Novel strain of Limosilactobacillus reuteri and uses thereof

Abstract

The present invention relates to the immunomodulatory effects of a novel Limosilactobacillus reuteri ACBB31 strain and its uses. The novel ACBB31 strain of the present invention induced a reduction in colitis-related symptoms in a mouse model of inflammatory bowel disease and exhibited an effect of lowering the overall expression of inflammatory factors in the body. Furthermore, when the heat-killed strain was mixed and fed to weaning pigs, the frequency of diarrhea decreased, growth increased, the overall expression of inflammatory factors in the body was lowered, and the health of the small intestine was morphologically improved. Therefore, the present invention can provide excellent immunomodulatory or anti-inflammatory effects.

Description

Technology Field

[0001] The present invention is a novel Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri This concerns the immunomodulatory effects of the ACBB31 strain and its uses. Background Technology

[0003] Probiotics have long been utilized as a primary strategy to improve gut health and regulate the balance of gut microorganisms in various animals, including pigs. In particular, as the use of antibiotic growth promoters has become restricted, the use of probiotics as an alternative to antibiotics has garnered significant attention. With advancements in related research revealing that postbiotics exhibit effects similar to probiotics, their functions are also receiving renewed interest. Postbiotics consist of the dead forms, components, or metabolites of microorganisms rather than living ones, and are characterized by their ability to produce health-promoting effects similar to those of probiotics. Compared to probiotics, postbiotics have the advantage of being free from issues such as the risk of infection, the potential for antibiotic resistance gene transfer, and concerns regarding product quality and stability.

[0004] Since microbial preparations can exhibit different effects depending on the strain even within the same species, postbiotics similarly show varying effects based on the strain; therefore, individual studies are required to elucidate their characteristics. Although research on postbiotic preparations is not yet as active as that on probiotic preparations, research and development on postbiotics are necessary to utilize the unique advantages they possess over probiotics. Prior art literature

[0006] KR 10-2022-0122338 (2022-09-27) The problem to be solved

[0007] As a result of diligent efforts to provide a new beneficial bacterium that can be utilized as a postbiotic, the inventors have found, among lactic acid bacteria, Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri The immunomodulatory or anti-inflammatory effect of the ACBB31 strain was confirmed, and the present invention was completed.

[0008] Therefore, the objective of the present invention is a novel Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri The purpose is to provide the immunomodulatory effects of the ACBB31 strain and its uses. means of solving the problem

[0010] The present invention relates to Limosil Lactobacillus reuteri ( Limosilactobacillus reuteri ) Provides an immunomodulatory composition comprising the ACBB31 strain.

[0011] According to a preferred embodiment of the present invention, the strain is the strain deposited under accession number KCTC 15647BP.

[0012] According to a preferred embodiment of the present invention, the strain is a heat-killed strain.

[0013] According to a preferred embodiment of the present invention, the strain comprises the rRNA sequence of SEQ ID NO. 1.

[0014] According to a preferred embodiment of the present invention, the immune modulation inhibits inflammation and T cell activation.

[0015] According to a preferred embodiment of the present invention, the immune modulation inhibits one or more selected from the group consisting of NO, IL-1b, IL-6, COX2, IL-1β, and PGE2.

[0016] According to a preferred embodiment of the present invention, the immune modulation inhibits CD4 T cells and CD8 T cells.

[0017] In addition, the present invention relates to Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteriProvides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the ACBB31 strain.

[0018] According to a preferred embodiment of the present invention, the inflammatory disease is one or more selected from the group consisting of inflammatory bowel disease, leaky gut syndrome, and irritable bowel syndrome.

[0019] In addition, the present invention relates to Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri ) Provides a health functional food composition for the prevention or improvement of inflammatory diseases containing the ACBB31 strain.

[0020] According to a preferred embodiment of the present invention, Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri ) Provides a postbiotics composition containing the ACBB31 strain.

[0021] According to a preferred embodiment of the present invention, i) Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri ) A step of culturing the ACBB31 strain on a plate medium at 35–40℃ for 20–25 hours;

[0022] ii) a step of shaking culture the cultured strain at 35~40℃ for 20~25 hours; and

[0023] iii) A step of inactivating the cultured strain by heating it at 90–110°C for 10–20 minutes;

[0024] Limosilactobacillus reuteri containing Limosilactobacillus reuteri ) Provides a method for producing postbiotics of the ACBB31 strain. Effects of the invention

