A novel strain of Lactipruncibacillus plantarum isolated from tea leaves

Abstract

To provide a novel strain of Lactiplantibacillus plantarum having high immunostimulation activity.SOLUTION: The present invention provides a Lactiplantibacillus plantarum LOC1 strain derived from tea leaves, the strain having higher immunostimulation activity than that of other strains of Lactiplantibacillus plantarum.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to a novel strain of the genus Lactipruncibacillus plantarum derived from tea leaves, and uses thereof. [Background technology]

[0002] Lactic acid bacteria are highly safe microorganisms that are widely used in the production of fermented foods, etc. In recent years, it has been reported that certain types of lactic acid bacteria act on macrophages and dendritic cells to enhance the secretion of interleukin 12 (IL-12), and activate natural killer cells via interferon-γ production from CD4 T cells, thereby enhancing the host's defense capabilities.

[0003] Lactiplantibacillus plantarum is a plant-derived lactic acid bacterium, and its type strain, ATCC14917, is known to have high immunostimulatory activity (Japanese Patent Publication No. 2012-213352).

[0004] Lactiprantibacillus plantarum is a lactic acid bacterium that was previously called Lactobacillus plantarum before the reclassification of the Lactobacillus genus. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-213352 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a novel strain of the genus Lactiplantibacillus plantarum, which has high immunostimulatory activity. [Means for solving the problem]

[0007] The inventors searched for lactic acid bacteria contained in tea leaves that enhance IL-12 production in macrophages and analyzed their mechanism of action, and identified a novel strain of the genus Lactiplantibacillus plantarum that significantly increases the mRNA expression level of IL-12.

[0008] That is, the present invention includes the following inventions. [1] Lactiplantibacillus plantarum LOC1 strain is derived from tea leaves and has higher immunostimulatory activity than other strains of Lactiplantibacillus plantarum. [2] The Lactiprantibacillus plantarum LOC1 strain described in [1], wherein the Lactiprantibacillus plantarum LOC1 strain is a strain identified by accession number NITE AP-03527. [3] An immunostimulant comprising the Lactiplantibacillus plantarum LOC1 strain described in [1] or [2] as an active ingredient. [4] The immunostimulant according to [3], for enhancing the expression of interleukin 12 (IL-12). [5] A composition comprising the Lactiplantibacillus plantarum LOC1 strain according to [1] or [2], a bacterial component thereof, a culture or a processed product thereof. [6] The composition described in [5], which is in the form of a beverage, food, supplement or pharmaceutical. [7] The composition according to [5] or [6], which is in the form of a liquid, powder, tablet or capsule. [Effects of the Invention]

[0009] The LOC1 strain has higher immunostimulatory activity than the ATCC14917 strain, which is the reference strain of Lactiplantibacillus plantarum. [Brief explanation of the drawings]

[0010] [Figure 1] Electropherogram of PCR products amplified with Lactiplantibacillus genus-specific primers. M: 100 bp DNA ladder; 1: strain LOC1; 2: Lactiplantibacillus plantarum ATCC 14917T; 3: Lactiplantibacillus plantarum ATCC 8014; 4: Bifidobacterium lactis Bb12; 5: negative control (distilled water). [Figure 2] Electropherogram of PCR products amplified with species-specific primers for Lactipranchibacillus plantarum. M: 100 bp DNA ladder; 1: strain LOC1; 2: Lactipranchibacillus plantarum ATCC 14917T; 3: Lactipranchibacillus plantarum ATCC 8014; 4: L. acidophilus La5; 5: negative control (distilled water). [Figure 3] The effect of Lactiplantibacillus plantarum on the transepithelial electrical resistance (TEER) of Caco-2 / HT29-MTX (100:0, 90:10, 75:25, and 0:100 ratios). All cells were cultured for 24 hours in unsupplemented medium or in medium supplemented with bacteria. After measuring the TEER values, the cells were treated with medium containing dextran sulfate sodium (DSS) (1% vol / wt) for 6 hours. The change in TEER after 6 hours compared to the initial TEER value was expressed as a percentage change. Values ​​are the mean (±SEM) of three experiments (three samples per treatment per experiment). Asterisks indicate significant differences between each treatment and the untreated control (*: P<0.05). Control = no treatment; LOC1 = LOC1 strain; ATCC14917 = Lactipranchibacillus plantarum ATCC 14917T; ATCC8014 = Lactipranchibacillus plantarum ATCC 8014. [Figure 4]Fold changes in TJP1, TJP2, and OCLN mRNA in Caco-2 / HT29-MTX (90:10 ratio, 75:25 ratio) after 48 hours of incubation with bacteria. Data are means (±SEM) of triplicate experiments (three samples per treatment per experiment). Asterisks indicate significant differences between each treatment and the untreated control (**: P<0.01). Control = untreated, LOC1 = strain LOC1, ATCC14917 = Lactiplantibacillus plantarum ATCC 14917T, ATCC8014 = Lactiplantibacillus plantarum ATCC 8014. [Figure 5] Fold changes in MUC2, MUC5AC, and MUC4 mRNA in Caco-2 / HT29-MTX (90:10 ratio, 75:25 ratio) after 48 hours of incubation with bacteria. Data are means (±SEM) of triplicate experiments (three samples per treatment per experiment). Asterisks indicate significant differences between each treatment and the untreated control (*: P<0.05, **: P<0.01). Control = untreated, LOC1 = strain LOC1, ATCC14917 = Lactiplantibacillus plantarum ATCC 14917T, ATCC8014 = Lactiplantibacillus plantarum ATCC 8014. [Figure 6] Analysis of the effect of lactic acid bacteria on IL-12 mRNA expression and secretion in J774.1 cells. N = untreated. LPS = lipopolysaccharide (positive control). DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments or implementations of the present invention will be described in detail, but the present invention is not limited to these and various modifications are possible within the scope of the gist thereof.

