JP7225364B1 - Lactic acid bacteria - Google Patents

Abstract

An object of the present invention is to provide a novel lactic acid bacterium. The lactic acid bacterium is Lactiplantibacillus plantarum strain S25 (acceptance number: NITE ABP-03571). [Selection figure] None

Description

NPMD NITE BP-03571

The present disclosure relates to novel lactic acid bacteria and the like. It should be noted that the contents of all documents mentioned herein are hereby incorporated by reference.

Various effects have been studied by lactic acid bacteria fermented products. For example, it is known that the intestinal barrier is improved (Patent Document 1), or the skin is improved (Patent Document 2), and useful lactic acid bacteria are being searched for.

In addition, IFN-γ and IL-12 produced by Th1 cells are known to enhance cell-mediated immunity such as activation of NK cells and cytotoxic T cells, and are particularly important in initial immune responses. On the other hand, IL-6 and other cytokines produced by Th2 cells are known to activate humoral immunity mediated by B cells.
Cytokines produced by Th1 cells (also called Th1-type cytokines) suppress cytokine production by Th2 cells, and cytokines produced by Th2 cells (also called Th2-type cytokines) suppress cytokine production by Th1 cells. suppress It is said that the balance between the two is important in the immune response, and the recent increase in allergic diseases is thought to be caused by the Th1-type cytokine/Th2-type cytokine balance leaning towards the Th2-type dominance.

Japanese Patent Application Laid-Open No. 2019-011315 Japanese Patent Application Laid-Open No. 2019-011316

An object of the present invention is to provide lactic acid bacteria having a high ability to induce cytokine production by Th1 cells.

The present inventors found that the Lactiplantibacillus plantarum strain S25 (acceptance number: NITE ABP-03571) has a high ability to induce IL-12 production, and made further improvements.

The disclosure includes, for example, subject matter described in the following sections.
Item 1.
A lactic acid bacterium, which is Lactiplantibacillus plantarum strain S25 (acceptance number: NITE ABP-03571).
Section 2.
Item 1. An oral composition comprising the lactic acid bacterium according to item 1.
Item 3.
Item 1. A food/beverage composition or pharmaceutical composition comprising the lactic acid bacterium according to item 1.
Section 4.
Item 4. The composition according to item 2 or 3, which is for immunoregulation or cell-mediated immunity enhancement.

A novel Lactiplantibacillus plantarum S25 strain is provided, which has a high ability to induce IL-12 production.

Fig. 2 shows IL-12 production-inducing ability (absorbance at OD450) of various plant-derived lactic acid bacteria. The IL-6 concentration (pg/ml) in the culture supernatant when Lactobacillus plantarum S25 fermented broccoli was added is shown. The IL-10 concentration (pg/ml) in the culture supernatant when Lactobacillus plantarum S25 fermented broccoli was added is shown. The IFN-γ concentration (pg/ml) in the culture supernatant when various fermented broccoli was added is shown. IL-12 concentrations (pg/ml) in culture supernatants to which various fermented broccoli were added are shown. FIG. 2 shows the Th1-type cytokine/Th2-type cytokine ratio (IFN-γ/IL-6) in the culture supernatant when various fermented broccoli were added. FIG. 2 shows the Th1-type cytokine/Th2-type cytokine ratio (IL-12/IL-10) in the culture supernatant when various fermented broccoli were added. NK activity by various vegetable lactic acid bacteria is shown.

Each embodiment included in the present disclosure will be described in further detail below.
A lactic acid bacterium encompassed by the present disclosure is Lactiplantibacillus plantarum S25 strain (accession number: NITE ABP-03571). In this specification, the lactic acid bacterium is sometimes referred to as "the lactic acid bacterium of the present disclosure".

The S25 strain of lactiplantibacillus plantarum was received on December 15, 2021 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), receipt number: Received under NITE ABP-03571.

The lactic acid bacteria of the present disclosure are vegetable lactic acid bacteria isolated from Suguki, a Kyoto pickle.

The lactic acid bacterium of the present disclosure has a high IL-12 production-inducing ability compared to Lactiplantibacillus plantalum isolated from shibazuke and Lactiprantibacillus pentosus isolated from shibazuke. have. IL-12 is a cytokine mainly produced by Th1 cells.

When broccoli is fermented using the lactic acid bacteria of the present disclosure, Lactipranchibacillus plantarum isolated from shibazuke, Lactipranchibacillus pentosus isolated from shibazuke, other lactiplanchibacillus isolated from suguki The production of IFN-γ can be promoted as compared with the case of using Pentosus and Bifidobacterium longum. IFN-γ is a cytokine mainly produced by Th1 cells.

