JP7803679B2 - A novel strain of Lactiprunchbacillus plantarum isolated from tea leaves - Google Patents

Description

NPMD NITE P-03527

Article 30, Paragraph 2 of the Patent Act applies. This was unveiled at the 33rd International Conference of the Japanese Society for Animal Cell Engineering, held from November 17th to November 20th, 2020.

Article 30, Paragraph 2 of the Patent Act applies. This was unveiled at the 2020 Joint Conference of the Japanese Society of Food Factors and the Western Japan Branch of the Agricultural Chemical Society of Japan, held from November 28th to 29th, 2020.

Article 30, Paragraph 2 of the Patent Act applies. Disclosed at the 2021 Annual Meeting of the Society of Agricultural Chemical Science of Japan, held from March 18th to March 21st, 2021.

Application of Article 30, Paragraph 2 of the Patent Act On August 6, 2021, Bioscience of Microbiota, Food and Health, Vol. 40(4), 186-195, published in 2021.

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

Lactic acid bacteria are highly safe microorganisms that are widely used in the production of fermented foods. 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 through the production of interferon-γ from CD4 T cells, thereby enhancing the body's defense capabilities.

Lactiplantibacillus plantarum is one type of lactic acid bacterium derived from plant materials. Its type strain, ATCC14917, is known to have high immunostimulatory activity (Japanese Patent Publication No. 2012-213352).

Note that Lactiprantibacillus plantarum is a lactic acid bacterium that was previously known as Lactobacillus plantarum before the reclassification of the Lactobacillus genus.

Japanese Patent Application Laid-Open No. 2012-213352

The present invention aims to provide a novel strain of the genus Lactiplantibacillus plantarum that has high immunostimulatory activity.

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 expression level of IL-12 mRNA.

That is, the present invention includes the following inventions.
[1]
1. A Lactiplantibacillus plantarum LOC1 strain derived from tea leaves, which has higher immunostimulatory activity than other strains of Lactiplantibacillus plantarum.
[2]
The Lactiprantibacillus plantarum LOC1 strain according to [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 according to [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, culture or processed product thereof.
[6]
The composition according to [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.

The LOC1 strain has higher immunostimulatory activity than the ATCC 14917 strain, the reference strain of Lactiprantibacillus plantarum.

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). Electropherogram of PCR products amplified with species-specific primers for L. plantarum. M: 100 bp DNA ladder; 1: strain LOC1; 2: L. plantarum ATCC 14917T; 3: L. plantarum ATCC 8014; 4: L. acidophilus La5; 5: negative control (distilled water). 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 medium supplemented with bacteria. TEER values were measured after incubation, and then 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 = strain LOC1; ATCC14917 = Lactipranchibacillus plantarum ATCC 14917T; ATCC8014 = Lactipranchibacillus plantarum ATCC 8014. 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. 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. 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).

The following describes in detail embodiments or implementations of the present invention, but the present invention is not limited to these and various modifications are possible without departing from the spirit of the invention.

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

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 type depending on the species (homolactic fermentation or heterolactic fermentation), but the fermentation of the LOC1 strain is considered to be homolactic based on the genes it possesses and the odor of the fermentation. While 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, within the range of pH 5.7 to 9.3.

The LOC1 strain can be used in a live state, but it is preferable to use it after heat sterilization in order to obtain the desired effects, such as immunostimulatory activity.

The immunostimulatory activity is not particularly limited as long as it has a beneficial effect of activating the immune system, and examples include the activity of promoting cytokine production. Such cytokines include interleukins such as interleukin 12 (IL-12) and interferons such as interferon gamma (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.

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 viral and bacterial infections and anti-tumor effects in the host, and is also known to have anti-tumor and anti-metastatic effects, effects against infectious diseases such as opportunistic infections, and preventive and therapeutic effects against allergic diseases such as asthma.

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 refers to an activity that 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.

The Lactiplantibacillus plantarum LOC1 strain was deposited at the International 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.

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. Furthermore, the LOC1 strain may also include mutant strains created from the deposited parent strain, as long as they have high immunostimulatory activity.

(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.

The LOC1 strain contained as an active ingredient may include mutant strains created from the deposited parent strain, so long as they have high immunostimulatory activity. Furthermore, the active ingredient may not only be the bacterial cells, but also components derived from the bacterial cells, or cultures or processed products of the bacterial cells. These preferably contain nucleic acid, particularly RNA, of the LOC1 strain.