[0012] (Lactipranthibacillus plantarum LOC1 strain) In one embodiment, there is provided a Lactipranchibacillus plantarum LOC1 strain derived from tea leaves, which has higher immunostimulatory activity than other strains of Lactipranchibacillus plantarum.

[0013] The Lactiplantibacillus plantarum LOC1 strain is a gram-positive bacillus derived from tea leaves and can grow at around room temperature, for example, between 10°C and 40°C. Lactic acid bacteria vary in fermentation mode depending on the species (homolactic fermentation or heterolactic fermentation), but the fermentation mode of the LOC1 strain is considered to be homolactic based on the genes it possesses and the odor of the fermentation. Although the LOC1 strain can grow under conditions other than anaerobic, it is preferable to grow it under anaerobic conditions. In one embodiment, the culture is carried out by adjusting the initial pH to a weakly acidic to weakly basic range, for example, between pH 5.7 and 9.3.

[0014] The LOC1 strain can be used in a viable state, but it is preferably heat-sterilized before use in order to obtain the desired effects such as immunostimulatory activity.

[0015] The immunostimulatory activity is not particularly limited as long as it has a beneficial effect of activating the immune system, and examples thereof include the activity of promoting cytokine production. Such cytokines include interleukins such as interleukin 12 (IL-12) and interferons such as interferon-γ (IFN-γ), which are produced by immune cells such as B cells, T cells, macrophages, natural killer (NK) cells, and dendritic cells. The immunostimulatory activity is preferably the activity of promoting IL-12 expression.

[0016] IL-12 is a type of cytokine secreted by antigen-presenting cells such as dendritic cells and macrophages, and has the effects of inducing differentiation into type 1 helper T cells (Th1), activating natural killer cells (NK cells), and enhancing the phagocytic activity of cells such as macrophages. IL-12 is thought to be involved in defense against infections caused by viruses and bacteria and in the host's antitumor effects, and is also known to have antitumor, antimetastatic, and anti-opportunistic infection effects, as well as preventive and therapeutic effects against allergic diseases such as asthma.

[0017] The Lactiplantibacillus plantarum LOC1 strain has higher immunostimulatory activity than other strains of the genus Lactiplantibacillus plantarum. As used herein, "high" immunostimulatory activity means that the desired activity is increased compared to a control, such as another strain of Lactiplantibacillus plantarum, and means that the activity is at least about 1%, 5%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or more higher than the control.

[0018] The Lactiplantibacillus plantarum LOC1 strain was deposited at the Patent Organism Depositary of the National Institute of Technology and Evaluation on August 31, 2021, and was assigned the accession number NITE AP-03527 on September 1 of the same year.

[0019] After culturing the LOC1 strain, the resulting culture may be used as is, or may be diluted or concentrated before use, or bacterial cells recovered from the culture may be used. The LOC1 strain may also include mutant strains prepared from the deposited parent strain, as long as they have high immunostimulatory activity.