[0026] The novel Limosil Lactobacillus reuteri of the present invention ( Limosilactobacillus reuteriThe ACBB31 strain (KCTC 15647BP) induced a reduction in colitis-related symptoms in a mouse model of inflammatory bowel disease and showed an effect of lowering the overall expression of inflammatory factors in the body. In addition, when the heat-killed strain was mixed and fed to weaning pigs, the frequency of diarrhea decreased, growth increased, the overall expression of inflammatory factors in the body decreased, and the health of the small intestine improved morphologically. Therefore, the present invention can provide excellent immunomodulatory or anti-inflammatory effects. Brief explanation of the drawing

[0028] FIG. 1a is Limosyl Lactobacillus reuteri of the present invention ( Limosilactobacillus reuteri ) Shows the culture results of the ACBB31 strain (KCTC 15647BP). FIG. 1b is Limosyl Lactobacillus reuteri of the present invention ( Limosilactobacillus reuteri ) Shows the 16s rRNA sequence (Sequence No. 1) of the ACBB31 strain (KCTC 15647BP). Figure 2 shows the ability of newly isolated lactic acid bacteria to inhibit nitric oxide (NO) production in LPS-treated RAW 264.7 cells. RAW 264.7 macrophages were stimulated with LPS and treated with 27 newly isolated HK LABs (20 μg / mL) for 24 hours. NO production was evaluated by the GRIESS assay. Figure 3 shows heat-killed (HK) lactic acid bacteria (ACBB31, ACBB36, LGG) on LPS-treated RAW 264.7 cells. Lactobacillus rhamnosusResults of inflammatory response regulation after treatment with GG)) are shown. (A) Viability of RAW 264.7 cells. (B) NO production rate. (C) Gene expression of IL-1β, IL-6, and COX2 in RAW 264.7 cells treated with the HK selected strain was measured by qRT-PCR. Relative mRNA levels were normalized to GAPDH. Treatment with the HK selected strain reduced inflammatory cytokines IL-1β and IL-6 and the inflammatory mediator COX2, and the HK selected strain at 500 μg / ml showed a significant effect. (D) Protein levels of IL-1β, IL-6, and PGE2 in the culture supernatant of RAW 264.7 cells treated with the HK selected strain (500 μg / mL) were measured by ELISA. *, p<0.05; **, p<0.01; ***, p<0.001. Results are expressed as mean ± standard error (SEM). Figure 4 shows heat-killed (HK) lactic acid bacteria (ACBB31, ACBB36, LGG ( Lactobacillus rhamnosus This study illustrates the immunosuppressive effect of GG on T cell proliferation. HK LAB was treated to CFSE-labeled human monocytes (MNCs) activated by concanavalin A. After 72 hours of culture, T cell proliferation rates were measured using a flow cytometer. The proliferation of total, CD4+, and CD8+ T cells was quantified. **, p<0.01; ***, p<0.001. Results are expressed as mean ± standard error (SEM). Figure 5 shows heat-killed (HK) lactic acid bacteria (ACBB31, ACBB36, LGG ( Lactobacillus rhamnosusThis shows the effect of ACBB31 on improving colitis. (A) Experimental plan for DSS-induced colitis. HK LAB (10 mg / kg) was administered orally daily, and recombinant CX3CL1 was injected intraperitoneally on days 0 and 4 while 3% DSS was administered for 7 days. Mouse (B) body weight and (C) Disease Activity Index (DAI) scores were monitored. Treatment with HK ACBB31 resulted in body weight loss and improvement in DAI scores. The DAI score included body weight loss (0-4), stool consistency (0-4), bloody stool (0-4), rectal prolapse (0-3), and bedding contamination (0-2). (D, E) Colon length was measured after sacrificing the mice on day 10. Colitis mice treated with HK ACBB31 showed increased colon length. (F, G) H&E staining results and histological scores of colon sections (n ​​= 3 mice per group). The scale bar represents 100 μm. Data are expressed as mean ± standard deviation. *, p<0.05; **, p<0.01; ***, p<0.001. Figure 6 shows heat-killed (HK) lactic acid bacteria (ACBB31, ACBB36, LGG ( Lactobacillus rhamnosus This shows the in vivo M2 polarization-inducing effect of GG). Cells isolated from the colon were analyzed and quantified using flow