Immunostimulator and method for producing the same

Abstract

To provide an immunostimulatory that contains microorganism-derived endotoxin and can induce production of interleukin.SOLUTION: A immunostimulatory contains endotoxin derived from halophilic bacteria belonging to Halomonas and induces production of interleukin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an immunostimulant and a method for producing the same. [Background technology]

[0002] With the recent spread of the novel coronavirus, there has been growing interest in preventing viral infections. One known preventative measure against viral infections is the intake of foods or supplements containing drugs that improve the body's immune function. As such drugs that improve the body's immune function, for example, an immunostimulant containing a drug that induces the production of interferon α, a type of cytokine, has been proposed (Patent Document 1).

[0003] The immunostimulant described in Patent Document 1 contains, as an active ingredient, a lactic acid bacterium that has a high ability to induce the production of interferon α, and nucleic acid from this lactic acid bacterium. This immunostimulant induces the production of interferon α, thereby enhancing the body's immune function and suppressing viral infections. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5950827 Summary of the Invention [Problem to be solved by the invention]

[0005] The above-mentioned Patent Document 1 reveals that the production of interferon α is induced by including lactic acid bacteria or their nucleic acids as active ingredients. However, it is not clear whether cytokine production is induced when bacteria other than lactic acid bacteria are used, or about immunostimulants containing microbial-derived components that can induce the production of other cytokines.

[0006] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an immunostimulant that contains endotoxin derived from a microorganism and is capable of inducing the production of interleukin, and a method for producing the same. [Means for solving the problem]

[0007] The immunostimulating agent according to the present invention for achieving the above object has the following characteristic features: Contains endotoxin derived from halophilic bacteria belonging to the genus Halomonas, which induces interleukin production death, The halophilic bacterium is Halomonas sp. KM-1 strain. It's at the point.

[0008] As a result of extensive research, the inventors of the present invention have discovered that halophilic bacteria belonging to the genus Halomonas Halomonas sp. KM-1 strain The present inventors have found that by culturing the above strain under specified conditions and treating the resulting culture medium, an immunostimulant containing endotoxin that induces interleukin production can be obtained, and have completed the present invention.

[0009] That is, according to the above characteristic configuration, the production of interleukins is induced by taking an immunostimulant, thereby enhancing the immune function of the living body and suppressing viral infections.

[0010] Further, a further characteristic configuration of the immunostimulant according to the present invention is It induces the production of interleukins by macrophages.

[0011] The present inventors have confirmed that by culturing macrophages with the addition of the immunostimulant according to the present invention, the endotoxin acts on the macrophages, causing the macrophages to produce interleukins.

[0012] Further, a further characteristic configuration of the immunostimulant according to the present invention is The interleukin is interleukin-12.

[0013] According to the above characteristic configuration, the production of interleukin 12 as an interleukin is induced, and as a result, the immune function of the living body is enhanced and viral infection can be suppressed.

[0015] The present inventors have experimentally confirmed that when the halophilic bacterium is Halomonas sp. KM-1 strain, endotoxin derived from the Halomonas sp. KM-1 strain induces the production of interleukin-12.

[0016] The method for producing an immunostimulant of the present invention for achieving the above object is characterized by the following features: A method for producing an immunostimulant that contains endotoxin derived from a halophilic bacterium belonging to the genus Halomonas and induces interleukin production, comprising: an aerobic culturing step of aerobically culturing the halophilic bacterium belonging to the genus Halomonas in a medium; a microaerobic culture step of culturing the halophilic bacterium in a medium while adjusting the pH, after the aerobic culture step; a removal step of removing bacterial cells from the medium after the microaerobic culture step; a fractionation step of subjecting the medium after the removal step to molecular weight fractionation; and a recovery step of recovering the high molecular weight fraction obtained in the fractionation step. Furthermore, the method for producing an immunostimulant of the present invention for achieving the above object is characterized by the following features: A method for producing an immunostimulant that contains endotoxin derived from a halophilic bacterium belonging to the genus Halomonas and induces interleukin production, comprising: an aerobic culturing step of aerobically culturing the halophilic bacterium belonging to the genus Halomonas in a medium; a microaerobic culture step of culturing the halophilic bacterium in a medium while adjusting the pH, after the aerobic culture step; A fractionation treatment step of subjecting the medium after the microaerobic culture step to molecular weight fractionation treatment; a removal step of removing bacterial cells from the high molecular weight fraction obtained in the fractionation step; and a recovery step of recovering the high molecular weight fraction after the removal step.