[0020] (immunostimulant) In one embodiment, there is provided an immunostimulant comprising, as an active ingredient, the Lactipranchibacillus plantarum LOC1 strain, which is derived from tea leaves and has higher immunostimulatory activity than other strains of Lactipranchibacillus plantarum.

[0021] The LOC1 strain contained as an active ingredient may be a mutant strain prepared from the deposited parent strain, as long as it has high immunostimulatory activity. The active ingredient may be not only the bacterial cells, but also a component derived from the bacterial cells, a culture of the bacterial cells, or a processed product thereof. These preferably contain nucleic acid, particularly RNA, of the LOC1 strain.

[0022] The immunostimulant can be provided as a pharmaceutical, quasi-drug, food or drink, supplement, etc. The dosage form is not particularly limited, and may be oral powder, granules, tablets, sugar-coated tablets, capsules, syrup, pills, suspensions, liquids, emulsions, parenteral injections, suppositories, etc. When provided as a food or drink, the immunostimulant may be provided not only in the form of a drink or food, but also in the form of a hard capsule, soft capsule, tablet, granule, etc.

[0023] The immunostimulant may contain other ingredients known to have immunostimulatory effects, and, if necessary, any ingredients such as excipients, stabilizers, preservatives, binders, disintegrants, pH adjusters, preservatives, and fragrances used in conventional pharmaceuticals, quasi-drugs, and food and beverages, within the scope that does not impair the effects of the LOC1 strain.

[0024] The immunostimulatory activity of the LOC1 strain is not limited as long as it has a beneficial effect of activating immunity. The immunostimulatory activity is preferably the activity of promoting cytokine production, particularly the activity of enhancing IL-12 expression.

[0025] The activity of enhancing IL-12 expression can also be used as an antiviral agent. The virus in question is not particularly limited as long as it can be prevented or treated by enhancing IL-12 expression, and an example thereof is influenza virus.

[0026] (composition) In one embodiment, there is provided a composition comprising Lactipranchibacillus plantarum LOC1 strain, which is derived from tea leaves and has higher immunostimulatory activity than other strains of Lactipranchibacillus plantarum, a bacterial component thereof, a culture, or a processed product thereof.

[0027] The LOC1 strain contained in the composition may be a mutant strain prepared from the deposited parent strain, as long as it has high immunostimulatory activity. The active ingredient may be not only the bacterial cells, but also a component derived from the bacterial cells, a culture of the bacterial cells, or a processed product of the bacterial cells. These preferably contain nucleic acid, particularly RNA, of the LOC1 strain.

[0028] Although not intended to be limiting, examples of processed products of the LOC1 strain include those that have been subjected to known physical treatments such as grinding or disruption of live or killed LOC1 cells, or other treatments, such as enzymatic treatment. Specific examples include ground products, disrupted products, liquid products (extracts, etc.), concentrates, pastes, dried products (spray-dried products, freeze-dried products, vacuum-dried products, drum-dried products, etc.), diluted products, enzymatically decomposed products, etc.

[0029] The culture of the LOC1 strain may include a suspension of bacterial cells after culturing the cells, a mixture of bacterial cells (including a fermented product), a residue after culture, etc. The culture may also include a cytoplasm or cell wall fraction obtained by processing the bacterial cell culture.

[0030] The medium for culturing the fungus is not particularly limited, and any medium suitable for culturing Lactiplantibacillus plantarum can be used. For example, the medium may be a natural medium, a synthetic medium, a semi-synthetic medium, or the like.

[0031] The culture conditions (culture temperature, pH, culture period, etc.) may be the same as those typically used for culturing Lactiplantibacillus plantarum. Although not intended to be limiting, the culture temperature may be, for example, typically about 10 to 40°C, preferably about 20 to 40°C, and the culture period may be, for example, about 15 to 50 hours, preferably about 16 to 24 hours.

[0032] The LOC1 strain is added to food and drink by sprinkling it on the surface of the food and drink, placing it on top, or mixing it into the food and drink.

[0033] As used herein, "food and drink" refers to processed foods, beverages, fruits and vegetables, and other items that are consumed. Examples of beverages include, but are not limited to, tea drinks, milk drinks, lactic acid bacteria drinks, soy milk, soft drinks (including flavored water such as near water), nutritional drinks, sports drinks, fruit drinks, vegetable juices, dairy drinks, alcoholic drinks, jelly drinks, carbonated drinks, and other beverages and health supplements.

