TW202348241A - Immunostimulating macrophage inducer, cancer microenvironment improving agent and cancer apoptosis inducer clarifying novel properties of the bacterial cells of Apilactobacillus kosoi strain 10H and the processed product of the bacterial cells - Google Patents

Abstract

The subject of the present invention is to clarify novel properties of the bacterial cells of Apilactobacillus kosoi strain 10H and the processed product of the bacterial cells, and to provide new uses based on this. The immunostimulating macrophage inducer, a cancer microenvironment improving agent, and a cancer apoptosis inducer of the present invention contain the bacterial cells or the processed bacterial cell product of the Apilactobacillus kosoi strain 10H as the active ingredients.

Description

Immunostimulating macrophage inducer, cancer microenvironment improving agent and cancer apoptosis inducer

The present invention relates to an immunostimulating macrophage inducer, a cancer microenvironment improving agent, and a cancer apoptosis inducer containing cells or a processed product of the cells of Lactobacillus meli 10H strain, which is an enzyme of lactic acid bacteria.

It has been previously known that lactic acid bacteria or fermentation products thereof have various physiological functions. For example, it is known that the enzyme Apilactobacillus kosoi 10H strain, which is a fructophilic lactic acid bacterium isolated from vegetable brown sugar fermentation broth, has excellent IgA production promoting effect (and immune activating effect) (see Patent Document 1; further, In recent years, the genus Lactobacillus has been reclassified, and the names of many lactic acid bacteria belonging to the old genus Lactobacillus have been changed; "Lactobacillus kosoi" in Patent Document 1 is the same as "Lactobacillus apiensis" in this specification). [Prior technical literature] [Patent Document]

[Patent Document 1] Japanese Patent Application Publication No. 2020-92704

[Problem to be solved by the invention]

The enzyme Lactobacillus apiensis 10H strain described in Patent Document 1 has the following characteristics: it has a relatively small genome compared to lactic acid bacteria, and has a low ability to anabolize glucose, and anabolizes fructose as a good carbon source. Although it was revealed that this strain promotes the production of IgA in the intestinal mucosa and has excellent immune activating effects, other effects are not yet clear.

The present invention was completed in view of the above-mentioned circumstances, and its object is to clarify the novel properties of the enzyme Lactobacillus apiensis 10H strain and provide new uses based on the novel properties. [Technical means to solve problems]

In order to solve the above-mentioned problems, the present inventors conducted intensive research on the new usefulness of the enzyme Lactobacillus apiensis 10H strain. As a result, they found that the cells and bacterial cell treatments of the enzyme Lactobacillus apiensis 10H strain have immune-activating macrophages. The present invention was completed through cell induction effect, cancer microenvironment improvement effect and cancer apoptosis induction effect. That is, the present invention includes the following embodiments.

(1) An immune-stimulating macrophage inducing agent containing cells or a processed product of the enzyme Lactobacillus kosoi 10H strain as an active ingredient. (2) The immunoactive macrophage inducer as described in (1) above, which is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition blended therein. (3) A cancer microenvironment-improving agent containing cells or a processed product of the enzyme Apilactobacillus kosoi 10H strain as an active ingredient. (4) The cancer microenvironment improving agent as described in the above (3), which is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition blended therein. (5) A cancer apoptosis inducing agent containing the enzyme cell or cell processed product of Apilactobacillus kosoi strain 10H as an active ingredient. (6) The cancer apoptosis inducing agent as described in the above (5), which is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition blended therein. (7) The use of enzyme cells or cell processed products of Apilactobacillus kosoi 10H strain, which are used to induce immunoactive macrophages in humans or non-human animals and to improve the microenvironment of cancer. Or pharmaceuticals, foods, and feeds for inducing cancer apoptosis, or active ingredient compositions formulated therein. [Effects of the invention]

According to the present invention, as a novel use of the enzyme Lactobacillus apiensis 10H strain, it is possible to provide an immune-stimulating macrophage inducer, a cancer microenvironment improving agent and a cancer apoptosis inducer containing the bacterial cells or bacterial cell treatments. .

Hereinafter, embodiments of the present invention will be described. Furthermore, the embodiments described below do not limit the invention within the scope of the patent application, and not all of the elements and their combinations described in the embodiments are necessary for the solution of the present invention.