[0017] As a result of extensive research into methods for producing immunostimulants, the inventors of the present application discovered that when 3-hydroxybutyric acid is produced using halophilic bacteria belonging to the genus Halomonas, 3-hydroxybutyric acid is present in the low molecular weight fraction obtained through an aerobic culture process, a microaerobic culture process, and a fractionation process, and that endotoxin, which induces the production of interleukins, is contained in the high molecular weight fraction obtained as a by-product, thereby completing the present invention.

[0018] That is, according to the above-described characteristic configuration, the immunostimulant of the present invention can be produced by performing an aerobic culture step, a microaerobic culture step, and a fractionation treatment step, removing the bacterial cells from the medium after the microaerobic culture step, or by removing the bacterial cells from the high molecular weight fraction obtained in the fractionation treatment step, and recovering the final high molecular weight fraction. The immunostimulant produced in this manner can be obtained as a by-product during the production of 3-hydroxybutyric acid using halophilic bacteria belonging to the genus Halomonas, and can be supplied inexpensively and stably. Furthermore, since the bacterial cells are removed from the immunostimulant produced through the removal step, it can be used relatively stably as a food or supplement. DETAILED DESCRIPTION OF THE INVENTION

[0019] The immunostimulant and its manufacturing method according to the present invention will be described below. Preferred examples are described below, but these examples are described to more specifically illustrate the present invention, and various modifications are possible within the scope of the present invention, and the present invention is not limited to the following descriptions.

[0020] [Immunostimulants] The immunostimulant of the present invention contains endotoxin derived from a halophilic bacterium belonging to the genus Halomonas, and induces the production of interleukin.

[0021] Specifically, when the halophilic bacterium belonging to the genus Halomonas is Halomonas sp. KM-1 strain, endotoxin derived from the Halomonas sp. KM-1 strain acts on macrophages to induce the production of interleukin-12.

[0022] That is, according to the immunostimulant of the present invention, endotoxin derived from halophilic bacteria acts on immune cells such as monocytes and macrophages, promoting the production of interleukins by the immune cells, thereby enhancing the immune function of the body and suppressing viral infection.

[0023] The immunostimulant of the present invention is obtained by treating the culture medium obtained by culturing a halophilic bacterium belonging to the genus Halomonas, and can be used in the form of foods, beverages, cosmetics, pharmaceuticals, or quasi-drugs.

[0024] Furthermore, the immunostimulant of the present invention is produced through the removal process described below, and is almost free of bacterial contamination or contains only a small amount of bacterial contamination, making it relatively stable for use as a food or supplement.

[0025] [Outline of the method for producing immunostimulants] The method for producing an immunostimulant of the present invention is a method in which a culture medium in which a halophilic bacterium belonging to the genus Halomonas has been cultured is subjected to a predetermined treatment, and a high molecular weight fraction containing endotoxin derived from the halophilic bacterium is recovered as an immunostimulant.

[0026] Specifically, the method for producing an immunostimulant of the present invention involves the following steps. (1-1) An aerobic culturing step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium. (1-2) After the aerobic culture step, a microaerobic culture step is performed in which halophilic bacteria are cultured in a medium under microaerobic conditions while adjusting the pH. (1-3) A removal step of removing bacterial cells from the medium after the microaerobic culture step. (1-4) A fractionation step in which the medium after the removal step is subjected to molecular weight fractionation. (1-5) A recovery step of recovering the high molecular weight fraction obtained in the fractionation step. Alternatively, the following steps are performed. (2-1) An aerobic culturing step in which a halophilic bacterium belonging to the genus Halomonas is aerobically cultured in a medium. (2-2) A microaerobic culture step in which, after the aerobic culture step, halophilic bacteria are cultured in a medium under microaerobic conditions while adjusting the pH. (2-3) A fractionation treatment step in which the medium after the microaerobic culture step is subjected to molecular weight fractionation treatment. (2-4) A removal step of removing bacterial cells from the high molecular weight fraction obtained in the fractionation step. (2-5) A recovery step of recovering the high molecular weight fraction after the removal step.