[0034] Examples of containers for filling the beverage include, but are not limited to, commonly used containers such as plastic bottles, cans, paper bottles, jars, etc. It is preferable to use a container that can be sealed after being filled.

[0035] Examples of foods include, but are not limited to, sweets such as cookies, biscuits, chocolate, cake, pudding, ice cream, sherbet, waffles, wafers, pancakes, donuts, popcorn, castella, caramel, candy, chewing gum, and Japanese sweets; breads such as white bread, sweet bread, and other breads; noodles such as udon, hiyamugi, somen, buckwheat, Chinese noodles, spaghetti, macaroni, rice vermicelli, and harusame; processed meat foods such as hamburger steak, ham, bacon, and sausage; and instant foods such as curry, ramen, and soup.

[0036] The content of Lactiplantibacillus plantarum LOC1 strain in the composition is determined appropriately depending on the dosage form of the composition, the method of use, the age and sex of the patient, the type of disease, the degree of disease, and other conditions, but is usually 5 × 10 4 ~5×10 8 cfu / ml, preferably in the range of 5 x 10 5 ~5×10 7 More preferably, it is in the range of cfu / ml.

[0037] When the composition is administered as a pharmaceutical composition, the dosage form can be determined depending on the formulation, the patient's age, sex, other conditions, the severity of the patient's symptoms, etc. The timing of administration is also not limited, and in any case, the composition can be administered once a day or in divided doses, or once every few days or weeks.

[0038] Medical applications include the treatment or prevention of diseases that can be treated by increased expression of IL-12, such as infectious diseases, tumors, allergies, etc.

[0039] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples. [Example]

[0040] Isolation and identification of Lactipranchibacillus strains Tea leaves collected from tea fields in Kagoshima Prefecture were soaked in 10 ml of phosphate-buffered saline (PBS) and crushed in a mortar and pestle at room temperature. 50 μl of this solution was plated on MRS (De Man, Rogosa, and Sharp) agar medium supplemented with sodium carbonate (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 1%. The mixture was then anaerobically cultured at 37°C for 48 hours using an AnaeroPack-Anaero (Mitsubishi Gas Chemical Company, Inc.).

[0041] One strain (LOC1) was randomly selected from the samples and inoculated into a screw-capped test tube containing 10 ml of MRS broth. The LOC1 strain was then cultured at 37°C for 1-2 days. The stock culture was stored at -80°C in MRS broth containing 30% glycerol. The genus and species of the LOC1 strain were then identified by PCR. Total genomic DNA of the LOC1 strain was prepared according to the procedure described by Marmur et al. (Marmur J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3: 208-218). The primers used in this example are listed in Table 1. [Table 1]

[0042] The PCR reaction mixture consisted of 250 ng of genomic DNA as a template, 100 pmol of each primer, 5 U of ExTaq DNA polymerase (Takara Bio Inc.), 12.5 mM MgCl2, and 200 mM dNTPs dissolved in deionized water to a final volume of 50 mL. Standard strains of Lactiplantibacillus and Lactiplantibacillus plantarum were included as positive and negative controls in each set of PCR amplification.

[0043] The PCR conditions were as follows: for Lactiplantibacillus, one cycle of 95°C for 5 minutes, 30 cycles of 95°C for 15 seconds, 58°C for 20 seconds, and 72°C for 45 seconds, followed by one cycle of 72°C for 5 minutes, were performed using a SimpliAmp thermal cycler (Eppendorf). For Lactiplantibacillus plantarum, one cycle of 95°C for 15 minutes, 40 cycles of 94°C for 20 seconds, 60°C for 20 seconds, and 72°C for 50 seconds, followed by one cycle of 72°C for 5 minutes, were performed using a SimpliAmp thermal cycler (Eppendorf). PCR products were confirmed on a 1% agarose gel. The gel was stained with 0.5 g L-1 (w / v) ethidium bromide and imaged using a FluoroPhoreStar 3000 (Anatech, Tokyo, Japan). The size of the PCR products was determined using a 100-bp DNA ladder (Takara Bio) as a control.

[0044] Co-culture conditions Human colon adenocarcinoma cell lines, Caco-2 and HT29-MTX, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Caco-2 cells were used after 28–33 passages, and HT29-MTX cells were used after 18–25 passages.