(I) Enzyme Lactobacillus apis strain 10H The active ingredient in one embodiment of the present invention is the bacterial cells of a specific lactic acid strain and its mutant strains isolated from the vegetable brown sugar fermentation broth or the processed bacterial cells. Preferably, it is a bacterial cell or bacterial cell process of a bacterium belonging to the genus Lactobacillus apiensis obtained from a vegetable brown sugar fermentation liquid prepared under the trade name "Georina (registered trademark) enzyme", and more preferably the enzyme Lactobacillus apiensis ( Apilactobacillus kosoi) 10H strain or its mutant strain or bacterial cell processed product. "Mutation strain" is intended to include those that have been mutated by the industry using methods well known to the industry within the scope of not changing its main properties, or those that the industry can confirm are equivalent.

Furthermore, the enzyme Lactobacillus kosoi (Apilactobacillus kosoi) 10H strain (at the time of deposit, it was "the enzyme Lactobacillus kosoi (Lactobacillus kosoi) 10H strain") was deposited at the independent administrative agency Product Evaluation on November 7, 2018 (original date of deposit) Technology Base Organization Patent Microorganism Storage Center (〒292-0818 Room 2-5-8, Kamazu Kamazu, Kisarazu City, Chiba Prefecture, Japan). The registration number is NITE BP-02811 (hereinafter, this strain may also be referred to as "10H strain"). In addition, the taxonomic properties of this 10H strain are described in a paper published by the inventors (Chiou T-Y et al., Antonie van Leeuwenhoek (2018), 111: 1149 - 1156) and Patent Document 1, the entire contents of which are hereby incorporated by reference. incorporated into this manual.

(Characteristics of Lactobacillus apiensis 10H strain) This strain is a recently discovered fructophilic lactic acid bacteria (FLAB) and is believed to have evolved from previous lactic acid bacteria (Filannino et al. "Fructose-rich niches traced the evolution of lactic acid bacteria toward fructophilic species" Critical Reviews in Microbiology, Vol.45, No.1, 2019, pp.65 - 81). FLAB survives in fructose-rich environments such as flowers or fruits, fermented foods, and the digestive tracts of insects that feed on fructose. FLAB is thought to be a heterofermentative lactic acid bacterium that prefers fructose rather than glucose as a carbon source, but it promotes growth in the presence of glucose by adding electron acceptors such as oxygen. Compared with other FLAB and lactic acid bacteria, strain 10H has a relatively smaller genome size and lower GC content (see Figure 3 of Filannino et al.).

"Bacteria" in this manual includes both living and dead bacteria. The bacterial cells can also be frozen or dried. In addition, the "microbial cell treated material" in this specification refers to a substance containing components derived from microbial cells obtained by performing a process to destroy microbial cells or a process to extract components from microbial cells. The treated bacterial cells include not only those obtained by physically treating the bacterial cells and containing part or all of the bacterial cells such as cell lipids or cell wall distinguishing parts, but also include extracts derived from the bacterial cells (see Examples to be described later). 2) Generally does not contain the bacteria themselves.

(II) Immunoactive macrophage inducer, cancer microenvironment improving agent and cancer apoptosis inducer In this specification, "immune active macrophage inducer" refers to an agent that induces immunosuppressive macrophages (M2 type macrophages) into immune active macrophages (M1 type macrophages). Macrophages are a type of white blood cell and are cells that play an important role in the infection defense mechanism of animals. Normally, macrophages function as immune-activated macrophages with anti-tumor effects such as attacking cancer cells and inducing apoptosis. On the other hand, if cancer cells are produced and increase, macrophages are attracted to the cancer site and differentiate into immunosuppressive cells by immunosuppressive macrophage-inducing factors (IL-6, IL-10, etc.) secreted from cancer cells. macrophages. Immunosuppressive macrophages promote angiogenesis through the expression of vascular endothelial growth factor (VEGF), thereby creating an environment in which cancer cells are prone to proliferate. Therefore, it is considered important to induce immune-active macrophages and suppress (reduce) immune-suppressive macrophages in order to suppress the proliferation of cancer cells.

The immune-stimulating macrophage inducer of the present invention contains the bacterial cells or processed bacterial cells of the enzyme Apilactobacillus kosoi 10H strain as an active ingredient. As shown in Example 3 described later, the bacterial cells and the processed bacterial cells have the inducing effect of immunostimulatory macrophages and the inhibitory effect of immunosuppressive macrophages.

In this specification, a "cancer microenvironment improving agent" refers to an agent that improves the cancer microenvironment, which is the environment around cancer cells, in a direction that can inhibit the proliferation of cancer cells. The cancer microenvironment improving agent of the present invention contains the bacterial cells or bacterial cell processed product of the enzyme Apilactobacillus kosoi 10H strain as an active ingredient. It is thought that the bacterial cells and the processed bacterial cells exert a cancer microenvironment-improving effect by inducing immune-stimulating macrophages and suppressing immunosuppressive macrophages, as described in Example 3 described below.