[0027] [Aerobic cultivation process] The aerobic culturing step in the production method of the present invention is a step of aerobically culturing halophilic bacteria belonging to the genus Halomonas (hereinafter also simply referred to as halophilic bacteria) in a medium to allow the halophilic bacteria to accumulate poly-3-hydroxybutyrate (PHB).

[0028] The halophilic bacteria used in the aerobic culture process grow aerobically in a medium containing inorganic salts and one or more organic carbon sources, and have the property of accumulating PHB within their own cells. Furthermore, these halophilic bacteria have an optimum salt concentration for growth of 0.1 to 1.0 M, and are particularly capable of growing in a salt-free medium. The above-mentioned halophilic bacteria belonging to the genus Halomonas usually grow in a medium with a pH of about 5 to 12.

[0029] An example of such a halophilic bacterium is the Halomonas sp. KM-1 strain. Halomonas sp. KM-1 was deposited on July 10, 2007, at the National Institute of Advanced Industrial Science and Technology (National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, 305-8566, Japan) under accession number FERM P-21316. This strain has also been transferred to international depository status under accession number FERM BP-10995. The 16S rRNA gene of the Halomonas sp. KM-1 strain has been registered with DDBJ under accession number AB477015. The treated culture product (high molecular weight fraction recovered in the recovery process) obtained by culturing Halomonas sp. KM-1 contains endotoxin, an interleukin that induces the production of interleukin-12.

[0030] Furthermore, in view of the growth characteristics of the above-mentioned halophilic bacteria, the halophilic bacteria used in the aerobic culture step of the present invention are not limited to Halomonas sp. KM-1 strain, as long as they accumulate poly-3-hydroxybutyrate and then secrete 3-hydroxybutyrate in the microaerobic culture step described below. Examples of such halophilic bacteria belonging to the genus Halomonas include Halomonas pantelleriensis (ATCC 700273) and Halomonas campisalis (ATCC 7000597).

[0031] Furthermore, analysis of the 16S ribosomal RNA sequence has revealed that, in addition to the above-mentioned halophilic bacteria, Halomonas nitritophilus, Halomonas alimentaria, etc. may also be used as halophilic bacteria belonging to the genus Halomonas in the aerobic culture step.

[0032] A gene may be introduced into the halophilic bacterium belonging to the genus Halomonas. The gene to be introduced is not particularly limited as long as it reduces the amount of endotoxin contained in the high molecular weight fraction finally obtained in the production method of the present invention. Various general methods can be used to introduce recombinant DNA into the bacteria and to transform them using the recombinant DNA.

[0033] The medium used in the aerobic culture step contains inorganic salts and an organic carbon source. The pH of the medium is not particularly limited as long as it satisfies the growth conditions of the halophilic bacteria, but specifically, it should be about pH 5 to 12, and more preferably pH 8.8 to 12. It is preferable to use an alkaline medium, as this effectively prevents contamination with other bacteria.

[0034] The medium may be a liquid medium or a solid medium.

[0035] The inorganic salts to be added to the medium used in the aerobic culture step are not particularly limited, and examples thereof include phosphates, nitrates, carbonates, sulfates, and metal salts such as sodium, magnesium, potassium, manganese, iron, zinc, copper, and cobalt.

[0036] For example, when sodium is used as an inorganic salt, NaCl, NaNO3, NaHCO3, Na2CO3, etc. may be used.

[0037] It is preferable to use compounds that serve as nitrogen and phosphorus sources for the halophilic bacteria as these inorganic salts.