[0045] Caco-2 cells and HT29-MTX cells were cultured in a 75cm culture medium containing high glucose and L-glutamine DMEM supplemented with 10% FBS (Daiichi Chemical Co., Ltd.), 1% non-essential amino acids (NEAA; Gibco BRL, Grand Island, NY, USA), 1% penicillin and streptomycin, and 2.5% HEPES. 2 0.5 x 10 in flask 5 The cells were cultured at a density of 1000 × 1000 in a humidified incubator containing 5% CO2 at 37°C. Before reaching the required passage number for the study, the cells were subcultured using trypsin-EDTA solution (0.25%), and the medium was changed every other day.

[0046] Bacterial strains and culture conditions The strain used in this study was LOC1, Lactipranthibacillus plantarum ATCC 14917. T and Lactiplantibacillus plantarum ATCC 8014. All strains were stored in MRS (Man-Rogosa-Sharpe) broth (Oxoid, Basingstoke, United Kingdom) at -80°C and subcultured twice in MRS broth before use. All strains were grown anaerobically in MRS broth at 37°C overnight. Bacterial strains were used in all tests in stationary phase.

[0047] Preparation of bacterial cells Stationary-phase cells were collected by centrifugation at 8,000 × g and then washed twice with phosphate-buffered saline (PBS). The bacterial count was determined using a hemocytometer (Brightline, Hausser Scientific) to be 1 × 10 9 After adjusting the concentration to cells / mL, the cells were autoclaved at 121°C for 15 minutes. 8 The cells were added.

[0048] statistical analysis Data were expressed as mean values ​​with standard deviations. Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) and Dunnett's test. Asterisks (*, **) indicate statistical differences, with significance levels of p<0.05 and p<0.01, respectively.

[0049] result When the solution containing crushed tea leaves was cultured on MRS agar medium supplemented with sodium carbonate, several colonies were observed. One of these was randomly selected and designated strain LOC1. PCR was performed to confirm the genus and species of the strain using genus-specific primers and species-specific primers, respectively. PCR using primers targeting the genus Lactiplantibacillus confirmed that strain LOC1 was a Lactiplantibacillus (Figure 1). Subsequently, PCR using primers targeting Lactiplantibacillus plantarum confirmed that strain LOC1 was a Lactiplantibacillus plantarum (Figure 2).

[0050] LOC1 strain suppresses the decrease in transepithelial electrical resistance (TEER) induced by dextran sulfate sodium (DSS) Transepithelial electrical resistance (TEER) is an important parameter for evaluating intestinal barrier function. The initial TEER values ​​(before DSS treatment) for the 100:0, 90:10, 75:25, and 0:100 Caco-2 / HT29-MTX mixtures were 470–510 Ω × cm, respectively. 2 , 440~480 Ω × cm 2 , 340~370 Ω × cm 2 , 100~140Ω × cm 2 It was.

[0051] Dextran sodium sulfate (DSS), a heparin-like polysaccharide containing up to three sulfates per glucose molecule and approximately 17% sulfur, has been used as a model to induce experimental colitis and colon cancer and to reduce the TEER of intestinal epithelial cells. After 6 hours of treatment with DSS, the TEER values ​​of all co-cultured cells decreased (data not shown). Meanwhile, the LOC1 strain significantly inhibited (p<0.05) the DSS-induced decrease in TEER in Caco-2 / HT29-MTX (100:0 and 90:10 ratios), but not in Lactiplantibacillus plantarum ATCC 14917T or Lactiplantibacillus plantarum ATCC 8014 (Figures 3a and 3b). In Caco-2 / HT29-MTX (75:25 ratio, 0:100 ratio), the addition of bacteria did not significantly suppress the DSS-induced decrease in TEER value (FIGS. 3c and 3d).

[0052] Effect of strain LOC1 on tight junction-related gene expression The effects of strain LOC1 on the expression of three tight junction (TJ)-related genes were compared using quantitative PCR in Caco-2 / HT29-MTX (90:10 and 75:25 ratios) with those of Lactipranchibacillus plantarum ATCC 14917T and Lactipranchibacillus plantarum ATCC 8014. In Caco-2 / HT29-MTX (90:10 ratio), the addition of strain LOC1 significantly increased OCLN expression compared to the control, whereas the expression of TJP1 and TJP2 was unaffected (p < 0.01) (Fig. 4a). In Caco-2 / HT29-MTX (75:25 ratio), the addition of strain LOC1 and Lactipranchibacillus plantarum ATCC 8014 significantly increased OCLN mRNA expression (p < 0.01) (Fig. 4b). On the other hand, no significant effect of bacteria on the expression of TJP1 and TJP2 was observed.