cytometry (A) (B). The ratio of F4 / 80+CD11b+ macrophages was measured, and the expression of iNOS (C) and Arginase 1 (D) in the F4 / 80+CD11b+ ratio was further analyzed. Results are expressed as mean ± standard deviation. p<0.05 compared to the control group; *, p<0.05; **, p<0.01; ***, p<0.001 compared to the DSS group. Figure 7 shows heat-killed (HK) lactic acid bacteria (ACBB31, ACBB36, LGG ( Lactobacillus rhamnosusShows the intestinal cytoprotective effect of GG). (A) Colon sections were stained with PAS to visualize the mucus layer. The scale bar represents 100 μm. (BD) Gene expression of MUC2, Claudin3, and Ocludin in colon tissue lysates was measured by qRT-PCR. Relative mRNA levels were normalized to GAPDH. Results are expressed as mean ± standard error (SEM). #, p<0.05 vs. control; **, p<0.01; ***, p<0.001 vs. DSS group. Figure 8 shows the growth and inflammation-regulating effects of live or heat-killed (HK) lactic acid bacteria (ACBB31) on pigs. (A) Pig feeding experiment plan. A total of 96 weaned piglets [(Yorkshire × Landrace) × Duroc] with an average body weight of 7.57 ± 0.07 kg were assigned to a 5-week feeding experiment. Pigs were randomly distributed into three dietary treatments, with 4 replicates per treatment in a randomized complete block design (RCBD) - 1) CON: Basal feed (corn-soybean meal), 2) Live L. reuteri : 0.05% L. reuteri Basic feed supplemented with, 3) HK L. reuteri : 0.05% HK L. reuteri (B) Average daily gain was measured based on the body weight of pigs in W0, W2, and W5. (C) The incidence of diarrhea was scored on a 5-point scale at a fixed time daily. (DK) Villi length and ridge depth were measured in W2 and W5. (D, H) Yellow arrows indicate villi depth and green arrows indicate villi length. (D) The intestines of W2 and (H) W5 were measured by H&E staining. (E, I) Villi length and (F, J) crypt depth were measured, and (G, K) villi height and crypt depth were calculated. (L) IL-1β, (M) IL-6, and (N) TNF-α protein levels in the serum of W5 pigs were Live and HK L. reuteriMeasured by ELISA with or without supplementation. *, p<0.05; **, p<0.01; ***, p<0.001; 0.05 ≤ p < 0.1 was considered a trend. Results were expressed as mean ± standard error. Figure 9 shows live or heat-killed (HK) lactic acid bacteria (ACBB31). Shows the effects on intestinal cells, microbiota, and metabolites when taken as a dietary supplement. (A) Live and HK L. reuteri (B) Optical microscope images of intestinal organoids treated or untreated (scale bar: 100 μm). Organoid formation efficiency was determined by comparing the number of inoculated crypts with the number of successfully formed organoids. (C) Transcription levels of LGR5, MUC2, and LYZ1 were measured by qRT-PCR. Relative mRNA levels were normalized to GAPDH. *, p<0.05; **, p<0.01; ***, p<0.001. Results are expressed as mean ± standard error (SEM). Fig. 10 shows live or heat-killed (HK) lactic acid bacteria (ACBB31). This shows the effects on intestinal cells, microbiota, and metabolites when consumed as a dietary supplement. (F) Stacked bar graph showing the relative abundance of the top 30 genera and other genera classified as "others" in 16S rRNA sequencing data. Relative abundance was averaged within each treatment group (Control, HK, Probiotics) and visualized through genus-level composition. (G) Principal Coordinate Analysis (PCoA) plot based on Bray-Curtis dissimilarity of genus-level microbiota composition. Each point in the plot represents a sample, and colored ellipses represent the 95% confidence interval for each group. (H, I) Control group (Con), Live L. reuteri and HK L. reuteriAs an alpha diversity analysis, a comparative analysis of (H) the whole tree of phylogenetic diversity (PD) and (I) amplicon sequence variation (ASV) was performed between the groups. (J, K) SCFA levels in W2 and W5 colonic digestive fluids were measured by HPLC. *, p<0.05; **, p<0.01; ***, p<0.001. Results were expressed as mean ± standard error (SEM). Specific details for implementing the invention