[0038] The nitrogen source may be, but is not limited to, nitrates, nitrites, urea, ammonium salts, etc., and may be compounds such as NaNO3, NaNO2, NH4Cl, etc.

[0039] The amount of nitrogen source used may be appropriately set within a range that does not affect the growth of the bacterial cells and achieves the purpose of producing an immunostimulant. Specifically, the amount of nitrate used is usually about 500 mg or more per 100 ml of medium at the initial stage of culture, more preferably about 1000 mg or more, and even more preferably about 1250 mg or more.

[0040] The phosphorus source may be a phosphate, monohydrogen phosphate, dihydrogen phosphate, or the like, and is not particularly limited, but may be, for example, a compound such as K2HPO4 or KH2PO4.

[0041] The amount of phosphorus source used may be determined appropriately from the same viewpoint as the amount of nitrogen source used. Specifically, the amount of dihydrogen phosphate used is usually about 50 to 400 mg, more preferably about 100 to 200 mg, per 100 ml of medium.

[0042] These inorganic salts may be used alone or in combination of two or more.

[0043] The organic carbon source to be added to the medium used in the aerobic culture step is not particularly limited and includes, for example, tryptone, yeast extract, soluble starch, ethanol, n-propanol, acetic acid, sodium acetate, propionic acid, waste glycerol, waste molasses, wood saccharification solution, hexoses such as psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, gulose, idose, galactose, and talose, pentoses such as ribulose, xylulose, ribose, arabinose, xylose, lyxose, and deoxyribose, disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose, and sugar alcohols such as erythritol, glycerin, mannitol, sorbitol, and xylitol.

[0044] The concentration of the organic carbon source may be appropriately set within a range in which accumulation of PHB progresses and the objective of producing an immunostimulant is achieved.

[0045] In the production method of the present invention, halophilic bacteria belonging to the genus Halomonas are cultured in a medium with a relatively high salt concentration, so there is almost no risk of contamination or proliferation of other bacteria, etc. Therefore, the medium may or may not be sterilized, and the culture can be carried out using simple equipment.

[0046] The aerobic culture conditions are not particularly limited as long as the halophilic bacteria grow and a significant amount of PHB accumulates in the bacteria.

[0047] Specifically, the halophilic bacteria are inoculated into approximately 5 ml of medium and pre-cultured overnight with shaking at a predetermined stirring speed and temperature. The bacterial cells obtained from the pre-culture are then diluted approximately 100-fold in medium contained in an Erlenmeyer flask, fermenter, jar fermenter, or the like, and main culture (corresponding to aerobic culture in this application) is carried out.

[0048] The culture temperature for the main culture can usually be set within a range of about 20 to 45°C, but is preferably set within a range of about 30 to 37°C. Furthermore, when an Erlenmeyer flask is used, the stirring speed can usually be set within a range of about 120 to 250 rpm, but is preferably set within a range of about 120 to 180 rpm. When a fermenter or jar fermenter is used, it is preferable to supply oxygen at an oxygen supply rate comparable to the above. Furthermore, the culture time for the main culture is not particularly limited as long as it is a time period during which PHB accumulation occurs, but is preferably a time period during which the amount of PHB accumulated within the halophilic bacterium becomes approximately constant, for example, 10 to 60 hours.

[0049] In the aerobic culture step, halophilic bacteria belonging to the genus Halomonas may be aerobically cultured under these culture conditions. Specifically, the dissolved oxygen concentration in the medium during aerobic culture is not particularly limited, but is usually 2 mg / L in the absence of bacterial cells, and preferably 5 mg / L or more.

[0050] The culture method in the aerobic culture step includes, but is not limited to, batch culture, semi-batch culture, continuous culture, and the like. However, considering that the halophilic bacteria used in the production method of the present invention are highly unlikely to be contaminated with other bacteria, long-term continuous culture is also possible. The culture environment may be one in which the medium is exposed to air, and may be adjusted by actively blowing an oxygen-containing gas onto the surface of the medium or by blowing such a gas into the medium. The culture environment may be either a non-sterile environment or a sterile environment.