Immunostimulants can be provided as pharmaceuticals, quasi-drugs, foods and beverages, supplements, etc. The dosage form is not particularly limited, and may be oral powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions, liquids, emulsions, parenteral injections, suppositories, etc. When provided as foods and beverages, immunostimulants can be provided not only in the form of beverages and foods, but also in the form of hard capsules, soft capsules, tablets, granules, etc.

The immunostimulant may contain other ingredients known to have immunostimulating effects, as well as optional ingredients such as excipients, stabilizers, preservatives, binders, disintegrants, pH adjusters, antiseptics, and flavorings used in conventional pharmaceuticals, quasi-drugs, and foods and beverages, as long as they do not impair the effects of the LOC1 strain.

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

The activity of enhancing IL-12 expression can also be used as an antiviral agent. There are no particular limitations on such viruses, as long as they can be prevented or treated by enhancing IL-12 expression, but one example is influenza virus.

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

The LOC1 strain contained in the composition may include mutant strains created from the deposited parent strain, so long as they have high immunostimulatory activity. Furthermore, the active ingredient may not only be the bacterial cells, but also components derived from the bacterial cells, or cultures or processed products of the bacterial cells. These preferably contain nucleic acid, particularly RNA, of the LOC1 strain.

While not intended to be limiting, examples of processed products of the LOC1 strain include those obtained by subjecting live or dead LOC1 strain cells to known physical treatments such as grinding or crushing, or other treatments, such as enzymatic treatment. Specific examples include ground products, crushed 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.

The culture of the LOC1 strain may include a suspension of bacterial cells after culturing the cells, a mixture of bacterial cells (including fermented products), and residues after culturing. The culture may also include a cytoplasmic or cell wall fraction obtained by processing the bacterial cell culture.

The medium for culturing the fungus is not particularly limited, and any medium that is used for culturing Lactiplantibacillus plantarum can be suitably used. For example, the medium may be a natural medium, a synthetic medium, a semi-synthetic medium, etc.

The culture conditions (culture temperature, pH during culture, culture period, etc.) can be the same as those typically used to culture Lactiplantibacillus plantarum. While 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.

The LOC1 strain can be added to food and beverages by sprinkling it on the surface of the food or beverage, or by mixing it into the food or beverage.

As used herein, "food and beverage" 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.

Examples of containers for filling beverages include commonly used containers such as plastic bottles, cans, paper bottles, and glass bottles, but are not limited to these. It is preferable to use containers that can be sealed after filling.

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 buns, and other breads; noodles such as udon, hiyamugi, somen, buckwheat, Chinese noodles, spaghetti, macaroni, rice vermicelli, and harusame; processed meat foods such as hamburger steaks, ham, bacon, and sausages; and instant foods such as curry, ramen, and soup.

The content of Lactiplantibacillus plantarum LOC1 strain in the composition is appropriately determined depending on the dosage form of the composition, the method of use, the age and sex of the patient, the type and severity of the disease, and other conditions, but is generally preferably within the range of 5 × ¹⁰ to 5 × ¹⁰ cfu/ml, and more preferably within the range of 5 × ¹⁰ to 5 × ¹⁰ cfu/ml.

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, and the severity of the patient's symptoms. The timing of administration is also not limited; in either case, it can be administered once a day or in multiple divided doses, or it can be administered once every few days or weeks.

Medicinal uses include the treatment or prevention of diseases that can be treated by increasing IL-12 expression, such as infectious diseases, tumors, and allergies.

The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

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 agar medium was then anaerobically cultured at 37°C for 48 hours using an AnaeroPack-Anaero (Mitsubishi Gas Chemical Company, Inc.).

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.

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 amplifications.

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.

Co-culture conditions: Human colon adenocarcinoma cell lines, Caco-2 and HT29-MTX cells, 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.

Caco-2 and HT29-MTX cells were cultured in high-glucose, L-glutamine DMEM medium 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 at a density of 0.5 × ¹⁰ cells in 75 ^cm flasks at 37°C in a humidified incubator containing 5% _CO . 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.

