. In addition, during drinking (2), the proportion of regulatory T cells in the test beverage group was significantly higher than that in the placebo group.

Example 4
Preparation of fermented lactic acid bacteria juice:
A lactic acid bacterium fermented fruit juice was produced by the same method as in Example 1 except that the Satsuma mandarin juice was replaced with Valencia orange. Each 125 mL of this was filled in a paper carton.

Example 5
Preparation of lactic acid bacteria-containing fruit juice beverage:
Brix8 juice (manufactured by Nankai Fruit Factory Co., Ltd.) of Satsuma mandarin (scientific name: Citrus unshiu) with 3% fructose and 0.4% Lactiplantibacillus plantarum YIT 0132 (FERM BP-11349). The juice added so as to be sterilized by heating at 100 ° C. for 35 seconds to obtain a fruit juice beverage containing lactic acid bacteria. Each 125 mL of this was filled in a paper carton.

The regulatory T cell regulators of the present invention can be used for the prevention and / or treatment of autoimmune diseases.

Claims (7)

A regulatory T cell regulator containing lactic acid bacteria as an active ingredient. The regulatory T cell regulator according to claim 1, wherein the lactic acid bacterium is Lactic acid bacillus plantarum. The regulatory T cell regulator according to claim 1, wherein the lactic acid bacterium is Lactiplanti Bacillus plantarum YIT 0132 (FERM BP-11349). A regulatory T cell regulator containing lactic acid bacterium fermented fruit juice using the lactic acid bacterium according to any one of claims 1 to 3 as an active ingredient. The regulatory T cell regulator according to claim 4, wherein the juice is citrus juice. The regulatory T cell regulator according to claim 4, wherein the fruit juice is the fruit juice of a citrus of the genus Citrus. A prophylactic and / or therapeutic agent for an autoimmune disease, which comprises the regulatory T cell regulator according to any one of claims 1 to 6.

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