[0053] Effect of LOC1 strain on mucin expression Mucins play an important role in the protective function of the intestinal barrier. Mucins secreted by intestinal goblet cells constitute the main component of the intestinal mucus layer. Together with water, they form a mucus layer that covers the free epithelial surface, providing lubrication and preventing pathogenic bacteria from adhering to and invading the intestinal tract (Antonissen G, Van Immerseel F, Pasmans F, Ducatelle R, Janssens GP, De Baere S, Mountzouris KC, Su S, Wong EA, De Meulenaer B et al. 2015. Mycotoxins deoxynivalenol and fumonisins alter the extrinsic component of the intestinal barrier in broiler chickens. J Agric Food Chem 63: 10846-10855.). We evaluated the effect of bacteria on the expression of the mucin genes MUC2, MUC4, and MUC5AC. In Caco-2 / HT29-MTX (90:10 ratio), the addition of strain LOC1 and Lactipranchibacillus plantarum ATCC 8014 significantly increased the expression of MUC2 and MUC4 compared to the control (Fig. 5a). In Caco-2 / HT29-MTX (75:25 ratio), the addition of bacteria significantly increased MUC2 expression compared to the control (Fig. 5b). Meanwhile, the addition of strain LOC1 and Lactipranchibacillus plantarum ATCC 8014 significantly increased MUC4 expression compared to the control. No significant change in MUC5AC expression was observed after the addition of bacteria compared to the control.

[0054] Consideration Strain LOC1 isolated from tea leaves was identified as Lactiplantibacillus plantarum by PCR. Lactobacillus plantarum is present in the digestive tract of plants and animals and is known as one of the probiotic species of lactic acid bacteria (Sadeghi-Aliabadi H, Mohammadi F, Fazeli H, Mirlohi M. 2014. Effects of Lactobacillus plantarum A7 with probiotic potential on colon cancer and normal cell proliferation in comparison with a commercial strain. Iran J Basic Med Sci 17: 815-819; Eom JS, Song J, Choi HS. 2015. Protective Effects of a Novel Probiotic Strain of Lactobacillus plantarum JSA22 from Traditional Fermented Soybean Food Against Infection by Salmonella enterica Serovar Typhimurium. J Microbiol Biotechnol 25: 479-491; Ren D, Li C, Qin Y, Yin R, Du S, Liu H, Zhang Y, Wang C, Rong F, Jin N. 2015. Evaluation of immunomodulatory activity of two potential probiotic Lactobacillus strains by in vivo tests. Anaerobe 35: 22-27.).For example, several strains of Lactobacillus plantarum have been reported to alleviate irritable bowel syndrome (Molin G. 2001. Probiotics in foods not containing milk or milk constituents, with special reference to Lactobacillus plantarum 299v. Am J Clin Nutr 73: 380S-385S; Niedzielin K, Kordecki H, Birkenfeld B. 2001. A controlled, double-blind, randomized study on the efficacy of Lactobacillus plantarum 299V in patients with irritable bowel syndrome. Eur J Gastroenterol Hepatol 13: 1143-1147). Lactibranchibacillus plantarum JSA22 strain inhibits infection of intestinal epithelial cells by Salmonella Typhimurium (Ren D et al., supra). Lactipranchibacillus plantarum CICC 23174 has immunostimulatory effects, such as enhancing the phagocytic activity of macrophages (Ren D et al. (supra)). Lactipranchibacillus plantarum can alleviate soybean allergy (Frias J, Song YS, Martinez-Villaluenga C, Gonzalez de Mejia E, Vidal-Valverde C. 2008. Immunoreactivity and amino acid content of fermented soybean products. J Agric Food Chem 56: 99-105.).Furthermore, some strains of Lactobacillus plantarum produce bacteriocins (Song DF, Zhu MY, Gu Q. 2014. Purification and characterization of Plantaricin ZJ5, a new bacteriocin produced by Lactobacillus plantarum ZJ5. PLoS One 9: e105549; Zhu X, Zhao Y, Sun Y, Gu Q. 2014. Purification and characterization of plantaricin ZJ008, a novel bacteriocin against Staphylococcus spp. from Lactobacillus plantarum ZJ008. Food Chem 165: 216-223.). For example, plantaricin KL-1Y from Lactobacillus plantarum KL-1 exhibits bactericidal activity against Bacillus cereus and growth inhibitory activity against Listeria innocua, Staphylococcus aureus, and Escherichia coli (Rumjuankiat K, Perez RH, Pilasombut K, Keawsompong S, Zendo T, Sonomoto K, Nitisinprasert S. 2015. Purification and characterization of a novel plantaricin, KL-1Y, from Lactobacillus plantarum KL-1. World J Microbiol Biotechnol 31: 983-994.).Extracellular proteins from Lactobacillus plantarum BMCM12 inhibit the adhesion of Escherichia coli and Salmonella (S. enterica subsp. enterica) to mucin (Sanchez B, Urdaci MC. 2012. Extracellular proteins from Lactobacillus plantarum BMCM12 prevent adhesion of enteropathogens to mucin. Curr Microbiol 64: 592-596.). Thus, Lactobacillus plantarum is one of the beneficial lactic acid bacteria species that can be used as a probiotic. In this example, the LOC1 strain was more effective at enhancing intestinal barrier function than the reference strain of Lactobacillus plantarum, suggesting that tea leaves can be used as a source of probiotics.