[0029] The present invention will be described in more detail below.

[0031] The term 'postbiotics' of the present invention may refer to a formulation containing useful microbial bioactive substances, metabolites, or by-products generated during the growth, death, or fermentation process of beneficial microorganisms (probiotics). Postbiotics may include, but are not limited to, forms such as dead cells, cell components, fermented products, short-chain fatty acids, or peptides.

[0032] The 'strain' of the present invention may be included in the form of a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof. In this case, "strain lysate" refers to a product obtained by culturing a strain and mechanically or chemically crushing it, and may include all products that have undergone additional processes such as extraction, dilution, concentration, and purification therefrom. "Strain culture" may refer to a medium (solid, liquid, etc.) containing the strain, the medium (solid, liquid, etc.) from which the strain has been isolated after culturing the strain, or the supernatant thereof. "Strain culture concentrate" refers to a product that has been purified from the strain culture through ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or a culture concentrate obtained through ultrafiltration, concentration, etc. "Strain culture extract" refers to a product extracted from the culture medium or its concentrate, and may include an extract, a diluted or concentrated extract of the extract, a dried product obtained by drying the extract, a modified or purified product thereof, or a fraction obtained by fractionating the same. The above drying form may include a freeze-drying form.

[0033] The term 'prevention' of the present invention may refer to any act that causes inflammatory diseases or related diseases to be suppressed or their onset to be delayed due to the ACBB31 strain of the present invention.

[0034] The term 'improvement' or 'treatment' of the present invention may refer to any act that causes parameters related to inflammatory diseases or related diseases, such as the degree of symptoms, to be improved or beneficial due to the ACBB31 strain of the present invention.

[0036] Conventional microbial feed additives pose risks such as opportunistic infections and quality stability issues due to the use of live microorganisms. To overcome these limitations, various studies are being conducted on postbiotics that do not use live microorganisms.

[0037] In the present invention, a novel Limosil Lactobacillus reuteri having excellent immunomodulatory ability ( Limosilactobacillus reuteri The ACBB31 strain (KCTC 15647BP) was selected. When the above strain is heat-killed, its activity is lost during the process, so it does not carry the inherent risks associated with live bacteria, such as opportunistic infection or sepsis, and thus has high biocompatibility. In addition, it can suppress the inflammatory response of activated macrophages to inhibit the secretion of inflammatory factors and inhibit cell proliferation caused by the activation of T cells, which are immune cells involved in inflammation, thereby providing an immunomodulatory effect on excessively activated immune cells. Furthermore, by suppressing inflammation, it can induce the improvement of intestinal tissue function that may be damaged by inflammation.

[0038] Furthermore, since the present invention utilizes existing bacterial culture systems as they are, there is no need for additional facility construction, and production using existing equipment is possible. In addition, the novel strain of the present invention is used as a postbiotic preparation, thereby minimizing the risks and stability issues associated with existing microbial preparations, making industrial application easier.

[0040] Therefore, the present invention relates to Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri An immunomodulatory composition comprising the ACBB31 strain can be provided.

[0041] According to a preferred embodiment of the present invention, the strain may be the strain deposited under accession number KCTC 15647BP.

[0042] According to a preferred embodiment of the present invention, the strain may be a heat-killed strain.

[0043] According to a preferred embodiment of the present invention, the strain may include the rRNA sequence of SEQ ID NO. 1.

[0044] According to a preferred embodiment of the present invention, the immune modulation may be one that inhibits inflammation and T cell activation.

[0045] According to a preferred embodiment of the present invention, the immune modulation may be one or more selected from the group consisting of NO, IL-1b, IL-6, COX2, IL-1β, and PGE2.

[0046] According to a preferred embodiment of the present invention, the immune modulation may be to inhibit CD4 T cells and CD8 T cells.

[0048] In addition, the present invention relates to Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri A pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the ACBB31 strain can be provided.

[0049] According to a preferred embodiment of the present invention, the strain may be the strain deposited under accession number KCTC 15647BP.

[0050] According to a preferred embodiment of the present invention, the strain may be a heat-killed strain.

[0051] According to a preferred embodiment of the present invention, the strain may include the rRNA sequence of SEQ ID NO. 1.

[0052] According to a preferred embodiment of the present invention, the inflammatory disease may be one or more selected from the group consisting of inflammatory bowel disease, leaky gut syndrome, and irritable bowel syndrome.

[0053] The pharmaceutical composition of the present invention may be in various oral or parenteral formulations. When formulating the composition, it may be prepared using one or more buffers (e.g., saline solution or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

[0054] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient with one or more compounds, for example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, or gelatin, etc. For example, tablets or sugar-coated tablets can be obtained by combining an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and processing it into a granular mixture.

[0055] In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, or syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, or preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants, and may additionally include anticoagulants, flavorings, emulsifiers, solubilizers, dispersants, flavorings, antioxidants, packaging agents, pigments, and preservatives.

[0056] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, or suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used as bases for suppositories.

[0057] The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. That is, the total effective amount of the composition of the present invention may be administered to the patient as a single dose, or administered via a fractionated treatment protocol involving long-term administration of multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the aforementioned factors, and this can be easily determined by a person skilled in the art.

[0058] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease.

[0059] The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0061] In addition, the present invention relates to Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri A health functional food composition for preventing or improving inflammatory diseases containing the ACBB31 strain can be provided.

[0062] According to a preferred embodiment of the present invention, the strain may be the strain deposited under accession number KCTC 15647BP.

[0063] According to a preferred embodiment of the present invention, the strain may be a heat-killed strain.

[0064] According to a preferred embodiment of the present invention, the strain may include the rRNA sequence of SEQ ID NO. 1.

[0065] According to a preferred embodiment of the present invention, the inflammatory disease may be one or more selected from the group consisting of inflammatory bowel disease, leaky gut syndrome, and irritable bowel syndrome.

