JP7723917B2 - Method for producing toll-like receptor 2 binding substance - Google Patents

Description

The present invention relates to a method for producing a Toll-like receptor 2 binding substance.

The epidermal barrier function is important for maintaining healthy skin. Tight junctions between keratinocytes play a part in this function. Non-Patent Document 1 discloses that TLR2 stimulation strengthens tight junctions between keratinocytes (Non-Patent Document 1).
Similar reports have also been published in the intestinal tract, suggesting that stimulation of TLR2 in primary immune cells in the intestinal lamina propria promotes the production of IL-10, an anti-inflammatory cytokine, and suppresses the onset of enteritis.Furthermore, it has been reported that stimulation of TLR2 on intestinal epithelial cells, rather than on immunocompetent cells, strengthens tight junctions (Non-Patent Document 2).

Journal of Immunology, 2011 (USA), Vol. 187, pp. 3230-3237 Mucosal Immunology, 2008 (UK), Vol. 1, Suppl 1, S62-6

The inventors hypothesized that strengthening tight junctions leads to strengthening of cell sheets (strengthening of the barrier), which in the skin prevents moisture loss and foreign pathogens from entering the body. Furthermore, in the intestinal tract, it is believed that strengthening of tight junctions prevents lipopolysaccharides (LPS) derived from gram-negative bacteria present in large numbers in the intestine from entering the blood vessels, thereby preventing microinflammation in the body.
Therefore, it was thought that a binding substance of Toll-like receptor 2 could be used in cosmetics, health foods, and pharmaceuticals. However, a method for easily producing a binding substance of Toll-like receptor 2 was not known.
Therefore, an object of the present invention is to provide a method for conveniently producing a binding substance for Toll-like receptor 2.

The inventors have conducted extensive research into a method for easily producing a binding substance for Toll-like receptor 2, and have surprisingly discovered that a substance that binds to Toll-like receptor 2 can be easily produced by culturing lactic acid bacteria with a plant extract and a milk component.
The present invention is based on this finding.
Therefore, the present invention provides
[1] A method for producing a Toll-like receptor 2 binding substance, comprising a step of culturing lactic acid bacteria in a medium containing a plant extract and a milk component;
[2] The method for producing the Toll-like receptor 2 binding substance according to [1], further comprising a step of heat-treating the obtained culture solution.
[3] The method for producing the Toll-like receptor 2 binding substance according to [1] or [2], wherein the milk component is whole milk, skim milk, or whey.
[4] The method for producing a Toll-like receptor 2 binding substance according to any one of [1] to [3], wherein the milk component is whole milk powder, skim milk powder, or whey powder.
[5] A Toll-like receptor 2 binding substance obtained by the production method according to any one of [1] to [4].
[6] A cosmetic composition comprising the toll-like receptor 2 binding substance according to [5].
[7] A pharmaceutical composition comprising the Toll-like receptor 2 binding substance according to [5], and [8] a food composition comprising the Toll-like receptor 2 binding substance according to [5].
Regarding.

The method for producing a Toll-like receptor 2 binding substance of the present invention allows for the easy production of a Toll-like receptor 2 binding substance.