In this specification, "cancer apoptosis inducer" refers to an agent that induces apoptosis of cancer cells. The cancer apoptosis inducing agent of the present invention contains the bacterial cells or bacterial cell processed product of the enzyme Lactobacillus kosoi (Apilactobacillus kosoi) 10H strain as an active ingredient. It is thought that the bacterial cells and the processed bacterial cells exert a cancer apoptosis-inducing effect through the induction of immunoactive macrophages and the inhibitory effect of immunosuppressive macrophages, as shown in Example 4 described below.

(III) Food, drink, medicine, feed, or active ingredient compositions formulated therein (active ingredient composition) The immune-stimulating macrophage inducer, cancer microenvironment improving agent, and cancer apoptosis inducer according to the present embodiment can be used in the form of an active ingredient composition formulated in foods, drinks, pharmaceuticals, or feeds. When used in the form of an active ingredient composition, not only the bacterial cells of the 10H strain or the processed bacterial cells as the active ingredient can be used directly, but the culture and fermentation liquid (including the culture supernatant) of the 10H strain can also be used. Or the crude or purified products. In addition, when the active ingredient is a treated substance of bacterial cells, an active ingredient composition containing a substance (for example, an extraction solvent) used for treating bacterial cells can also be used.

In addition, the bacterial cells may be not only live bacterial cells but also those sterilized by heat sterilization operation or the like (dead bacterial cells). The 10H strain is thought to have a solid cell surface structure because it grows under harsh growth conditions with high sugar concentration (high osmotic pressure). The conditions for causing the 10H strain to become dead cells by heat treatment are preferably set at 65 to 85°C for more than 1 minute, such as 10 minutes, 30 minutes, or about 6 minutes, and more preferably at 70 to 75 times for more than 1 minute, such as 5 minutes, 10 minutes, or about 30 minutes. As will be explained in the Examples described below, even heat-treated bacterial cells can be expected to induce immune-stimulating macrophages, improve cancer microenvironment, and induce cancer apoptosis. In addition, in the case of bacterial growth, morphological changes may occur during distribution or display after the product is manufactured, so dead microorganisms that have been heat-sterilized without further morphological changes can be preferably used.

Moreover, the active ingredient composition of this embodiment may further contain an appropriate amount of nutrients suitable for the maintenance, growth, etc. of the 10H strain, if necessary. Specific examples of the nutritional components include carbon sources such as fructose, glucose, sorbose, ribose, lyxose, xylose, arabinose, lactulose, and sucrose used in culture media for culturing microorganisms. For example, yeast extract, proteinaceous and other nitrogen sources, vitamins, minerals, trace metal elements, other nutrients and other ingredients. Examples of vitamins include vitamin B, vitamin D, vitamin C, vitamin E, vitamin K, and the like. Examples of trace metal elements include zinc, selenium, and the like. Examples of other nutritional components include various oligosaccharides such as lactulooligosaccharide, soybean oligosaccharide, lactulose, lactitol, fructooligosaccharide, and galactooligosaccharide. The compounding amount of these oligosaccharides is not particularly limited, but is generally preferably selected from the range of approximately 1 to 30% by weight in the composition of the present embodiment.

The amount of the 10H strain to be added to the active ingredient composition of the present embodiment can usually be appropriately selected so that the number of bacteria in 100 g of the composition is about 10 ⁸ to 10 ¹³ (it does not need to be the number of biotic bacteria). The bacterial count can be determined by the limiting dilution culture method using MRS liquid medium containing 10% fructose. Since the number of bacteria is related to the turbidity, if the correlation between the number of bacteria and the turbidity is determined in advance, the number of bacteria can be counted by measuring the turbidity instead of measuring the number of bacteria. The active ingredient composition of the present embodiment is preferably prepared in the form of foods, drinks, pharmaceuticals, etc. as described below by appropriately blending a suitable edible carrier (food material), a pharmaceutically acceptable carrier, etc.

(Pharmaceuticals) When the immunostimulating macrophage inducer, cancer microenvironment improving agent, and cancer apoptosis inducer of the present embodiment are in the form of pharmaceuticals, they are combined with the bacterial cells of the 10H strain or bacterial cells as active ingredients. The treated materials are prepared into the form of a pharmaceutical composition using appropriate preparation carriers that are acceptable in pharmaceuticals. Examples of the preparation carrier include diluents or excipients such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, and lubricants commonly used in this field.

As the dosage unit form of the pharmaceutical, various forms can be selected, and a preferred example is a preparation for oral administration. Representative oral administration preparations include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, and the like.