Bacterial strains and culture conditions: The strains used in this study were strain LOC1, Lactiplantibacillus plantarum ATCC ^14917T , 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 cultured anaerobically overnight at 37°C in MRS broth. Bacterial strains were used in stationary phase for all tests.

Stationary-phase bacterial cells were collected by centrifugation at 8,000 × g and then washed twice with phosphate-buffered saline (PBS). The bacterial count was measured using a hemocytometer (Brightline, Hausser Scientific) to obtain a cell density of 1 × 10 ⁹ cells/mL, and the cells were then autoclaved at 121°C for 15 minutes. Approximately 1 × 10 ⁸ bacterial cells were added to each well in all experiments.

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.

Results: 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 as strain LOC1, and PCR was performed to confirm its genus using genus-specific primers and its species using species-specific primers. 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).

The LOC1 strain inhibits the decrease in transepithelial electrical resistance (TEER) induced by dextran sulfate sodium (DSS). Transepithelial electrical resistance 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 cultures were 470-510 Ω × ^cm2 , 440-480 Ω × ^cm2 , 340-370 Ω × ^cm2 , and 100-140 Ω × ^cm2 , respectively.

Dextran sodium sulfate (DSS), a heparin-like polysaccharide containing up to three sulfates per glucose molecule and approximately 17% sulfur, is used as a model to induce experimental colitis and colon cancer and to decrease the TEER of intestinal epithelial cells. After 6 hours of treatment with DSS, the TEER values of all cocultured cells decreased (data not shown). Meanwhile, the LOC1 strain significantly inhibited the DSS-induced decrease in TEER in Caco-2/HT29-MTX (100:0 and 90:10 ratios) (p<0.05), but not in Lactipranchibacillus plantarum ATCC 14917T or Lactipranchibacillus 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 values (Figures 3c and 3d).

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.

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, which, together with water, forms 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 MUC2 and MUC4 expression compared to the control (Fig. 5a). In Caco-2/HT29-MTX (75:25 ratio), MUC2 expression increased after bacterial addition compared to the control (Fig. 5b). Meanwhile, MUC4 expression increased after bacterial addition compared to the control. MUC5AC expression did not change significantly after bacterial addition compared to the control.

Discussion The 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, some 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.). Lactipranchibacillus plantarum JSA22 strain suppresses 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 derived 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, Lactibranchibacillus 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 Lactibranchibacillus plantarum, suggesting that tea leaves can be used as a source of probiotics.

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 lactic acid bacteria and bifidobacteria, 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.). One such example has been shown to exert a barrier-maintaining effect upon heat killing. For example, heat-killed Lactobacillus rhamnosus OLL2838 strain prevents mucosal barrier permeability impairment 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 that it can be used as a safe probiotic.

TEER values were used to evaluate intestinal barrier function. The LOC1 strain inhibited the DSS-induced decrease in TEER in Caco-2/HT29-MTX (100:0 and 75:25 ratios), suggesting that the LOC1 strain plays an important role in altering the permeability of intestinal cells. 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.).

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, while 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, the suppression of bacterial-induced reduction in TEER may be due to alterations in other TJ components that were not measured in this study. For example, the increase in TEER of Caco-2 monolayers after treatment with Lactobacillus plantarum MB452 was shown to be due to TJP1 and cingulin, an intracellular plaque protein that 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 administered 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.).

However, no significant change in MUC5AC expression was observed after bacterial treatment. This may be because, unlike HT29-MTX cells, which are capable of secreting mucin, Caco-2 monolayers do not possess the full functionality of intestinal goblet cells (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, MUC5AC expression was low in Caco-2/HT29-MTX cocultures (90/10 and 75/25), with no significant difference observed between bacterial treatments and controls.

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

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. IL-12 mRNA expression and protein secretion in 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 listed in Table 2 below were used.

Analysis of the effects of lactic acid bacteria on IL-12 mRNA expression and secretion in J774.1 cells revealed that Lactiprantibacillus plantarum LOC1 significantly increased IL-12 mRNA expression and also promoted IL-12 secretion (Figure 6).

Furthermore, LOC1 was treated with different enzymes to examine the active components of lactic acid bacteria that enhance IL-12 expression. Results showed that IL-12 induction was not affected by DNase, but was significantly suppressed by RNase treatment. These results suggest that LOC1 RNA is partially involved in IL-12 induction.

Claims (5)

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