10 is a graph showing the TLR2 stimulating activity in a medium containing lactic acid bacteria cultured in a medium containing asparagus extract and whey powder. (TLR2 stimulating activity of asparagus extract fermentation liquid with 8.3% whey added. ●: sterilized at 110°C for 20 minutes before culture, no sterilization after culture; 〇: sterilized at 110°C for 20 minutes before culture, and sterilized at 110°C for 20 minutes after culture; ▲: sterilized at 75°C for 30 minutes before culture, no sterilization after culture; △: sterilized at 75°C for 30 minutes before culture, and sterilized at 110°C for 20 minutes after culture.) 10 is a graph showing the TLR2 stimulating activity in culture media when lactic acid bacteria were cultured in a medium containing asparagus extract and whey powder, a medium containing asparagus extract only, or a medium containing whey powder only (TLR2 stimulating activity of culture media under various conditions. ●: asparagus extract only, 〇: 8.3% whey only, ▲: asparagus extract + 8.3% whey, △: asparagus extract + whey, cultured, then sterilized at 110°C for 20 minutes). 10 is a graph showing the TLR2 stimulating activity in a medium containing asparagus extract and skim milk, when lactic acid bacteria were cultured in the medium. (TLR2 stimulating activity of asparagus extract fermentation liquid to which 3% skim milk was added. ●: 1% glucose added, sterilized at 110°C for 20 minutes before culture, and no sterilization after culture. 〇: 1% glucose added, sterilized at 110°C for 20 minutes before culture, and sterilized at 110°C for 20 minutes after culture. ▲: no glucose added, sterilized at 110°C for 20 minutes before culture, and no sterilization after culture. △: no glucose added, sterilized at 110°C for 20 minutes before culture, and sterilized at 110°C for 20 minutes after culture.) This is a graph showing the TLR2 stimulating activity in a medium containing lactic acid bacteria cultured in asparagus extract and soybean peptone. (TLR2 stimulating activity of asparagus extract fermentation liquid with 10% soybean peptone added. ●: 6% glucose added, sterilized at 110°C for 20 minutes before culture, no sterilization after culture. 〇: 6% glucose added, sterilized at 110°C for 20 minutes before culture, and also sterilized at 110°C for 20 minutes after culture.) This is a graph showing the TLR2 stimulating activity in a medium containing lactic acid bacteria cultured in a medium containing asparagus extract and yeast extract. (TLR2 stimulating activity of asparagus extract fermentation liquid with 10% yeast extract added. ●: 6% glucose added, sterilized at 110°C for 20 minutes before culture, no sterilization after culture. 〇: 6% glucose added, sterilized at 110°C for 20 minutes before culture, and also sterilized at 110°C for 20 minutes after culture.) This graph shows the TLR2 stimulating activity in the culture medium for lactic acid bacteria cultured in a medium containing asparagus extract, broccoli extract, cabbage extract, carrot extract, radish extract, Chinese cabbage extract, and skim milk, and then sterilized at 110°C for 20 minutes and then filtered, and for samples that were sterilized by filtration without heat treatment. (TLR2 stimulating activity of fermented liquids obtained by fermenting various plant extracts and skim milk. (A) Asparagus cuttings, (B) Broccoli (stalk), (C) Cabbage, (D) Carrot (root), (E) Radish (root), (F) Chinese cabbage. (◯) No heat treatment after culture, (●) Heat treatment at 110°C x 20°C after culture.) This graph shows the TLR2 stimulating activity in the culture medium for lactic acid bacteria cultured in a medium containing apple extract, eggplant extract, cucumber extract, lemon balm extract, mandarin orange extract, tomato extract, or grape extract and skim milk, and then sterilized at 110°C for 20 minutes and then filter-sterilized, as well as for cultures that were filter-sterilized without heat treatment. (TLR2 stimulating activity of fermented solutions of various plant extracts and skim milk. (G) Apple (fruit), (H) Eggplant (fruit), (I) Cucumber (fruit), (J) Lemon balm (leaf), (K) Mandarin orange (fruit), (L) Tomato (fruit), (M) Grape (fruit). (◯) No heat treatment after culture, (●) Heat treatment at 110°C x 20°C after culture.) This is a graph showing the TLR2 stimulating activity of lactic acid bacteria cultured in a medium containing cow skim milk powder, cow whole milk powder, sheep whole milk powder, goat whole milk powder, camel whole milk powder, cheese whey, or yogurt whey. (TLR2 stimulating activity of fermented liquids obtained by fermenting asparagus extract and various milk components. (●) cow skim milk, (〇) cow cheese whey, (▲) cow yogurt whey, (△) cow whole milk powder, (■) sheep whole milk powder, (□) goat whole milk powder, (◆) camel whole milk powder. All were heat-treated at 110°C x 20°C after culture.) This is a graph showing the TLR2 stimulating activity in the culture medium after culturing lactic acid bacteria using asparagus extract at 80°C for 3 hours. (Asparagus extract was extracted under three different conditions: 80°C for 3 hours (without compression) (●), 80°C for 3 hours (with compression) (〇), and 121°C for 15 minutes (with compression) (▲), and the TLR2 stimulating activity of the fermented liquid was then fermented under static conditions with the addition of 3% skim milk. All samples were heat-treated at 110°C x 20°C after cultivation.) 1 is a graph showing the change in intercellular adhesion determined by fluorescein permeability after the Toll-like receptor 2 binding substance of the present invention was allowed to act on a cell sheet of normal neonatal epidermal keratinocytes NHEK (NB).