[0051] [Microaerobic culture process] The microaerobic culture step in the production method of the present invention is a step in which, after the aerobic culture step, halophilic bacteria are cultured in a medium under microaerobic conditions while adjusting the pH, and poly-3-hydroxybutyrate accumulated in the cells of the halophilic bacteria is secreted into the medium as 3-hydroxybutyric acid.

[0052] Specifically, in the microaerobic culturing step, after the aerobic culturing step, aeration is stopped, a pH adjuster is added to adjust the pH to within a predetermined range, and the halophilic bacteria are cultured microaerobic.

[0053] The conditions for microaerobic culture are not particularly limited as long as they allow PHB accumulated within the bacterial cells to be secreted into the medium as 3HB.

[0054] When microaerobic cultivation is continued, the pH of the medium tends to decrease due to the production of organic acids. The pH of such a medium can be appropriately confirmed using a known pH measuring device or a jar fermenter equipped with such a device.

[0055] In the microaerobic culture step, the pH is adjusted and / or maintained within a predetermined range. Here, "adjusting and / or maintaining" means that the pH is maintained within the predetermined range by adding a pH adjuster while checking the pH, or that the pH is simply adjusted by adding a pH adjuster at the start of culture and then not adjusted thereafter.

[0056] The pH adjusted and maintained in the microaerobic culture step is preferably 7.5 or higher, more preferably 8.0 or higher, and even more preferably 8.5 or higher.

[0057] Halophilic bacteria belonging to the genus Halomonas can usually be cultured under moderately high salt concentrations and alkaline conditions, resulting in little contamination by contaminating bacteria. However, some lactic acid bacteria can grow in environments with moderately high salt concentrations and a pH of 8.4 or less. If such bacteria contaminate the culture system of the present invention, they may consume 3-hydroxybutyric acid or a salt thereof secreted by the halophilic bacteria belonging to the genus Halomonas as a substrate for lactic acid fermentation, and further decrease the pH of the medium.

[0058] Therefore, in the present invention, in order to culture halophilic bacteria belonging to the genus Halomonas without sterilizing the medium and / or in a non-sterile environment and secrete 3HB into the medium, it is preferable to adjust and maintain the pH of the medium at about 8.5 or higher during the microaerobic culture process.

[0059] The timing of adjusting the pH is not particularly limited as long as it is after the aerobic culture step, but it is preferably after the amount of PHB accumulated in the cells of the halophilic bacterium has become approximately constant.

[0060] [Fractionation process] The fractionation step in the production method of the present invention is a step of subjecting the medium after the removal step described below or the medium after the microaerobic culture step to molecular weight fractionation.

[0061] The molecular weight fractionation treatment is not particularly limited as long as it can separate a low molecular weight fraction containing 3-hydroxybutyric acid from a high molecular weight fraction containing endotoxin, and an example of such a method is a method using an ultrafiltration membrane.

[0062] [Removal process] The removal step in the production method of the present invention is a step of removing bacterial cells from the medium after the microaerobic culture step or from the high molecular weight fraction obtained in the fractionation step.

[0063] In the removal step, a known method is applied to the medium obtained after the microaerobic culture step is stopped or to the high molecular weight fraction obtained through the fractionation step, and the medium or the high molecular weight fraction is separated from the halophilic bacterial cells, thereby removing the bacterial cells from the medium or the high molecular weight fraction. For example, when the aerobic culture step and the microaerobic culture step are performed using a liquid medium, the culture in the microaerobic culture step is stopped, and the medium and the halophilic bacterial cells are separated by a separation means, thereby removing the bacterial cells from the medium.

[0064] Specific separation techniques that can be used include known solid-liquid separation procedures such as centrifugation and filtration. The method for terminating the culture is also not particularly limited. For example, a method in which the halophilic bacteria are sterilized by heating, acid treatment, or the like after the microaerobic culture step to terminate the culture, or a method in which the halophilic bacteria are separated from the medium by solid-liquid separation, thereby ultimately terminating the culture, can be used. [Example]

[0065] The present invention will be described in more detail below with reference to examples, although it goes without saying that the present invention is not limited to these examples.