[0066] The health functional food composition of the present invention can be prepared in various forms according to conventional methods known in the art. General foods may be prepared by adding the novel strain of the present invention to beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and its processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malt syrup, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, sauces, etc.), although not limited thereto. Additionally, nutritional supplements may be prepared by adding the novel strain of the present invention to capsules, tablets, pills, etc. In addition, as a health functional food, the novel strain of the present invention itself may be consumed by liquefying, granulating, encapsulating, and powdering it, for example, by manufacturing it in the form of tea, juice, and drink so that it can be consumed (health beverage). In addition, to use the novel strain of the present invention as a food additive, it may be manufactured and used in the form of a powder or concentrate. Furthermore, the novel strain of the present invention may be mixed with a known active ingredient known to have an effect of preventing or improving inflammatory diseases to be prepared in the form of a composition.

[0067] When the novel strain of the present invention is used as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates described above may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract; or synthetic sweeteners such as saccharin and aspartame may be used.

[0068] In addition, the novel strain of the present invention may be included as an active ingredient in a health functional food composition for the prevention or improvement of inflammatory diseases, and the amount may be included in an amount effective for achieving the action of preventing or improving inflammatory diseases. The health functional food composition of the present invention may be prepared by mixing the novel strain with other active ingredients known to be effective against inflammatory diseases.

[0069] In addition to the above, the health functional food of the present invention may contain various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. Furthermore, the health functional food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination.

[0071] In addition, the present invention relates to Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri A postbiotics composition containing the ACBB31 strain can be provided.

[0072] According to a preferred embodiment of the present invention, the strain may be the strain deposited under accession number KCTC 15647BP.

[0073] According to a preferred embodiment of the present invention, the strain may be a heat-killed strain.

[0074] According to a preferred embodiment of the present invention, the strain may include the rRNA sequence of SEQ ID NO. 1.

[0076] In addition, the present invention relates to i) Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri ) A step of culturing the ACBB31 strain on a plate medium at 35–40℃ for 20–25 hours;

[0077] ii) a step of shaking culture the cultured strain at 35~40℃ for 20~25 hours; and

[0078] iii) A step of inactivating the cultured strain by heating it at 90–110°C for 10–20 minutes;

[0079] Limosilactobacillus reuteri containing Limosilactobacillus reuteri A method for producing postbiotics of the ACBB31 strain can be provided.

[0080] According to a preferred embodiment of the present invention, the strain may be the strain deposited under accession number KCTC 15647BP.

[0081] According to a preferred embodiment of the present invention, the strain may be a heat-killed strain.

[0082] According to a preferred embodiment of the present invention, the strain may include the rRNA sequence of SEQ ID NO. 1.

[0083] The above manufacturing method preferably comprises i) Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri ) A step of culturing the ACBB31 strain on a plate medium at 36–38°C for 23–25 hours;

[0084] ii) a step of shaking culture the cultured strain at 36–38°C for 23–25 hours; and

[0085] iii) a step of inactivating the cultured strain by heating at 95–105°C for 13–17 minutes; comprising Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri ) This may be a method for producing postbiotics of the ACBB31 strain.

[0087] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0089] 이스한 균주 아니다

[0090] Fresh fecal matter from a beagle was collected and transported to the laboratory within 3 hours. 500 mg of fecal matter was placed in 5 mL of PBS and vortexed, then left at 4°C for 30 minutes to precipitate the particles. The supernatant was collected, serially diluted onto LBS agar medium, and incubated at 37°C for 24 hours. A single colony formed was isolated, inoculated onto MRS broth medium, and incubated with shaking at 37°C for 24 hours; the cultured strain was then stored at -80°C.

[0091] 균주 균주 명치 ACBB21 Enterococcus faecium ACBB22 Enterococcus faecium ACBB23 Enterococcus faecium ACBB24 Enterococcus gilvus ACBB25 Enterococcus faecium ACBB26 Enterococcus faecium ACBB27 Enterococcus faecium ACBB28 Enterococcus faecium ACBB29 Enterococcus faecium ACBB20 Enterococcus faecium ACBB31 Limosilactobacillus reuteri subsp. reuteri ACBB32 Limosilactobacillus reuteri subsp. reuteri ACBB33 Lactobacillus hominis DSM 23910 ACBB34 Enterococcus faecium ACBB35 Enterococcus faecium ACBB36 Enterococcus faecium ACBB37 Limosilactobacillus reuteri ACBB38 Limosilactobacillus reuteri subsp. reuteri ACBB39 Limosilactobacillus reuteri subsp. reuteri ACBB30 Limosilactobacillus reuteri ACBB41 Enterococcus faecium ACBB42 Lactobacillus johnsonii ACBB43 Lactobacillus gasseri ACBB44 Limosilactobacillus reuteri subsp. reuteri ACBB45 Lactobacillus psittaci ACBB46 Limosilactobacillus reuteri subsp. reuteri ACBB47 Lactobacillus gasseri