When broccoli is fermented using the lactic acid bacteria of the present disclosure, Lactipranchibacillus plantarum isolated from shibazuke, Lactipranchibacillus pentosus isolated from shibazuke, other lactiplanchibacillus isolated from suguki Compared to the case of using Pentosus and Bifidobacterium longum, IL-12 production can be promoted.

When broccoli is fermented using the lactic acid bacteria of the present disclosure, Lactipranchibacillus plantarum isolated from shibazuke, Lactipranchibacillus pentosus isolated from shibazuke, other lactiplanchibacillus isolated from suguki Compared to the case of using Pentosus and Bifidobacterium longum, the concentration ratio of IFN-γ/IL-6 shows a high value of 1.0 or more. IL-6 is a cytokine mainly produced by Th2 cells.
In addition, fermentation of broccoli using the lactic acid bacteria of the present disclosure can promote production of IL-6.

When broccoli is fermented using the lactic acid bacteria of the present disclosure, Lactipranchibacillus plantarum isolated from shibazuke, Lactipranchibacillus pentosa isolated from shibazuke, other lactiplanchibacillus isolated from suguki Compared to the case of using Pentosus and Bifidobacterium longum, the concentration ratio of IL-12/IL-10 shows a high value of 1.0 or more. IL-10 is a cytokine mainly produced by Th2 cells.
In addition, fermentation of broccoli using the lactic acid bacteria of the present disclosure can promote the production of IL-10.

The lactic acid bacterium of the present disclosure enhances the production of both Th1-type cytokines and Th2-type cytokines, and thus can stimulate the immunity of the whole organism. In particular, by inducing the production of IFN-γ and/or IL-12, cytokines produced by Th1 cells, the Th1-type/Th2-type cytokine ratio (eg, IFN-γ/IL-6, IL- 12/IL-10, etc.) can be increased.

The lactic acid bacteria of the present disclosure can enhance NK activity by inducing the production of IFN-γ and/or IL-12, which are cytokines produced by Th1 cells.

The present disclosure also includes oral compositions comprising the lactic acid bacteria.
The present disclosure also includes food and drink compositions containing the lactic acid bacteria.
The present disclosure also includes pharmaceutical compositions containing the lactic acid bacteria.
In this specification, these compositions may be collectively referred to as "the composition of the present disclosure".

In the case of food and drink compositions, for example, processed foods, beverages, health foods (foods with nutrient function claims, foods for specified health uses, etc.), supplements, food for sick people (hospital food, sick food, nursing care food, etc.), etc. may

The content of lactic acid bacteria in the composition of the present disclosure is not particularly limited, and is preferably, for example, about 0.1 to 100% by mass, about 1 to 99% by mass, about 2 to 90% by mass, or about 5 to 50% by mass. is more preferred.

In the composition of the present disclosure, lactic acid bacteria may be viable cells or dead cells.

In the composition of the present disclosure, lactic acid bacteria may or may not be subjected to, for example, crushing, heating, drying (freeze-drying, vacuum-drying, spray-drying, etc.), freezing, bacteriolysis, extraction, and the like.

The compositions of the present disclosure contain the lactic acid bacteria described above and may further contain other ingredients. Such other components include pharmacologically or food hygienically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, Examples include thickeners, antioxidants, preservatives, coating agents, colorants and the like. These components can be used individually by 1 type or in combination of 2 or more types.

The composition of the present disclosure can be prepared by a conventional method by combining the lactic acid bacteria described above and, if necessary, other ingredients.

The lactic acid bacteria of the present disclosure, for example, induce production of IL-12, induce production of IFN-γ, Th1-type cytokine/Th2-type cytokine ratio (e.g., IFN-γ/IL-6, IL-12 /IL-10, etc.) or enhance NK activity. Therefore, the composition of the present disclosure can be suitably used, for example, for immunoregulation, cell-mediated immunity enhancement, intestinal immunity adjustment, intestinal immunity enhancement, and the like. As used herein, "immunomodulation" means the dominance of Th1-type cytokines in the Th1-type/Th2-type cytokine balance. In addition, the composition of the present disclosure can, for example, alleviate the effects of external stimuli on the body and maintain a good physical condition on a daily basis. It can also be suitably used as a composition for increasing resistance) and maintaining a good general condition.