[1] Method for producing a Toll-like receptor 2 binding substance The method for producing a Toll-like receptor 2 binding substance of the present invention comprises a step of culturing lactic acid bacteria in a medium containing a plant extract and a milk component. Preferably, the method for producing a Toll-like receptor 2 binding substance of the present invention further comprises a step of heat-treating the obtained culture solution. The Toll-like receptor 2 binding substance is thought to be secreted into the medium by the lactic acid bacteria. Therefore, it is thought to be a soluble Toll-like receptor 2 binding substance.

《Culture step (1)》
In the culturing step, the lactic acid bacteria are cultured in a medium containing a plant extract and a milk component, whereby a Toll-like receptor 2 binding substance can be produced.

《Lactic acid bacteria》
The lactic acid bacteria used for culturing are not particularly limited, and include the genus Lactobacillus, Enterococcus, Lactococcus, Pediococcus, and Leuconostoc. In this specification, the genus Lactobacillus includes the genus Acetilactobacillus, the genus Agrilactobacillus, the genus Amylolactobacillus, the genus Apilactobacillus, the genus Bombilactobacillus, the genus Companilactobacillus, the genus Dellaglioa, the genus Fructilactobacillus, the genus Furfurilactobacillus, the genus Holzapfelia, the genus Lacticaseibacillus, the genus Lactiplantibacillus, the genus Lactic Lactic acid bacteria of the genera O. bacillus, Lapidilactobacillus, Latilactobacillus, Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolactobacillus, Paralactobacillus, Paucilactobacillus, Schleiferilactobacillus, and Secundilactobacillus can be cultured in a medium containing a plant extract and a milk component to produce a Toll-like receptor 2 binding substance.

《Milk ingredients》
The milk components used for the culture are not particularly limited, and either liquid or dried milk components can be used. Examples of liquid milk components include raw milk or processed milk. Examples of dried milk components include whole milk powder, skim milk powder, or whey powder. The milk components are not particularly limited as long as they are derived from mammals, and examples of mammals include cows, goats, sheep, deer, bison, buffalo, reindeer, donkeys, horses, camels, yaks, and water buffalo.

The amount of milk components added is not particularly limited as long as the effects of the present invention are achieved, but the lower limit is 0.1% by weight or more, preferably 0.2% by weight, more preferably 0.5% by weight, and even more preferably 0.7% by weight or more, in terms of protein content. The upper limit is 10% by weight or less, preferably 5% by weight or less, more preferably 3% by weight or less, and even more preferably 2% by weight or less, in terms of protein content. The above lower and upper limits can be combined as appropriate.

Plant extracts
The plant extract used for the culture is not particularly limited as long as it can obtain the effects of the present invention, and any plant extract containing no toxic components can be used, for example, an extract derived from an edible plant. If the extract is an edible plant, the obtained Toll-like receptor 2 binding substance can be safely used as an active ingredient in cosmetics, foods, or pharmaceuticals.
Examples of the edible plants include plants of the Asparagaceae family (e.g., asparagus), plants of the Brassicaceae family (e.g., radish, cabbage, Chinese cabbage, broccoli, kohlrabi, kale, cauliflower, Chinese broccoli, Chinese radish, turnip, rutabaga, bok choy, nozawana, komatsuna, rape, mizuna, and tatsai), plants of the Umbelliferae family (e.g., carrot, mitsuba, Japanese parsley, coriander, cumin, angelica tree, and celery), plants of the Asteraceae family (e.g., burdock, garland chrysanthemum, lettuce, stem lettuce, butterbur, and mugwort), plants of the Solanaceae family (e.g., eggplant, tomato, potato, bell pepper, chili pepper, paprika, and shishito pepper), plants of the Cucurbitaceae family (e.g., cucumber, watermelon, wax gourd, and turnip), Examples of suitable plants include: Amaranthaceae (spinach, beet), Legumes (for example, kidney bean, lima bean, scarlet bean, broad bean, soybean, pea, peanut, adzuki bean), Convolvulaceae (for example, sweet potato), Euphorbiaceae (for example, cassava), Araceae (for example, taro, taro root), Rosaceae (for example, apple, peach, strawberry, plum, pear, loquat, quince), Rutaceae (for example, mandarin orange, lemon, kumquat, Japanese pepper), Araliaceae (for example, ginseng, Panax notoginseng, American ginseng), Polygonaceae (for example, rhubarb), and Dioscoreaceae (for example, yam, Chinese yam).
The parts from which the plant extract is extracted include, but are not limited to, leaves, stems, roots, flowers, or fruits.