When it is formed into a tablet, excipients such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. can be used as the preparation carrier; water , ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone and other binding agents; carboxymethylcellulose sodium , carboxymethyl cellulose calcium, low-substitution hydroxypropyl cellulose, dry starch, sodium alginate, agar powder, lamina sugar powder, sodium bicarbonate, calcium carbonate and other disintegrants; polyoxyethylene sorbitan fatty acid ester Surfactants such as surfactants, sodium lauryl sulfate, monoglyceryl stearate, etc.; disintegration inhibitors such as sugar, stearin, cocoa butter, hydrogenated oil, etc.; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate, etc.; glycerin, Starch and other humectants; starch, lactose, kaolin, bentonite, colloidal silicic acid and other adsorbents; refined talc, stearate, boric acid powder, polyethylene glycol and other lubricants. Tablets may be coated with a conventional coating as needed, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, or double-layered tablets or multi-layered tablets.

When forming into pills, as preparation carriers, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc can be used; combined with gum arabic powder, tragacanth powder, gelatin, ethanol, etc. Agents; disintegrating agents such as lamina sugar, agar, etc.

Furthermore, pharmaceuticals may optionally contain coloring agents, preservatives, spices, flavoring agents, sweeteners, etc. or other pharmaceuticals.

The method of administering the pharmaceutical according to this embodiment is not particularly limited and is determined based on the form of the preparation, conditions such as age and gender of the patient, the degree of the disease, and the like. In addition, the dosage is appropriately determined depending on the usage, conditions such as the age and gender of the patient, the degree of the disease, etc. Generally, it is considered that the active ingredient composition can be about 0.5 to 100 mg per day per 1 kg of body weight. . Pharmaceuticals can be administered to humans 1 to 4 times a day.

(Food and drink) The term "food and drink" in this manual includes all forms (for example, drinks) that are used orally specifically for eating and drinking. Even if they are in the form of tablets, etc., as long as they are specifically used for eating and drinking, they are also included in the food and drink in this specification. Good quality. For example, health foods, health supplements, foods for patients, nutritional supplements, or health functional foods (foods for specific health uses, nutritional functional foods) specified by the Ministry of Health, Labor and Welfare of Japan are also included in the food and drink in this specification. Healthy food refers to food that has a more positive meaning than ordinary food and is intended for health care, health maintenance/enhancement, etc.

When the immunostimulating macrophage inducing agent, cancer microenvironment improving agent and cancer apoptosis inducing agent of the present embodiment are used as food or drink, for example, fermented milk, lactic acid bacteria beverage, fermented vegetable beverage, Fermented fruit drinks, fermented soy milk drinks, etc. "Fermented milk" refers to a paste or liquid obtained by fermenting milk or dairy products using lactic acid bacteria or yeast. Therefore, this fermented milk includes a beverage form and a yogurt form. In addition, "lactic acid bacteria drink" refers to a drink obtained by diluting a paste or liquid obtained by fermenting milk or dairy products with lactic acid bacteria or yeast as the main raw material.

Examples of other food and drink forms include fermented foods such as pickles, bean paste, fermented tea, and bread, foods for infants and young children such as weaning foods, milk powder, and baby food, foaming preparations, chewing gum, gummy candies, and puddings Nutritional supplements such as snacks, noodles, capsules, granules, powders, and tablets, and dairy products other than the above-mentioned fermented milk and lactic acid bacteria drinks.

The content of the active ingredient composition in the food and drink of this embodiment is not particularly limited and can be determined appropriately. From the viewpoint of exerting the effect of inducing immune-stimulating macrophages, improving the cancer microenvironment, and inducing cancer apoptosis, the total mass of each food and drink is, for example, preferably 0.001 mass % or more, more preferably 0.01 mass % or more. , more preferably 0.1% by mass or more. On the other hand, the upper limit of the content of the active ingredient composition in food and beverages is not particularly limited and can usually be appropriately adjusted according to the form of the food and beverages.

(feed) When the immunostimulating macrophage inducer, cancer microenvironment improving agent, and cancer apoptosis inducer of the present embodiment are in the form of feed, for example, they can be in the form of preparations for oral administration (aqueous solution, emulsified liquid, granules, powder, capsules, tablets, etc.).

[Example] Hereinafter, the present invention will be described in further detail with reference to examples, but the present invention is not limited by these examples in any way. In the following examples, the unit % used to express numerical values such as the amount of addition of various components means mass % unless otherwise stated.