[0066] 1. Culture medium for KM-1 strain The KM-1 strain was cultured in a medium based on SOT Modified 5 (Spirulina platensis Medium Modified 5) shown in Table 1. This medium was Spirulina platensis Medium (National Institute for Environmental Studies website), and the amounts of NaHCO3 and Na2CO3 were adjusted, and the nitrogen source, NaNO3, was increased five-fold, and the phosphorus source, K2HPO4, was increased four-fold. The pH of the medium after adjustment was 9.4 ± 0.1, and it was used as is without sterilization procedures such as autoclaving.

[0067] [Table 1]

[0068] During the culture, the above medium was supplemented with a 26% aqueous sucrose solution.

[0069] 2. Sample Solution Preparation 50 L of the medium supplemented with the above sucrose aqueous solution was placed in a 90 L jar fermenter, and Halomonas sp. KM-1 strain was aerobically cultured for 40 hours (aerobic culture process). After that, aeration was stopped, and microaerophilic culture was performed for 5 hours while adjusting the pH, and PHB accumulated within the cells was secreted outside the cells as 3HB (microaerobic culture process). Subsequently, the cells were removed from the culture solution using a microfiltration membrane. Next, the culture solution after cell removal was subjected to molecular weight fractionation using an ultrafiltration membrane, and fractionated into low molecular weight fractions and high molecular weight fractions, and the resulting high molecular weight fraction was obtained as an immunostimulant (hereinafter referred to as "stock solution"). The resulting stock solution was then added to 10 6 double, 10 5 double, 10 4 double, 10 3 double, 10 2 Six sample solutions A to F were prepared by diluting them 10 times and 10 times.

[0070] 3. Endotoxin Concentration Measurement The endotoxin concentration of the stock solution obtained as described above was measured using an LAL reagent (Pyrochrome (registered trademark) manufactured by Seikagaku Corporation) according to the procedure described in the attached instruction manual. As a result, the endotoxin concentration was 1 × 10 6 Therefore, the endotoxin concentrations of sample solutions A to F, which were obtained by diluting the stock solution, were 1 EtIU / mL, 1 x 10 EtIU / mL, and 1 x 10 2 EtIU / mL, 1 × 10 3 EtIU / mL, 1 × 10 4 EtIU / mL, 1 × 10 5 The mass / volume concentration of endotoxin in the stock solution is 100 μg / mL.

[0071] 4. Production induction confirmation test In the production induction confirmation test, J774.1 cells, a monocyte-macrophage-like cell line obtained from the JCRB Cell Bank, were used.

[0072] J774.1 cells were subcultured in DMEM medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin. T25 culture flasks were used for the culture, and 0.5 to 1 × 10 cells were added every 3 or 4 days. 5 The cells were subcultured at 1000 cells / mL and cultured in an incubator at 37°C in a 5% CO2 atmosphere.

[0073] Cells precultured in a T25 culture flask were detached from the flask wall by pipetting, and the resulting cell suspension was transferred to a 50 mL conical tube. The conical tube was then centrifuged (1000 rpm, 5 minutes) at room temperature, the supernatant was discarded by decantation, and the precipitated cells were recovered. After tapping, the cells were dissociated, 5 mL of the above-mentioned DMEM medium was added, and the cells were uniformly suspended by pipetting to obtain a cell suspension, which was used to confirm production induction. A portion (11 μL) of the resulting cell suspension was transferred to another conical tube, 11 μL of 0.5% trypan blue was added, and the number of cells per mL of cell suspension was counted using a hemocytometer to calculate the viability. Cell viability was confirmed to be high (95.7%).

[0074] Next, based on the measured cell number, the cell suspension was diluted with the above DMEM medium to a concentration of 1.6 × 10 6 The cell suspension was then dispensed in 100 μL portions into each well of a 96-well flat-bottom plate, and cultured at 37°C in a 5% CO2 incubator for 3 hours until the cells adhered to the bottom of the wells and spread.