[0093] Heat annihilation of lactic acid bacteria strains

[0094] Each lactic acid bacteria strain was cultured on MRS agar plates at 37°C for 24 hours. Colonies taken from the plates were cultured on MRS broth in a shaking incubator at 37°C for 24 hours. The lactic acid bacteria culture was resuspended in PBS and washed twice. The lactic acid bacteria pellet was resuspended in distilled water and inactivated by heating at 100°C for 15 minutes. The heat-killed lactic acid bacteria were freeze-dried and stored at -80°C prior to use.

[0096] Inhibitory effect of NO production by lactic acid bacteria strains

[0097] We aimed to confirm the inhibitory effect of heat-killed (HK) lactic acid bacteria (LAB) strains on nitric oxide (NO) production.

[0098] Specifically, 2×10 RAW 264.7 macrophages 5Cells were inoculated into a 24-well plate at a density of cells / cm² and stimulated with LPS. Subsequently, 27 newly isolated HK LAB cells (20 μg / mL) from <Example 1> were treated for 24 hours. NO production was evaluated by calculating nitrite concentration using a NaNO₂ standard curve via the GRIESS assay, and absorbance was measured at 530–540 nm (VersaMax, Molecular Devices).

[0099] As a result, as shown in [Figure 2], the inhibitory effect on NO production varied depending on the lactic acid bacteria strain. In particular, Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri It was confirmed that the ACBB31 strain exhibited the most superior NO production inhibition effect.

[0101] Regulating inflammatory response of lactic acid bacteria strains

[0102] We aimed to verify the inflammatory response regulating effects of heat-killed (HK) lactic acid bacteria (LAB) strains.

[0103] Specifically, HK selected strains (ACBB31, ACBB36, LGG) at concentrations of 50, 200, 500, or 2000 μg / mL ( Lactobacillus rhamnosus The viability of RAW 264.7 cells treated with GG was measured using the MTT assay.

[0104] The NO production rate was measured using the same method as in <Example 3> above.

[0105] mRNA levels of inflammatory factors (Il1b, Il6, and COX2) were measured by qRT-PCR, and relative mRNA levels were normalized to GAPDH. Protein levels of inflammatory factors (IL-1β, IL-6, and PGE2) were measured by ELISA of IL-1β, IL-6, and PGE2 in the culture supernatant of RAW 264.7 cells treated with the HK selected strain (500 μg / mL).

[0106] As a result, as shown in [Figure 3], the ACBB31 strain did not exhibit cytotoxicity, while the ACBB36 and LGG strains exhibited cytotoxicity when treated at 2000 μg / mL. Additionally, the HK selected strains reduced NO production. Treatment with the HK selected strains reduced the inflammatory cytokines Il1b and Il6 and the inflammatory mediator COX2, and the HK selected strain at 500 μg / mL showed a significant protein inhibitory effect.

[0108] T cell proliferation regulatory effects of lactic acid bacteria strains

[0109] We aimed to confirm the immunosuppressive effect of heat-killed (HK) lactic acid bacteria (LAB) strains on T cell proliferation.

[0110] Specifically, HK LAB was treated to CFSE-labeled human mononuclear cells (MNCs) activated by concanavalin A. After 72 hours of culture, T cell proliferation rates were measured using a flow cytometer, and the proliferation of total, CD4+ T cells, and CD8+ T cells was quantified.

[0111] As a result, as shown in [Figure 4], it was confirmed that the ACBB31 strain of the present invention exhibited the most superior T cell proliferation inhibitory effect.

[0113] Effects of lactic acid bacteria strains on improving colitis

[0114] We aimed to verify the effect of heat-killed (HK) lactic acid bacteria (LAB) strains on improving colitis among inflammatory diseases.

[0115] Specifically, HK LAB (10 mg / kg) was administered orally daily, and recombinant CX3CL1 was injected intraperitoneally on days 0 and 4 while 3% DSS was administered for 7 days; subsequently, mouse body weight and Disease Activity Index (DAI) scores were monitored. The DAI score included body weight loss (0-4), stool consistency (0-4), bloody stool (0-4), rectal prolapse (0-3), and bedding contamination (0-2). Colon length was measured after sacrificing the mice on day 10. Colon sections were examined by H&E staining, and histological scores were verified (n = 3 mice per group).