Subjects for ingestion of the composition of the present disclosure include, for example, humans and mammals other than humans (eg, rats, mice, rabbits, cows, pigs, dogs, cats, sheep, monkeys, etc.).
Examples of humans include humans in which Th2-type cytokines are dominant in the blood Th1-type cytokine/Th2-type cytokine balance; humans with allergic symptoms (e.g., atopic dermatitis, humans with hay fever, etc.); and weak intestinal immunity. (e.g., elderly people whose immunity weakens with age, people with intestinal inflammation due to excessive intake of high-fat foods, etc.); people prone to colds; people with bronchial asthma; chronic inflammatory conditions (e.g., ulcerative colitis, people who smoke/drink frequently, people who feel tired all the time, people who have disordered eating habits, people in stressful environments, lifestyle-related diseases such as obesity and diabetes) humans, etc.); Humans with diseases that are thought to be caused by an imbalance in the Th1-type cytokine/Th2-type cytokine balance in the intestinal tract and the whole body (such diseases include, for example, allergies (children and adults with atopic dermatitis, hay fever), inflammatory bowel disease (ulcerative colitis), etc.); and those who want to enhance their systemic immune function (NK cell activity) for the purpose of preventing cancer and viral infections such as the common cold. It can also be used prophylactically to maintain immune function even when these symptoms are not observed.

The amount of lactic acid bacteria to be ingested is not particularly limited, and is preferably 1 to 100 billion, more preferably 1 to 10 billion, per day (particularly per day for adults).
The amount of lactic acid bacteria contained in the composition of the present disclosure can also be set with reference to the daily intake of lactic acid bacteria. Since 1 g of the killed lactic acid bacteria powder usually contains about 1.0×10 ¹¹ to 1.0 to 10 ¹³ lactic acid bacteria, for example, with reference to this value, the killed lactic acid bacteria powder is added to the composition of the present disclosure. The composition can also be prepared so as to include the daily intake of lactic acid bacteria. Alternatively, the amount of lactic acid bacteria contained in the composition may be set to, for example, 1/2 or 1/3 of the daily intake of lactic acid bacteria, and the composition may be ingested several times (twice or three times) per day.

In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Also, the present disclosure encompasses any and all combinations of the constituent elements described herein.

Also, the various characteristics (property, structure, function, etc.) described for each of the embodiments of the disclosure described above may be combined in any way to identify subject matter encompassed by the disclosure. That is, the present disclosure encompasses all subject matter consisting of any and all possible combinations of the features described herein.

Hereinafter, the embodiments of the present disclosure will be described more specifically with examples, but the embodiments of the present disclosure are not limited to the following examples.

As test substances, 6 strains of plant-derived lactic acid bacteria shown in Table 1 were used.

Separation of human peripheral blood mononuclear cells (PBMC) Heparinized human peripheral blood was collected, separated into PBMCs by Ficoll-Hypaque specific gravity centrifugation (Nycomed Pharma, Oslo, Norway), and placed in RPMI 1640 medium (supplemented with 10% FCS, 5 mM). Hepes and antibiotics).
In Vitro Stimulation Conditions Human peripheral blood PBMC (1×10 ⁶ /ml) were stimulated in vitro with a test substance (1 μg dry weight, approximately 1×10 ⁶ cells) to examine changes in IL-12 levels. PBMC alone was used as a negative control, and PBMC stimulated with 10 µg/ml phytohemagglutinin (PHA) was used as a positive control.
Measurement of IL-12 production amount IL-12 production amount in the culture supernatant when PBMCs were stimulated in vitro with the test substance for 18 hours was measured by ELISA. The results are shown in FIG.

As shown in FIG. 1, it was confirmed that both the evaluated Lactiplantibacillus pentosus and the Lactiplantibacillus plantarum S25 strain have a high ability to induce IL-12 production.

The 6 strains of plant lactic acid bacteria shown in Table 1 and the Bifidobacterium longum strain purely cultured from Bifilon 50N were each inoculated into Lactobacilli MRS Broth prepared according to a conventional method. . The longum strain was cultured at 36°C for 20 hours.
On the other hand, 80 g of frozen broccoli puree was thawed and packed into transparent pouches, preheated appropriately in a microwave oven, sealed with a heat seal, and heat sterilized in a hot water bath (97° C. or higher) for 15 minutes. They were cooled to 30-40° C., opened and inoculated with 4% (3.2 g) of each Lactobacilli MRS Broth culture as a starter. After the pouch opening was sealed again by heat sealing, the pouch was well kneaded by hand to mix the contents. The strain longum was fermented at 36°C for 24 hours in a thermostat. After completion of fermentation and collection of test samples, the mixture was heat-sealed again, sterilized in a hot water bath at 90°C or higher for 10 minutes, and stored in a refrigerator until the cytokine induction test was started.