[0075] Next, 100 μL of each of the sample solutions A to F prepared as described above was dispensed into separate wells. After dispensing, sample solutions A to F were incubated for 24 hours in an incubator at 37°C in a 5% CO2 atmosphere. After incubation, 150 μL of the supernatant from each well was collected in a 1.5 mL tube as a measurement sample, and these were subjected to ELISA measurements. Furthermore, an LDH assay was performed using the collected supernatant to confirm that the ELISA results were not affected by cell damage. Furthermore, 50 μL of the above-mentioned DMEM medium was added to the flat-bottom plate after supernatant collection, and an MTS assay was performed to confirm that the ELISA results were not affected by cell growth inhibition.

[0076] The amount of interleukin-12 (p40) produced was measured using an ELISA kit (BioLegend IL-12 / IL-23 (p40) ELISA MAX™ Deluxe Sets) according to the procedure described in the attached instruction manual. For the measurements, the supernatant collected from each well was diluted 2-fold and 50-fold with diluent.

[0077] Prior to measuring the measurement samples, we confirmed that it was possible to evaluate whether an unknown substance has the ability to induce interleukin 12 production by measuring the amount of interleukin 12 produced by J774.1 cells. Specifically, six control test sample solutions A' to F' were prepared with different concentrations of lipopolysaccharide (LPSp; derived from Pantoea agglomerans), which is known to induce interleukin 12 production. The amount of interleukin 12 produced by J774.1 cells using these solutions was measured by ELISA, and it was confirmed that there was a concentration- (volume-) dependent increase in interleukin 12 production, as shown in Table 2 below.

[0078] [Table 2]

[0079] Table 3 shows the results of ELISA measurements for sample solutions A to F. As can be seen from Table 3, the amount of interleukin-12 produced increases as the dilution ratio of the sample solution decreases (the concentration increases). This confirms that the addition of a processed culture medium obtained by culturing Halomonas sp. KM-1, a basophilic bacterium belonging to the Halomonas genus, induces the production of interleukin-12 by macrophages. Furthermore, it was revealed that adding 100 pg / mL of the processed culture medium to the culture of a monocyte-macrophage-like cell line induces the production of interleukin-12 (p40) at levels of 30 pg / mL or more.

[0080] [Table 3]

Claims

1. It contains endotoxin derived from halophilic bacteria belonging to the genus Halomonas, and induces the production of interleukins. The immunostimulant, wherein the halophilic bacterium is Halomonas sp. KM-1 strain.

2. The immunostimulant according to claim 1, which induces the production of interleukin by macrophages.

3. The immunostimulant according to claim 1 or 2, wherein the interleukin is interleukin-12.

4. A method for producing an immunostimulant that contains endotoxin derived from a halophilic bacterium belonging to the genus Halomonas and induces interleukin production, comprising: an aerobic culturing step of aerobically culturing the halophilic bacterium belonging to the genus Halomonas in a medium; a microaerobic culture step of microaerophilically culturing the halophilic bacterium in a medium while adjusting the pH after the aerobic culture step; a removal step of removing bacterial cells from the medium after the microaerobic culture step; a fractionation step of subjecting the medium after the removal step to molecular weight fractionation; and a recovery step of recovering the high molecular weight fraction obtained in the fractionation step.

5. A method for producing an immunostimulant that contains endotoxin derived from a halophilic bacterium belonging to the genus Halomonas and induces interleukin production, comprising: an aerobic culturing step of aerobically culturing the halophilic bacterium belonging to the genus Halomonas in a medium; a microaerobic culture step of microaerophilically culturing the halophilic bacterium in a medium while adjusting the pH after the aerobic culture step; A fractionation treatment step of subjecting the medium after the microaerobic culture step to molecular weight fractionation treatment; a removal step of removing bacterial cells from the high molecular weight fraction obtained in the fractionation step; and a recovery step of recovering the high molecular weight fraction after the removal step.

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