[0116] As a result, as shown in [Figure 5], HK ACBB31 treatment showed weight loss and improved DAI scores in mice. Colitis mice administered HK ACBB31 had increased colon length and the lowest histological scores.

[0118] Effect of Lactic Acid Bacteria Strains on M2 Macrophage Polarization

[0119] To confirm the macrophage polarization-inducing effect of heat-killed (HK) lactic acid bacteria (LAB) strains, plaque cells isolated from the large intestine were analyzed and quantified using flow cytometry.

[0120] Specifically, to isolate lamina propria cells, C57BL / 6 mice were sacrificed, and the colon was excised and washed with cold PBS. The external mesentery and fat were removed, and the contents of the intestinal lumen were gently removed. The colon was longitudinally incised and cut into 3 × 3 mm sections, and epithelial cells were removed by shaking in an HBSS solution (EDTA solution) containing 5% FBS and 2 mM EDTA at 37°C and 220 rpm for 30 minutes. The removed tissue sections were washed with PBS and placed in a digestive solution containing collagenase D (1 mg / mL) and DNase I (0.1 mg / mL), and shaken incubated at 37°C and 220 rpm for 60 minutes. Single cells were separated from the cell suspension by passing it through a 40 μm cell strainer, centrifuged at 1700 rpm for 5 minutes at 4°C, and washed with PBS. Finally, the cells were resuspended in RPMI1640 medium containing 10% FBS.

[0121] After treating the isolated platelets with an Fc receptor blocker, macrophage markers and polarization markers were stained with the following antibodies: surface staining - F4 / 80, CD11b, intracellular staining - iNOS, Arginase 1. Staining was performed at 4°C for 30 minutes, and fixation and permeabilization were performed using a permeabilization buffer.

[0122] Afterwards, the ratio of F4 / 80+CD11b+ macrophages was measured by flow cytometry, and the expression of iNOS and arginase 1 in the F4 / 80+CD11b+ ratio was further analyzed.

[0123] As a result, as shown in [Figure 6], it was confirmed that the HK ACBB31 treatment of the present invention induces polarization of M2 macrophages by significantly increasing arginase 1+ cells among DSS-treated macrophages.

[0125] Protective effect of lactic acid bacteria strains on intestinal cells

[0126] We aimed to confirm the protective effect of heat-killed (HK) lactic acid bacteria (LAB) strains on intestinal cells.

[0127] Specifically, the colon was excised from mice with inflammatory bowel disease (DSS-induced colitis model) at the end of the experiment (Day 10). The excised colons were separated for section and tissue lysate analysis and fixed or frozen, respectively. Paraffin blocks were prepared from the colon tissue sections, and staining was performed on the sections. After removing the paraffin, Periodic Acid-Schiff (PAS) staining was performed on the sections to visualize the presence and distribution of the mucus layer. The results of the PAS staining were observed and photographed using an optical microscope. Total RNA was extracted from separately collected colon tissues using Trizol reagent, and cDNA was synthesized. The synthesized cDNA was used to analyze the gene expression of MUC2, Claudin3, and Ocludin via qRT-PCR. The relative expression levels of each gene were normalized using GAPDH as an internal control and calculated using the ΔΔCt method.

[0128] As a result, as shown in [Fig. 7], it was confirmed that the HK ACBB31 treatment of the present invention provides an excellent intestinal cell protective effect.

[0130] Growth and inflammation-regulating effects of the ACBB31 strain

[0131] We aimed to investigate the growth and inflammation control effects of pigs when live or heat-killed ACBB31 strains were fed as pig feed.

[0132] Specifically, a total of 96 weaned piglets [(Yorkshire × Landrace) × Duroc] with an average body weight of 7.57 ± 0.07 kg were assigned to a 5-week feeding experiment. Pigs were randomly distributed into three dietary treatments, repeated 4 times per treatment in a randomized complete block design (RCBD) - 1) CON: Basal feed (corn-soybean meal), 2) Live L. reuteri : 0.05% L. reuteri Basic feed supplemented with, 3) HK L. reuteri : 0.05% HK L. reuteri Basal feed supplemented with. Average daily gain was measured based on pig body weight at week 0 (W0), week 2 (W2), and week 5 (W5). The incidence of diarrhea was scored on a 5-point scale at a fixed time daily. The length and depth of villi and crypts were measured in W2 and W5. The intestines of W2 and W5 were examined by H&E staining. IL-1β, IL-6, and TNF-α protein levels in the serum of W5 pigs were measured using Live and HK L. reuteri It was measured by ELISA with or without supplementation.

[0133] As a result, as shown in [Fig. 8], it was confirmed that the ACBB31 strain of the present invention provides superior effects on growth and inflammation control in pigs when heat-treated compared to when alive.