[Example 1] Culture of enzyme Lactobacillus apiensis 10H strain and preparation of dead cells The preserved strain of Lactobacillus apiensis 10H strain from Arsoa Keio Group Co., Ltd. was used in MRS medium (Gibco) supplemented with 10% (w/v) D(-)-fructose (Fujifilm Wako Pure Chemical Industries, Ltd.). The culture medium was cultured statically at 30°C for 48 hours while the 500 mL culture medium bottle (AGC Techno Glass Co., Ltd.) was tightly connected. After the culture, the culture solution was treated with a centrifuge (Kubota Shoji Co., Ltd., model 6800) at 8000 rpm for 20 minutes to remove the MRS medium and obtain a bacterial cell slurry. The obtained bacterial cell slurry was repeatedly suspended in DPBS (Thermo Fisher Scientific Inc.) and centrifuged (8000 rpm, 20 minutes) three times to obtain a washed bacterial cell slurry. A part of the washed bacterial cell slurry was processed in an autoclave at 70° C. for 30 minutes to prepare dead bacterial cells, and then freeze-dried using a freeze dryer (Tokyo Rika Instruments Co., Ltd., FDU-2200). In Examples 3 and 4 described below, a suspension of dead cells of the 10H strain in DPBS at a concentration of 40 mg/mL was used.

[Example 2] Preparation of enzyme extract of Lactobacillus apiensis 10H strain (cell treated product) 19 mg of the washed bacterial cell slurry of the 10H strain obtained in Example 1 was suspended in 100 μL of 0.1 M sodium acetate buffer (Fuji Film Wako Pure Chemical Industries, Ltd., pH = 4.7), and transferred to EZ-Beads (AMR Co., Ltd., 76813M), and processed using a vortex mixer (M&S Instruments Co., Ltd.) for 2 minutes and 30 seconds. Thereafter, 100 μL of n-butanol (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was crushed using a vortex mixer for 30 minutes. Then, 100 μL of 0.1 M sodium acetate buffer (pH value = 4.7) and 100 μL of n-butanol were added, and the mixture was treated with a vortex mixer for 30 minutes. Next, use centrifugation at 13,000 rpm for 5 minutes to separate into two layers. The upper layer (n-butanol phase) and the lower layer (water phase) were respectively recovered into different tubes for freeze-drying. Hereinafter, the extract of the upper layer (n-butanol phase) will be described as "oil-soluble extract", and the extract of the lower layer (water phase) will be described as "water-soluble extract". In the experiment described below (see Example 3), an oil-soluble extract dissolved in methanol (Fujifilm Wako Pure Chemical Industries, Ltd.) so as to have a concentration of 10 mg/mL was used, and a water-soluble extract was used. The sexual extract is dissolved in DPBS.

[Example 3] Evaluation of induction of immune-competent macrophages (M1 type macrophages) As described in the description of "Immunoactive Macrophage Inducer", it is considered important to induce immune-active macrophages (M1 type macrophages) and suppress immunosuppressive macrophages in order to inhibit the proliferation of cancer cells. cells (M2 macrophages). Therefore, in Example 3, it was evaluated whether the dead cells of the 10H strain, the water-soluble extract, and the oil-soluble extract could induce immune-active macrophages from immunosuppressive macrophages.

(Preparation of immunosuppressive macrophages (M2 type macrophages)) The human monocytic leukemia cell line THP-1 (JCRB Cell Bank) was transformed into 2× using RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd.) 10 ⁵ cells/well were seeded in a 24-well plate (Nunc). Next, add phorbol 12-myristate 13-acetate (Sigma) diluted in RPMI 1640 medium so that the final concentration becomes 100 ng/mL, and use CO ₂ at 37°C and 5% CO ₂ Macrophages were induced by culturing in an incubator (Wakenbtech, WKN-MC35) for 72 hours. Furthermore, the obtained macrophages were washed three times with RPMI 1640 medium and cultured for 72 hours with RPMI 1640 medium supplemented with human IL-6 recombinant protein (Proteintech) at a final concentration of 12.5 ng/mL to differentiate into immune cells. Suppressive macrophages (M2 macrophages).

(Induction from immunosuppressive macrophages to immunocompetent macrophages) Immunosuppressive macrophages adhered to a 24-well plate were added with 10H dead cells, water-soluble extract, or oil-soluble extract at a final concentration of 2 μg/mL or 20 μg/mL, and then cultured for 24 hours. After the cultured cells were washed once with DPBS, 0.3 mL of 0.5 μM EDTA (Nacalai Tesque Co., Ltd.) was added and kept for 10 minutes to peel the cells from the plate. Furthermore, 0.3 mL of RPMI 1640 medium was added, and the cells were collected into a 1.5 mL microtube (VIOLAMO) by pipetting. The wells were washed again with 0.3 mL of DPBS, and the washing solution was collected into the same tube. Next, the supernatant was removed by centrifugation using a centrifuge (TOMY SEIKO Co., Ltd., MX-301) at 5000 rpm, 5 minutes, and 4°C to obtain cell pellets.