The extraction solvent for the plant extract is not particularly limited, but examples include aqueous solvents and organic solvents. However, aqueous solvents are preferred because they are used to cultivate lactic acid bacteria. Examples of aqueous solvents include water or aqueous buffer solutions (e.g., phosphate buffer). Alcohols (e.g., ethanol, butanol, 1,3-butylene glycol, glycerin), or a mixture of water and alcohol, can also be used. The extraction temperature is also not particularly limited, and extraction is possible at, for example, 4°C to 130°C. Although extraction can also be performed at low temperatures, extraction at relatively high temperatures is preferred due to the improved extraction efficiency and sterilization (disinfection). For example, extraction temperatures of 50°C to 130°C, preferably 60°C to 130°C, more preferably 70°C to 130°C, and even more preferably 80°C to 130°C. The extraction time is also not particularly limited, but for example, a longer extraction time is preferable at low temperatures, while a shorter extraction time may be acceptable at high temperatures. For example, extraction at 50°C to 130°C can be performed for 10 to 10 hours.
The ratio of the solvent to the plant during extraction is not particularly limited, but is generally 0.1 to 2 parts by weight, preferably 0.2 to 1 part by weight, and more preferably 0.3 to 0.8 parts by weight, of the plant relative to 1 part by weight of the solvent.

Other ingredients
The medium may consist solely of milk components and food extracts, but may also contain other ingredients such as water, sugars (e.g., glucose), vegetable proteins (e.g., soybean peptone), salts (e.g., sodium chloride, sodium phosphate, potassium phosphate, manganese chloride, etc.), and vitamins.

The medium containing the plant extract and milk component is preferably pasteurized or sterilized, although it is not particularly limited. The heating temperature is not particularly limited, but is, for example, 60°C to 130°C, preferably 80°C to 130°C, and more preferably 100°C to 130°C. The heating time is also not particularly limited, but is 5 to 60 minutes, preferably 10 to 30 minutes. If the temperature is low, a longer heating time is sufficient, and if the temperature is high, the heating time can be shortened.
The culture temperature in the culture can be appropriately determined depending on the optimal growth temperature of the lactic acid bacteria, and is, for example, 25° C. to 40° C., and preferably 30° C. to 38° C. The culture time can also be appropriately determined depending on the lactic acid bacteria, and is, for example, 2 to 72 hours, preferably 6 to 48 hours, and more preferably 12 to 36 hours.
The pH of the culture medium during the culture does not need to be particularly controlled, but depending on the properties of the lactic acid bacteria used, it may be set to an optimum condition by adding, for example, a sodium hydroxide solution or a hydrochloric acid solution, and/or cultured while maintaining the optimum condition.

Toll-like receptor 2 binding substance
Toll-like receptor 2 (hereinafter sometimes referred to as TLR2) is one of the human homologs of Toll, which was discovered as a gene necessary for normal development in Drosophila. Ten Toll-like receptor homologs have been discovered in humans. TLR2 forms heterodimers with TLR1 or TLR6, and uses peptideglycans, lipoproteins, etc., which are characteristic of Gram-positive bacteria, as ligands.
It has been reported that stimulation of TLR2 strengthens tight junctions between keratinocytes (Non-Patent Document 1). It has also been reported that stimulation of TLR2 on intestinal epithelial cells strengthens tight junctions (Non-Patent Document 2). Therefore, it is believed that stimulating TLR2 strengthens the barrier function of the skin or intestinal tract against pathogens, etc. The Toll-like receptor 2 binding substance is not limited, but is preferably a Toll-like receptor 2 stimulator.

On the other hand, Toll-like receptor 4 (hereinafter sometimes referred to as TLR4) uses lipopolysaccharide (LPS), which is characteristic of gram-negative bacteria, as its ligand. However, the TLR4 ligand LPS does not strengthen tight junctions. It has also been reported that stimulation of TLR4 loosens the intestinal barrier, inducing so-called leaky gut. Therefore, it is thought that stimulating TLR4 weakens the barrier function of the skin or intestinal tract against pathogens, etc.

Heat Treatment Step (2)
The method for producing a Toll-like receptor 2 binding substance of the present invention preferably further comprises a step (2) of heat-treating the obtained culture medium. The obtained culture medium may contain Toll-like receptor 4 (TLR4)-stimulating activity in some cases. Heat treatment after culture can kill remaining lactic acid bacteria and simultaneously inactivate the TLR4-stimulating activity. Furthermore, surprisingly, heat treatment improves the TLR2-stimulating activity. The heating temperature is not particularly limited, but is, for example, 60°C to 130°C, preferably 80°C to 130°C, and more preferably 100°C to 130°C. The heating time is also not particularly limited, but is 5 to 60 minutes, preferably 10 to 30 minutes. A longer heating time is sufficient when the temperature is low, and a shorter heating time is possible when the temperature is high.