[0135] Effects of ACBB31 strain on intestinal cells, microbiota, and metabolites

[0136] We wanted to determine the effects on intestinal cells, microbiota, and metabolites when live or heat-killed ACBB31 strains were provided as a dietary supplement.

[0137] First, to prepare intestinal organoids, the small intestine was isolated from pigs undergoing feeding experiments, and after removing adipose tissue and internally washing, the intestinal villi were removed. The tissue was washed several times with 30 mL of ice-cold PBS+P / S, and crypts were isolated using GCDR (Gentle Cell Dissociation Reagent). The isolated crypts were filtered through a 70 μm cell strut, centrifuged, washed with AdDMEM, and resuspended in 100 μL. The number of crypts was counted, and Matrigel and growth medium were mixed in a 1:1 ratio and seeded in a dome shape in a 48-well plate. Microscopic images were taken on the 5th day after culture, and efficiency was calculated based on the number of organoids produced relative to the seeded crypts.

[0138] Total RNA was extracted from organoids cultured for 5 days using TRIzol, and after cDNA synthesis, the transcription levels of LGR5, MUC2, and LYZ1 were analyzed by qRT-PCR to examine the expression of target genes. Gapdh was used as the housekeeping gene, and relative expression levels were calculated using the ΔΔCt method.

[0139] Digestive tissue from the large intestines of experimental animals was collected, and DNA was extracted using a fecal DNA extraction kit. Based on the extracted DNA, the 16S rRNA region was amplified and sequenced, and genus-level composition was confirmed through taxonomic analysis. Alpha diversity (α-diversity) and beta diversity (β-diversity) analyses were performed, and the results were visualized as stacked bar plots, PCoA plots, whole trees of phylogenetic diversity (PD), and amplicon sequence variations (ASV).

[0140] For the analysis of SCFA (Short chain fatty acids), stool was suspended in a 0.01 N sulfuric acid (H2SO4) solution at a ratio of 1:6 (w / v). After vortexing for 1 minute, the sample was centrifuged at 10,000 × g for 10 minutes, and the supernatant was filtered through a 0.2 μm cellulose acetate membrane. The filtered sample was stored at -20°C, and the concentrations of SCFAs, including acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid, were quantified by HPLC analysis.

[0141] As a result, HK L., as shown in [Fig. 9] and [Fig. 10]. reuteri Pigs treated with [the substance] showed increased PD total tree index and ASV. In addition, in W2, SCFAs including butyric acid, isovaleric acid, and valeric acid, and in W5, SCFAs including butyric acid and valeric acid, HK L. reuteri It was possible to confirm that it increased during processing.

[0143] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resources Center (KCTC) Trustee Number: KCTC15647BP Date of Deposit: 2023-10-19

Claims

Claim 1 Limosilactobacillus reuteri ( Limosilactobacillus reuteri Immunomodulatory composition comprising the ACBB31 strain. Claim 2 A composition according to claim 1, characterized in that the strain is a strain deposited under accession number KCTC 15647BP. Claim 3 A composition according to claim 1, characterized in that the strain is a heat-killed strain. Claim 4 A composition according to claim 1, characterized in that the strain comprises the rRNA sequence of SEQ ID NO.

1. Claim 5 A composition according to claim 1, wherein the immune modulation is characterized by inhibiting inflammation and T cell activation. Claim 6 A composition according to claim 1, wherein the immune modulation is characterized by inhibiting one or more selected from the group consisting of NO, IL-1b, IL-6, COX2, IL-1β, and PGE2. Claim 7 A composition according to claim 1, wherein the immune modulation is characterized by inhibiting CD4 T cells and CD8 T cells. Claim 8 Limosilactobacillus reuteri ( Limosilactobacillus reuteri A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the ACBB31 strain. Claim 9 A composition according to claim 8, characterized in that the inflammatory disease is one or more selected from the group consisting of inflammatory bowel disease, leaky gut syndrome, and irritable bowel syndrome. Claim 10 Limosilactobacillus reuteri ( Limosilactobacillus reuteri A health functional food composition for the prevention or improvement of inflammatory diseases containing the ACBB31 strain. Claim 11 Limosilactobacillus reuteri ( Limosilactobacillus reuteri A postbiotics composition containing the ACBB31 strain. Claim 12 i) Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri ) a step of culturing the ACBB31 strain on a plate medium at 35–40°C for 20–25 hours; ii) a step of shaking the cultured strain at 35–40°C for 20–25 hours; and iii) a step of inactivating the cultured strain by heating at 90–110°C for 10–20 minutes; comprising Limosyl Lactobacillus reuteri ( Limosilactobacillus reuteri Method for producing postbiotics of the ACBB31 strain.

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