Each 1.0 ml of the PBMC cell preparation prepared by the same method as described above was seeded on a 12-well plate (1.0×10 ⁶ cells/well). The above-described fermented sample was added to 1 mg/ml, and an equal amount of PBS(-) was added as a control and cultured for 24 hours. After 24 hours, the culture supernatant was collected and centrifuged, and IL-6, IL-10, IL-12 and IFN-γ in the supernatant were measured using the Bio-Plex Suspension Array System. The results are shown in FIGS. 2A, B, C and D as concentrations (pg/ml) of IL-6, IL-10, IFN-γ and IL-12 in the culture supernatant 24 hours after administration of the sample. 2E and F show the Th1-type cytokine/Th2-type cytokine ratio (IFN-γ/IL-6 concentration ratio, IL-12/IL-10 concentration ratio) calculated from the cytokine concentration in the supernatant.

As shown in Figures 2A and B, L. It was confirmed that the addition of broccoli fermented using the planta rum S25 strain promoted the production of IL-6 and IL-10.
As shown in Figures 2C and D, L. It was confirmed that the addition of broccoli fermented using the planta rum S25 strain promoted the production of IFN-γ and IL-12.
As shown in Figures 2E and F, L. By adding broccoli fermented using the planta rum S25 strain, the Th1 type cytokine/Th2 type cytokine ratio (IFN-γ/IL-6, IL-12/IL-10) increased to a high value of 1.0 or more. It was confirmed that

Commercially available cryopreserved human peripheral blood mononuclear cells (Cellular Technology USA) were used. The medium for thawing frozen PBMCs is the manufacturer's recommended medium CTL-Anti-Aggregate Wash Supplement. One vial of frozen PBMCs is rapidly thawed at 37°C, washed with CTL-Anti-Aggregate Wash Supplement, and centrifuged to remove PBMCs. Obtained.
The thawed PBMCs were adjusted to a concentration of 3×10 ⁶ /ml with RPMI1640 medium containing 10% FCS, and 500 μl of each was plated on a 24-well plate. Next, 50 μl of Lactiplantibacillus plantar um strain S25 (acceptance number: NITE ABP-03571) or Lactobacillus acidophilus isolated from yogurt was added and cultured at 37° C. under 5% CO ₂ for 20 hours. There was also a test segment where PBS was added as a control.
After the culture was completed, large debris was removed using a tube with a cell strainer cap, followed by centrifugation at 2000 rpm for 5 minutes, and the sedimented PBMC was used for NK activity measurement.

PBMCs were used as effector cells, and K562 cells, a human NK cell-sensitive strain, were used as target cells. K562 cells were adjusted to 1×10 ⁶ cells/ml with PBS, then added with a hydrophobic cyanine membrane-based fluorescent dye DiO to a final concentration of 8 μg/ml, and stained at 37° C. for 10 minutes. Then, the cells were washed once with PBS, suspended in RPMI1640 medium containing 10% FCS, and adjusted to a concentration of 5×10 ⁴ cells/ml.
To each well of a 96-well U-bottom microculture plate was added the membrane-impermeable nucleic acid-binding fluorescent dye PI to a final concentration of 25 μg/ml. 100 μl of PBMC (concentration of 2×10 ⁶ cells/ml) suspended in RPMI1640 medium was dispensed thereon, and then 100 μl of K562 cells were dispensed (E:T ratio=40:1). The microculture plate was centrifuged at 2000 rpm for 5 minutes, reacted at 37° C. for 2 hours, and NK activity was measured using a flow cytometer (Beckman Coulter). The results are shown in FIG.
The NK activity was calculated by the following formula.
NK activity (%) = number of DiO-positive PI-positive cells/(number of tens of DiO-positive PI-negative cells/number of DiO-positive PI-positive cells) x 100-spontaneous lysis.

As shown in FIG. The planta rum S25 strain was confirmed to exhibit high NK activity.

Claims (5)

A lactic acid bacterium which is Lactiplantibacillus plantarum strain S25 (acceptance number: NITE ABP-03571). An oral composition comprising the lactic acid bacterium according to claim 1. A food/beverage composition or pharmaceutical composition comprising the lactic acid bacterium according to claim 1. An oral composition comprising a lactic acid fermented product of broccoli,
An oral composition, wherein the lactic acid bacterium is the lactic acid bacterium of claim 1. The composition according to any one of claims 2 to 4 , which is for immunoregulation or cell-mediated immunity enhancement.

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