[0055] After heat treatment, industrially grown probiotic bacteria, including bacterial extracts and supernatants, often retain important probiotic properties, allowing for the development of safe formulations with more optimal pharmaceutical properties (e.g., extended shelf life). (Taverniti V, Guglielmetti S. 2011. The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: Proposal of paraprobiotic concept). Genes Nutr 6: 261-274.; Canducci F, Armuzzi A, Cremonini F, Cammarota G, Bartolozzi F, Pola P, Gasbarrini G, Gasbarrini A. 2000. A lyophilized and inactivated culture of Lactobacillus acidophilus increases Helicobacter pylori eradication rates. Aliment Pharmacol Ther 14: 1625-1629.; Lee SH, Yoon JM, Kim YH, Jeong DG, Park S, Kang DJ. 2016. Therapeutic effect of tyndallized Lactobacillus rhamnosus IDCC 3201 on atopic dermatitis mediated by down-regulation of immunoglobulin E in NC / Nga mice. Microbiol Immunol 60: 468-476).Furthermore, various strains of bacteria, including Lactobacillus and Bifidobacterium, can exert beneficial effects in heat-inactivated forms (Zaki MH, Boyd KL, Vogel P, Kastan MB, Lamkanfi M, Kanneganti TD. 2010. The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity 32: 379-391.), including a barrier-maintaining effect that is exerted by heat killing. For example, heat-killed Lactobacillus rhamnosus OLL2838 strain prevents mucosal barrier permeability dysfunction in mice with induced colitis (Miyauchi E, Morita H, Tanabe S. 2009. Lactobacillus rhamnosus alleviates intestinal barrier dysfunction in part by increasing expression of zonula occludens-1 and myosin light-chain kinase in vivo. J. Dairy Sci 92: 2400-2408).In Caco-2 / TC7 cell monolayers infected with diarrheagenic, diffusively adherent Afa / Dr Escherichia coli (E. coli) C1845, heat-killed Lactobacillus acidophilus LB and its culture medium inhibited the E. coli-induced increase in paracellular permeability (Lievin-Le Moal V, Sarrazin-Davila LE, Servin AL. 2007. An experimental study and a randomized, double-blind, placebo-controlled clinical trial to evaluate the antisecretory activity of Lactobacillus acidophilus strain LB against nonrotavirus diarrhea. Pediatrics 120: e795-e803.). This example demonstrates the beneficial effect of autoclaved LOC1 strain on the intestinal barrier, suggesting its potential use as a safe probiotic.

[0056] The intestinal barrier function was evaluated using TEER (metabolic enzyme response) values. LOC1 inhibited the DSS-induced decrease in TEER in Caco-2 / HT29-MTX (100:0 and 75:25 ratios), suggesting that LOC1 plays an important role in altering intestinal cell permeability. Alterations in intestinal permeability are widely known to be a major factor in predisposing to intestinal inflammatory diseases and diarrhea (Groschwitz KR, Hogan SP. 2009. Intestinal barrier function: Molecular regulation and disease pathogenesis. J Allergy Clin Immunol 124: 3-20).