[2] Toll-like receptor 2 binding substance The Toll-like receptor 2 binding substance of the present invention can be obtained by the above-mentioned production method. Although the structure of the Toll-like receptor 2 binding substance has not yet been identified, it is thought to be a soluble Toll-like receptor 2 binding substance produced by lactic acid bacteria using the production method of the present invention and secreted into the culture medium.

[3] Cosmetic Composition The cosmetic composition of the present invention contains the Toll-like receptor 2 binding substance. The inclusion of the Toll-like receptor 2 binding substance strengthens tight junctions between skin keratinocytes. Specific examples of the cosmetic composition include serums, lotions, cleansers, emulsions, creams, lipsticks, foundations, gels, packs, face powders, blushers, hair tonics, shampoos, rinses, sunscreens, facial cleansers, and lip balms.

The content of the Toll-like receptor 2 binding substance in the cosmetic composition of the present invention is not particularly limited as long as the effects of the present invention are obtained, but is, for example, 0.1 to 100% by weight, preferably 1 to 50% by weight, and more preferably 1 to 25% by weight.

The cosmetic composition of the present invention may contain moisturizers (e.g., trimethylglycine, N-[2-hydroxy-3-(trimethylammonio)propyl]chloride hydrolyzed wheat protein solution, hyaluronic acid, sodium pyrrolidonecarboxylate, betaine, jojoba oil, hydrolyzed keratin), colorants (e.g., pigments or dyes), viscosity modifiers (e.g., methylcellulose), emulsifiers (e.g., glyceryl monostearate), pearlescent agents (e.g., glycol distearate or ethylene glycol distearate), salts (e.g., sodium chloride), plant extracts, preservatives (e.g., methylparaben, propylparaben, butylparaben, 1,3-butylene glycol (1,3-butanediol), phenoxyethanol, or pentylene glycol (1,2-pentanediol)), vitamins, fragrances, ultraviolet absorbers, antioxidants, humectants, chelating agents, pH adjusters (e.g., citric acid or tartaric acid), and water, as long as the effects of the present invention are not impaired.

[4] Pharmaceutical Composition The pharmaceutical composition of the present invention contains the Toll-like receptor 2 binding substance. The content of the Toll-like receptor 2 binding substance in the pharmaceutical composition of the present invention is not particularly limited as long as the effects of the present invention are obtained, but is, for example, 0.1 to 100% by weight, preferably 1 to 50% by weight, and more preferably 1 to 25% by weight. It is desirable to determine the effective amount through animal experiments and/or clinical trials, etc.

The dosage form of the pharmaceutical composition is not particularly limited, and examples include oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, or parenteral preparations such as injections, topical liquids, ointments, suppositories, topical creams, or eye drops.

Oral preparations can be prepared according to conventional methods using excipients such as gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, silicic anhydride, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, flow enhancers, diluents, preservatives, colorants, flavorings, flavoring agents, stabilizers, moisturizers, antiseptics, or antioxidants. For parenteral administration, topical solutions, ointments, suppositories, and topical creams are suitable.

[5] Food Composition The food composition of the present invention contains the Toll-like receptor 2 binding substance. The food composition can be administered in various forms, such as a health food (e.g., a functional food or a supplement) or as feed. The food composition also includes a beverage composition.

For example, "health food" refers to food that has or is expected to have some effect on health, and "functional food" refers to the above-mentioned "health food" that has been designed and processed to fully exhibit various biological regulatory functions (for example, regulatory functions for physiological systems such as the digestive system, circulatory system, endocrine system, immune system, or nervous system). Because the food composition of the present invention contains a Toll-like receptor 2 binding substance, it strengthens the tight junctions of intestinal epithelial cells in the intestinal tract, preventing the invasion of pathogens, etc.

The food composition of the present invention can be prepared in the same manner as a general food composition, except that it contains a Toll-like receptor 2 binding substance as an active ingredient. For example, the food element of the present invention can be prepared by adding an effective amount of the active ingredient to a substance that can be ingested as food. The effective amount of the Toll-like receptor 2 binding substance can be determined appropriately depending on various factors, such as the shape or type of food, or the individual, age, and weight of the person consuming it.