(Preparation of antibody solution) First, Fc Blocker (BioLegend Co., Ltd.) was added at a 100-fold dilution in DPBS, 7-AAD (Beckman Coulter Co., Ltd.) and FITC-labeled anti-CD11b antibody ( BioLegend Co., Ltd.). The above solution was labeled with PE-labeled anti-CD163 antibody (BioLegend Co., Ltd.) and APC-labeled anti-CD209 antibody (BioLegend Co., Ltd.), or PE-labeled-anti-CD80 antibody (BioLegend Co., Ltd.) and APC-labeled-anti-HLA- For a combination of DR antibodies (BioLegend Co., Ltd.), each fluorescently labeled antibody was added in a 50-fold dilution to prepare an antibody solution.

(Measurement of expression rates of CD80, HLA-DR, CD163 and CD209) The prepared antibody solution was added to the above-mentioned cell pellets, and homogenized with a vortex mixer. The cell pellets to which fluorescently labeled antibodies were added were stained at 4°C for 15 minutes, and then analyzed using Cyto FLEX (Beckman Coulter Co., Ltd., B53015). In flow cytometry analysis, a gate is set for 7-AAD negative cells, and the expression rates of CD80, HLA-DR, CD163, and CD209 among CD11b-positive cells in the cells within the gate are quantified. Furthermore, the increase or decrease in the expression rate of CD80 and HLA-DR can be used as markers related to immune-active macrophages (hereinafter referred to as M1 markers), and CD163 and CD209 can be used as markers related to immune-suppressive macrophages. Cell-related markers (hereinafter referred to as M2 markers).

(Measurement of TNF-α production) For differentiated immunosuppressive macrophages, add 2 μg/mL or 20 μg/mL of 10H strain dead bacteria, water-soluble extract, or oil-soluble extract, then culture for 24 hours, and recover the culture supernatant. To measure the TNF-α production in the culture supernatant, a Duoset ELISA (R&D Systems Inc.) kit was used. All measurements are carried out according to the recommended operating procedures of the kit. Furthermore, TNF-α is one of the inflammatory cytokines and is produced by immune-activating macrophages but is rarely produced by immunosuppressive macrophages. Therefore, its production amount can be used as an M1 marker.

(experimental results) The results of the above experiments are shown in Figures 1 to 6. Figure 1 is a bar graph showing the induction of immunocompetent macrophages produced by dead bacteria of the 10H strain. Figure 1(a) is a bar graph showing the expression rate of CD80 (M1 marker), Figure 1(b) is a bar graph showing the expression rate of HLA-DR (M1 marker), and Figure 1(c) is A graph showing the production amount of TNF-α (M1 marker). Figure 2 is a bar graph showing the inhibitory effect of immunosuppressive macrophages produced by dead bacteria of the 10H strain. Figure 2(a) is a bar graph showing the expression rate of CD163 (M2 marker), and Figure 2(b) is a bar graph showing the expression rate of CD209 (M2 marker). Figure 3 is a bar graph showing the induction of immunocompetent macrophages by a water-soluble extract. Figure 3(a) is a bar graph showing the expression rate of CD80 (M1 marker), Figure 3(b) is a bar graph showing the expression rate of HLA-DR (M1 marker), and Figure 3(c) is A graph showing the production amount of TNF-α (M1 marker). Figure 4 is a bar graph showing the inhibitory effect on immunosuppressive macrophages produced by a water-soluble extract. Figure 4(a) is a bar graph showing the expression rate of CD163 (M2 marker), and Figure 4(b) is a bar graph showing the expression rate of CD209 (M2 marker). Figure 5 is a bar graph showing the induction of immunocompetent macrophages by oil-soluble extracts. Figure 5(a) is a bar graph showing the expression rate of CD80 (M1 marker), Figure 5(b) is a bar graph showing the expression rate of HLA-DR (M1 marker), and Figure 5(c) is A graph showing the production amount of TNF-α (M1 marker). Figure 6 is a bar graph showing the inhibitory effect on immunosuppressive macrophages produced by oil-soluble extracts. Figure 6(a) is a bar graph showing the expression rate of CD163 (M2 marker), and Figure 6(b) is a bar graph showing the expression rate of CD209 (M2 marker). In addition, the "*" mark shown on the bar graphs in Figures 1 to 6 indicates that it is consistent with the control (when the added amount of 10H strain dead cells, water-soluble extract, or oil-soluble extract is 0 μg/mL) The ratios are significantly different (Dunnett's test P<0.01). The same is true in Figure 7 described below.