[0057] Furthermore, in Caco-2 / HT29-MTX (100:0 and 75:25 ratios), occludin mRNA expression was significantly increased after the addition of LOC1 strain compared to control. Increased occludin expression is associated with epithelial barrier protection, whereas decreased occludin expression is associated with epithelial barrier dysfunction and increased epithelial permeability (Karczewski J, Troost FJ, Konings I, Dekker J, Kleerebezem M, Brummer RJ, Wells JM. 2010. Regulation of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective effects on the epithelial barrier. Am J Physiol Gastrointest Liver Physiol 298: G851-G859.). Meanwhile, TJP1 and TJP2 mRNA levels were unchanged by any bacterial treatment. Therefore, it is possible that the suppression of the decrease in TEER value caused by bacteria is due to changes in other TJ components that were not measured in this example.For example, it has been shown that the increase in TEER of Caco-2 monolayers after treatment with Lactobacillus plantarum MB452 was due to TJP1 and the intracellular plaque protein cingulin, which directly binds to cytoskeletal actin filaments (Robinson K, Deng Z, Hou Y, Zhang G. 2015. Regulation of the Intestinal Barrier Function by Host Defense Peptides. Front Vet Sci 2: 57.) (Anderson RC, Cookson AL, McNabb WC, Park Z, McCann MJ, Kelly WJ, Roy NC. 2010. Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation. BMC Microbiol 10: 316.). Claudin-1, a transmembrane TJ protein that directly interacts with TJP1, was increased in the jejunal epithelium of young piglets treated with Lactobacillus reuteri I5007 (Yang F, Wang A, Zeng X, Hou C, Liu H, Qiao S. 2015. Lactobacillus reuteri I5007 modulates tight junction protein expression in IPEC-J2 cells with LPS stimulation and in newborn piglets under normal conditions. BMC Microbiol 15: 32.).

[0058] However, no significant changes in MUC5AC expression were observed after bacterial treatment, likely because Caco-2 monolayers do not possess the full functionality of intestinal goblet cells, unlike HT29-MTX cells, which are capable of secreting mucins (Greenbaum D, Colangelo C, Williams K, Gerstein M. 2003. Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol 4: 117.; Vincent A, Perrais M, Desseyn JL, Aubert JP, Pigny P, Van Seuningen I. 2007. Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (muc2, muc5ac, muc5b, muc6) in epithelial cancer cells. Oncogene 26: 6566-6576.). Therefore, in Caco-2 / HT29-MTX cocultures, MUC5AC expression was low (90 / 10 and 75 / 25), with no significant difference between bacterial treatments and controls.

[0059] From the results obtained in this example, it can be concluded that Lactipranchibacillus plantarum LOC1 isolated from tea leaves can be used as a safer probiotic that beneficially contributes to the intestinal barrier.

[0060] Enhancement of IL-12 expression by probiotics in macrophage-like cells J774.1 cells, a mouse macrophage model, were used as macrophage-like cells. Lactic acid bacteria were suspended in PBS and heat-killed at 121°C for 15 minutes. The heat-treated lactic acid bacteria were added to J774.1 cells and incubated, after which the levels of IL-12 mRNA expression and protein secretion in the J774.1 cells were quantified by real-time PCR and ELISA, respectively. Furthermore, the heat-killed lactic acid bacteria were treated with DNase and RNase, and the effect on IL-12 expression was analyzed. In addition to the LOC1 strain, the lactic acid bacteria strains shown in Table 2 below were used. [Table 2]

[0061] We analyzed the effects of lactic acid bacteria on the expression and secretion of IL-12 mRNA in J774.1 cells and found that Lactiplantibacillus plantarum LOC1 significantly increased IL-12 mRNA expression and also promoted IL-12 secretion (Fig. 6).

[0062] Furthermore, we treated LOC1 with different enzymes to investigate the active components of LAB that enhance IL-12 expression. IL-12 induction was not affected by DNase treatment, but was significantly suppressed by RNase treatment. These results suggest that LOC1 RNA is partly involved in IL-12 induction.

Claims

1. A Lactiplantibacillus plantarum LOC1 strain derived from tea leaves, the Lactiplantibacillus plantarum LOC1 strain being a strain identified by accession number NITE AP-03527, and having higher immunostimulatory activity than other strains of Lactiplantibacillus plantarum.

2. An immunostimulant for enhancing expression of interleukin-12 (IL-12) in macrophages, comprising the RNA of the Lactiplantibacillus plantarum LOC1 strain according to claim 1 as an active ingredient.

3. A composition for enhancing expression of interleukin-12 (IL-12) in macrophages, comprising the RNA of the Lactiplantibacillus plantarum LOC1 strain according to claim 1 as an active ingredient.

4. The composition described in claim 3 in the form of a supplement or a pharmaceutical.

5. 5. The composition of claim 3 or 4, in the form of a liquid, powder, tablet or capsule.

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