The present invention will be explained in detail below using examples, but these are not intended to limit the scope of the present invention.

Example 1
In this example, 27 types of lactic acid bacteria were used and cultured in a medium containing asparagus extract and skim milk, and the TLR2 stimulating activity in the medium was measured.
Asparagus extract was prepared as follows. 1 kg of asparagus cut pieces was placed in a polypropylene dashi bag (Dashitori San-Ace, Asahi Kasei Spunbond) and placed in a 5 L beaker. 2 kg of purified water was added, and the beaker was covered with aluminum foil. High-pressure hot water extraction was performed at 121°C for 15 minutes using an autoclave. After cooling, the contents of the dashi bag were squeezed in a safety cabinet to obtain the asparagus extract.
0.9 g of skim milk was added to the asparagus extract (3 mL) and the mixture was sterilized by high-pressure steam (110°C x 20 minutes). Approximately 2% of a preculture solution (MRS Broth) of each lactic acid bacterium was added, and the mixture was statically cultured at 37°C for 24 hours. After sampling, the mixture was sterilized by high-pressure steam (110°C x 20 minutes) and sterilized using a 0.2 μm filter.

(Measurement of TLR2 stimulating activity)
TLR2 stimulatory activity was measured using InvivoGen's HEK Blue ^™ hTLR2 cells. These cells were human embryonic kidney cells 293 (HEK293) cotransfected with the human TLR2 gene and the SEAP (secreted embryonic alkaline phosphatase) gene. The SEAP reporter gene was placed under the control of the IFN-β minimal promoter fused with five NF-κB and AP-1 binding sites, and the CD14 co-receptor gene was also transfected to enhance TLR2 responses. When TLR2 is stimulated, NF-κB and AP-1 are activated in cells, promoting the production of SEAP, the amount of which can be measured using the HEKBlue ^™ Detection Kit, a substrate for alkaline phosphatase.
The medium used was DMEM high glucose medium (Sigma-Aldrich) containing 10% fetal bovine serum, supplemented with the indicated amounts of penicillin, streptomycin, normocin, and HEK-Blue ^™ Selection. 100 μL of HEK Blue ^™ hTLR2 was seeded at a concentration of 2 × 10 ⁵ cells/mL into each well of a 96-well microplate for cell culture (Iwaki tissue culture treated 3860-096). After approximately 48 hours of incubation at 37°C under 5% _CO2 and saturated water vapor conditions, 100 μL/well of test samples diluted with medium to various concentrations (sterile filtered through a 0.2 μm filter) was added, and incubation was continued for another 24 hours. As a positive control, 100 μL of 200 ng/mL Pam3CSK4 was added (final concentration: 100 ng/mL).
Binding activity was measured using the following procedure. After incubation, 20 μL of the culture medium was transferred to a 96-well SND2020-004-7 microplate, and 180 μL of Quanti Blue Solution (InvivoGen) was added to each well. The mixture was then incubated at 37°C for 30 minutes. Finally, the absorbance of each well at 655 nm was measured using a microplate reader. The results were expressed as a relative value to the positive control value after subtracting the negative control (medium) value from the measured value of each well.

As shown in Table 1, TLR2 stimulating activity was detected in the culture supernatants of all lactic acid bacteria.

Example 2
In this example, the lactic acid bacteria No. 1 of Example 1 was cultured in a medium containing asparagus extract and whey powder, and the TLR2 stimulating activity in the medium was measured.
Asparagus extract was prepared according to Example 1. 8.3 g of whey powder was added to the asparagus extract (100 mL), followed by autoclaving (110°C x 20 minutes) or heat sterilization (75°C x 30 minutes). The amount of whey powder added was such that the protein content was 1 wt%. Approximately 2% of a lactic acid bacteria preculture solution (MRS Broth) was added to the resulting medium, followed by static culture at 37°C for 24 hours. After sampling, either autoclaving (110°C x 20 minutes) or not was performed. Each culture solution was then sterilized using a 0.2 μm filter.
The TLR2 stimulating activity of the obtained samples was measured according to "Measurement of TLR2 stimulating activity" in Example 1. The results are shown in Figure 1.
Under conditions without sterilization after culture, the TLR2 stimulating activity was higher when sterilization before culture was performed at 75°C for 30 minutes than when sterilization before culture was performed at 110°C for 20 minutes, but sterilization after culture significantly increased the activity in both cases.