As shown in Figures 1, 3, and 5, all 10H strain dead cells, water-soluble extracts, and oil-soluble extracts were compared with the case where these were not added (0 μg/mL), and it was confirmed that they were M1 markers. The expression rate of CD80 and HLA-DR and the production of TNF-α are increased. Furthermore, as shown in Figures 2, 4, and 6, all 10H strain dead cells, water-soluble extracts, and oil-soluble extracts were compared with the case where these were not added (0 μg/mL), and it was confirmed that they were M2 The expression rates of markers CD163 and CD209 were reduced. From these results, it was found that the bacterial cells and bacterial cell processed products of Lactobacillus apiensis strain 10H induce immune-stimulating macrophages from immunosuppressive macrophages. Therefore, it is believed that the bacterial cells and bacterial cell treatments of Lactobacillus apiensis 10H strain can suppress (reduce) immunosuppressive macrophages and induce (increase) immune-stimulating macrophages in an environment where a large number of immunosuppressive macrophages exist. Phagocytosis can be expected to be an active ingredient as an immune-stimulating macrophage inducer and a cancer microenvironment improving agent.

[Example 4] Evaluation of cancer apoptosis induction effect In Example 4, immunosuppressive macrophages were cultured in the presence of dead bacterial cells of the 10H strain, a conditioned medium was prepared, and cancer cell lines were cultured in the presence of the conditioned medium to investigate whether cancer cell lines were induced. Apoptosis.

(Preparation of conditioned medium) First, immunosuppressive macrophages were induced by the same method as in Example 3. Dead bacterial cells of 10H strain (2 μg/mL) were added to the obtained immunosuppressive macrophages and cultured for 24 hours. In order to remove bacterial cells from the culture supernatant, centrifugation was performed at 8000 rpm, 5 minutes, and 4°C, and the supernatant was recovered as a conditioned medium (sample name: M2 A. kosoi-CM). In addition, an immunosuppressive macrophage culture medium that was treated in the same manner as above without adding dead cells of the 10H strain was used as a non-added control (sample name: M2 None-CM).

(Addition of conditioned medium to cancer cell lines and culture) The cancer cell lines used were colorectal cancer cell line HT-29 (KAC Co., Ltd.), lung cancer cell line A549 (JCRB Cell Bank), and breast cancer cell line MCF-7 (JCRB Cell Bank). ). Regarding the culture medium, RPMI 1640 medium was used for HT-29 cells, MEM medium (Nissui Pharmaceutical Co., Ltd.) was used for A549 cells, and DMEM medium (Nissui Pharmaceutical Co., Ltd.) was used for MCF-7 cells. Each cancer cell line was inoculated into 1.2×10 ⁴ cells/well and cultured for 24 hours to adhere to the bottom of the plate. Thereafter, 50% of the culture medium of each cancer cell line was replaced with the conditioned medium, and cultured for 48 hours (HT-29 cells and A549 cells) or 72 hours (MCF-7 cells). After culturing, each cell was washed once with DPBS, and 0.3 mL of TrypLE express (Theremo Fisher Scientific Inc.) diluted 2-fold with DPBS was added and kept for 5 minutes to detach the cells from the plate. Thereafter, each culture medium containing 0.3 mL of 10% fetal calf serum (v/v) was added to stop the enzyme reaction, and the cells were collected into a 1.5 mL microtube (VIOLAMO). Furthermore, the plate was washed once with 0.3 mL of DPBS to recover the cells. The recovered cells are centrifuged to obtain cell pellets. The centrifugal separation treatment was performed using a centrifugal separator (TOMY SEIKO Co., Ltd., MX-301) at 5000 rpm, 5 minutes, and 4°C.

(Calculation of cell staining and apoptosis ratio) A 7-AAD solution diluted 5 times with DPBS and a FITC-labeled annexin V solution diluted 25 times were prepared respectively. Add 13 μL of 7-AAD antibody solution to the cells washed with DPBS, stain at 4°C in the dark for 15 minutes, add 1 mL of DPBS, and obtain cell pellets under the same centrifugation conditions as above. After adding 20 μL of FITC-labeled Annexin V solution to the cell pellet, staining was performed in the dark at room temperature for 15 minutes. Furthermore, 200 μL of Annexin V binding buffer (BioLegend) was added, and the apoptotic cell ratio was analyzed using Cyto FLEX. The apoptosis ratio was calculated as the ratio of 7-AAD-positive and Annexin V-positive cells to all included cells.