Example 3 and Comparative Examples 1 and 2
In this example and comparative example, the lactic acid bacteria No. 1 was cultured in a medium containing asparagus extract and whey powder, a medium containing only asparagus extract, or a medium containing only whey powder, and the TLR2 stimulating activity in the medium was measured.
The procedure of Example 2 was repeated, except that a medium containing asparagus extract and whey powder, a medium containing only asparagus extract, or a medium containing only whey powder was used. The medium before culturing was autoclaved (110°C x 20 minutes), and after sampling, either one was autoclaved (110°C x 20 minutes) or one was not autoclaved. The TLR2 stimulating activity of the obtained samples was measured according to the "Measurement of TLR2 stimulating activity" section of Example 1. The results are shown in Figure 2. Compared to the medium containing only asparagus extract (Comparative Example 1) or the medium containing only whey powder (Comparative Example 2), the TLR2 stimulating activity of the medium containing asparagus extract and whey powder (Example 3) was higher.

Example 4
In this example, the lactic acid bacteria No. 1 of Example 1 was cultured in a medium containing asparagus extract and skim milk, and the TLR2 stimulating activity in the medium was measured.
The procedure of Example 2 was repeated, except that 3.0 g of skim milk was used instead of 8.3 g of whey powder. An example was also carried out in which a medium containing 1 g of glucose was added. The amount of skim milk added was such that the protein content was 1% by weight. The results are shown in Figure 3.
The addition of 1% glucose did not significantly affect the activity. As with whey, the TLR2 stimulating activity was significantly increased by sterilization after the end of the culture, and the strength was equivalent to that of whey sterilized at 75°C for 30 minutes before the culture.

Comparative Example 3
In this comparative example, lactic acid bacteria were cultured in a medium containing asparagus extract and soybean peptone, and the TLR2 stimulating activity in the medium was measured.
The procedure of Example 2 was repeated, except that 10.0 g of soybean peptone was used instead of 8.3 g of whey powder, and 6.0 g of glucose was added as the carbon source. The results are shown in Figure 4.
Under these conditions, no sufficient TLR2 stimulating activity was observed.

Comparative Example 4
In this comparative example, lactic acid bacteria were cultured in a medium containing asparagus extract and yeast extract, and the TLR2 stimulating activity in the medium was measured.
The procedure of Example 2 was repeated, except that 10.0 g of yeast extract was used instead of 8.3 g of whey powder, and 6.0 g of glucose was added as the carbon source. The results are shown in Figure 5.
Under these conditions, no sufficient TLR2 stimulating activity was observed.

Example 5
In this example, lactic acid bacteria were cultured in a medium containing asparagus extract (cuttings), radish extract (roots), carrot extract (roots), cabbage extract (leaves), Chinese cabbage extract (leaves), broccoli extract (stalks), tomato extract (fruit), eggplant extract (fruit), apple extract (fruit), lemon balm extract (leaves), cucumber extract (fruit), mandarin orange (fruit), or grape (fruit) and skim milk, and the TLR2 stimulating activity in the medium was measured.
The procedure of Example 4 was repeated, except that radish, carrot, cabbage, Chinese cabbage, broccoli, tomato, eggplant, apple, lemon balm extract, cucumber, mandarin orange, or grape was used instead of asparagus. After incubation, the TLR2 stimulating activity was measured for samples that were sterilized at 110°C for 20 minutes and then filter-sterilized, as well as samples that were filter-sterilized without heat treatment. The results are shown in Figure 6.
All of the plant extracts exhibited high TLR2 stimulating activity, similar to that of the asparagus extract, and the activity was increased by heat treatment.

Example 6
In this example, the cells were cultured in a medium containing skim milk powder from cows, whole milk powder from cows, whole milk powder from sheep, whole milk powder from goats, whole milk powder from camels, cheese whey, or yogurt whey, and the TLR2 stimulating activity in the medium was measured.
The procedure of Example 2 was repeated, except that cow's skim milk powder (3 g), cow's whole milk powder (3 g), sheep's whole milk powder (3 g), goat's whole milk powder (3 g), camel's whole milk powder (3 g), cheese whey (8.3 g), and yogurt whey (8.3 g) were used. The results are shown in Figure 7. Production of TLR2-stimulating components was observed with all milk components.