(experimental results) The results of the above experiments are shown in Figure 7. Figure 7 is a bar graph showing the induction of cancer apoptosis by dead cells of the 10H strain. Figure 7(a) is a bar graph showing the induction of cancer apoptosis in HT-29 cells. Figure 7(b) is a bar graph showing the induction of cancer apoptosis in A549 cells. Figure 7(c) ) is a bar graph showing the induction of cancer apoptosis in MCF-7 cells.

As shown in Figure 7, it was confirmed for all three types of cancer cell lines used in the experiment that the addition of conditioned medium (M2 A. kosoi- CM), whereby the apoptotic ratio was significantly increased compared to the unadded control (M2 None-CM). Based on these results, it is believed that the Lactobacillus apiensis 10H strain can induce cancer by inhibiting immunosuppressive macrophages and inducing immune-promoting macrophages in an environment where a large number of immunosuppressive macrophages exist. It can be expected to act as an active ingredient in inducing cancer apoptosis due to cell apoptosis. In addition, referring to the results of Example 3, it is considered that the bacterial cell treatment (extract) of the enzyme Lactobacillus meli 10H strain can also induce immunostimulating macrophages and suppress immunosuppressive macrophages. It induces apoptosis of cancer cells in the same way as bacteria. Therefore, the bacterial cell processed product (extract) can also be expected to serve as an active ingredient of a cancer apoptosis inducer.

(Formulation example 1 tablet) According to the usual method, mix the following ingredients to make a tablet. In addition, the active ingredient composition used the freeze-dried cells of the enzyme Lactobacillus apiensis 10H strain obtained in Example 1. composition Active ingredient composition (Example 1) 150 mg Cellulose 80 mg Starch 20 mg Sucrose fatty acid ester 2 mg The above ingredients are mixed and tableted to obtain a tablet.

(Formulation Example 2 Capsules) According to the usual method, mix the following ingredients to obtain soft capsules. In addition, the active ingredient composition used the freeze-dried cells of the enzyme Lactobacillus apiensis 10H strain obtained in Example 1. composition Active ingredient composition (Example 1) 100 mg Beeswax 10 mg Grape seed oil 110 mg Mix the above ingredients and fill them into a capsule base mixed with gelatin and glycerin to obtain a soft capsule.

Figures 1 (a) to (c) are bar graphs showing the induction of immunocompetent macrophages produced by dead bacteria of the 10H strain. Figure 2 (a) and (b) are bar graphs showing the inhibitory effect of immunosuppressive macrophages produced by dead bacteria of the 10H strain. Figures 3 (a) to (c) are bar graphs showing the induction of immunocompetent macrophages by the water-soluble extract. Figures 4(a) and (b) are bar graphs showing the inhibitory effect of the water-soluble extract on immunosuppressive macrophages. Figures 5(a) to (c) are bar graphs showing the induction of immunocompetent macrophages produced by the oil-soluble extract. Figures 6(a) and (b) are bar graphs showing the inhibitory effect of the oil-soluble extract on immunosuppressive macrophages. Figures 7 (a) to (c) are bar graphs showing the induction of cancer apoptosis by dead cells of the 10H strain.

Claims (9)

An immune-stimulating macrophage inducing agent contains cells or a processed product of the enzyme Apilactobacillus kosoi 10H strain as an active ingredient. The immune-stimulating macrophage inducer of Claim 1 is in the form of a food, drink, medicine, feed, or an active ingredient composition formulated therein. A cancer microenvironment improving agent, which contains the bacterial cells or bacterial cell processing of the enzyme Apilactobacillus kosoi (Apilactobacillus kosoi) 10H strain as an active ingredient. The cancer microenvironment improving agent of claim 3 is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition blended therein. A cancer apoptosis inducer, which contains cells or a processed product of the enzyme Lactobacillus kosoi 10H strain as an active ingredient. The cancer apoptosis inducing agent of claim 5 is in the form of a food, drink, pharmaceutical, feed, or an active ingredient composition blended therein. The use of enzyme cells or cell processed products of Apilactobacillus kosoi 10H strain, which are used to produce pharmaceuticals, foods, feeds, or feeds for inducing immune-activating macrophages in humans or non-human animals. The active ingredient composition formulated in these. The use of enzyme cells or cell processed products of Apilactobacillus kosoi 10H strain, which are used to produce pharmaceuticals, food and beverages, feeds for improving the microenvironment of cancer in humans or non-human animals, or to be formulated in the same The active ingredient composition among others. The use of enzyme cells or cell processed products of the 10H strain of Lactobacillus bee (Apilactobacillus kosoi), which are used to produce pharmaceuticals, food and beverages, and feeds for inducing cancer apoptosis in humans or non-human animals, or to be formulated in the same The active ingredient composition among others.