Example 7
In this example, the TLR2 stimulating activity in the medium was measured using a plant extract extracted at a low temperature.
The procedure of Example 2 was repeated using the extract obtained by high-pressure hot water extraction at 121°C for 15 minutes, as well as the extract obtained by heat treatment at 80°C for 3 hours without squeezing the residue, or the extract obtained by thoroughly squeezing the residue. The results are shown in Figure 8.
Even under mild extraction conditions of 80°C for 3 hours, sufficient or even greater TLR2 stimulating activity was confirmed compared to high-pressure hot water extraction at 121°C for 15 minutes. There was no significant difference, at least in terms of TLR2 stimulating activity, whether the residue was squeezed or not.

Example 8
In this example, it was examined whether the Toll-like receptor 2 binding substance of the present invention actually strengthens tight junctions and affects intercellular adhesion.
Human epidermal keratinocytes used were normal neonatal epidermal keratinocytes (NHEK) (NB) from Kurabo Industries. Cells were cultured in Lifeline's DermaLife Basal Medium supplemented with cell growth factors (DermaLife K LifeFactors). Cells detached with trypsin-EDTA were seeded onto Corning BioCoat ^™ Control Culture Inserts (0.4 μm PET membrane, 24-well format) (100 μL of intra-well fluid, 600 μL of extra-well fluid) and cultured for 4 days until confluent. After 4 days, the medium inside and outside the wells was replaced with medium containing 1.8 mM _CaCl2 to induce differentiation. Simultaneously, asparagus/skim milk fermentation broth (sterilized at 110°C for 20 minutes after culture) obtained as described in Example 2 was added in the following conditions: undiluted, 25-fold diluted, and 50-fold diluted. After medium replacement, culture was continued for an additional 3 days. After 3 days, 1/50 volume of 1% uranine (fluorescein sodium salt) aqueous solution was added to the transwell (final concentration: 0.02%) and incubated for 30 minutes. After 30 minutes, the solution outside the well was collected and the fluorescence intensity was measured at a 10-fold dilution. The fluorescence intensity was measured using the fluorescence measurement function of Takara Bio's Thermal Cycler Dice Real-time System II.
Statistical analysis was performed to evaluate the significance of the difference between each group and the condition without the asparagus/skim milk fermented liquid using Dunnett's multiple comparison test. A value of p<0.05 was considered significant.
The results are shown in Figure 9. The n numbers for each group were 6, 4, and 6 for the no-addition, 25x, and 50x dilution conditions, respectively. The graph shows the average values along with the standard deviation. As shown in the figure, the addition of asparagus/skim milk fermentation broth significantly and dose-dependently reduced fluorescein permeability through the cell sheet. This suggests that TLR2 stimulation strengthened tight junctions.

The Toll-like receptor 2 binding substance obtained by the production method of the present invention can be used in skin cosmetics, foods such as supplements, or pharmaceuticals.

Claims (9)

Cultivating lactic acid bacteria selected from the group consisting of Lactobacillus delbrueckii subsp. Lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Pediococcus pentosaceus, Pediococcus dextrinicus (Lapidilactobacillus dextrinicus), Enterococcus faecium, Enterococcus faecalis, and Lactococcus casei subsp. casei (Lacticaseibacillus casei subsp. casei) in a medium containing a plant extract selected from the group consisting of asparagus extract, radish extract, carrot extract, cabbage extract, Chinese cabbage extract, broccoli extract, tomato extract, eggplant extract, apple extract, lemon balm extract, cucumber extract , mandarin orange extract, and grape extract , and a milk component with a protein content of 0.5% by weight or more ; and recovering the culture medium .
A method for producing a soluble Toll-like receptor 2 stimulator , comprising: The method for producing a soluble Toll-like receptor 2 stimulator according to claim 1 , wherein the lactic acid bacterium is Lactobacillus delbrueckii subsp. Lactis and the plant extract is asparagus extract. The method for producing a soluble Toll-like receptor 2 stimulator according to claim 1 or 2, further comprising a step of heat-treating the obtained culture solution. The method for producing a soluble Toll-like receptor 2 stimulator according to any one of claims 1 to 3 , wherein the milk component is whole milk, skim milk, or whey. The method for producing a soluble Toll-like receptor 2 stimulator according to any one of claims 1 to 4 , wherein the milk component is whole milk powder, skim milk powder, or whey powder. A soluble Toll-like receptor 2 stimulating substance obtained by the method according to any one of claims 1 to 5 . A cosmetic composition comprising the soluble Toll-like receptor 2 stimulator according to claim 6 . A pharmaceutical composition comprising the soluble Toll-like receptor 2 stimulator of claim 6 . A food composition comprising the soluble Toll-like receptor 2 stimulator of claim 6 .