JP2008179549A - Filamentous fungus-derived immunostimulant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high activity immunostimulator at a low cost and in a large amount.
An immunostimulant comprising a defatted microbial cell of a filamentous fungus belonging to the genus Mucor or Mortierella or an extract residue or extract thereof.
[Selection figure] None

Description

  The present invention relates to an immunostimulant obtained from fungal cells.

  Conventionally, extracts obtained from bacteria and fungi have been examined for composition and physiological activity. Proteins, polysaccharides, glycoproteins and the like are known as compositions. In terms of function, in addition to use as a thickener such as xanthan gum, an immunostimulatory effect is widely known. As a remarkable immunostimulant, an extract containing polysaccharides derived from basidiomycetes such as Agaricus and Maitake is widely used.

  Hot water extracts derived from basidiomycetes such as maitake and agaricus are widely used as functional agents having an immunostimulatory effect (see Patent Documents 1 and 2), but when using fruit bodies (mushrooms) as raw materials Takes a long time to form a fruiting body and is not productive. In addition, basidiomycetes also have a low growth rate when using mycelium by liquid culture. Therefore, the productivity is very poor and the product price is expensive.

  Β-glucan derived from Aspergillus has been confirmed to promote the production of cytokine interleukin-8 (IL-8) (see Patent Document 3), but it requires oxidative extraction under alkaline conditions in the production process. Yes, the manufacturing process is complicated.

  In the extract derived from Rhizopus, mitogenic activity (lymphocyte proliferation promoting action → immunostimulation) has been confirmed (refer to Patent Document 4), but the culture bed is limited to Husmar barley and is a solid culture. Slow growth and poor productivity.

  In addition, a method for obtaining unsaturated fatty acid-containing phospholipids from bacteria belonging to the genus Mucor or Conidiobolus by extraction with polar solvents and nonpolar solvents has been described (see Patent Document 5). It has not been known that a highly active immunostimulator can be obtained from residual defatted cells.

JP 09-238697 Japanese Patent Laid-Open No. 02-211847 JP 2003-176304 A JP 2001-078796 Japanese Patent Laid-Open No. 02-16989

  An object of the present invention is to provide a highly active immunostimulator at a low cost and in a large amount.

  The present invention provides an immunostimulant comprising a defatted microbial cell of a filamentous fungus belonging to the genus Mucor or Mortierella, or an extract residue or extract thereof. More specifically, the present invention is characterized by comprising a defatted microbial cell obtained by subjecting a fungal cell belonging to the genus Mucor or Mortierella to extraction with a nonpolar solvent, or an extraction residue or extract thereof. An immunostimulant is provided.

That is, the gist of the present invention is as follows.
(1) An immunostimulant comprising a defatted fungus body of a filamentous fungus belonging to the genus Mucor or Mortierella, or an extraction residue or extract thereof.
(2) The immunostimulant according to (1), comprising a defatted microbial cell obtained by extracting microbial cells belonging to the genus Mucor or Mortierella with a nonpolar solvent.
(3) It contains a polar solvent extraction residue obtained by further extracting a defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent, and further extracting with a polar solvent. The immunostimulant according to (1), characterized in that it is characterized.
(4) A defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent or a polar solvent extraction residue obtained by extracting the defatted microbial cell with a polar solvent The immunostimulant according to (1), comprising a hot water extract.
(5) A defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent, and further comprising a hot water extraction residue obtained by hot water extraction treatment The immunostimulant according to (1).
(6) Polarity of a defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent or a hot water extract obtained by subjecting the defatted microbial cell to a hot water extraction treatment The immunostimulant according to (1), comprising a solvent extract.
(7) characterized in that it comprises an extraction residue obtained by subjecting a defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent to a polar solvent and hot water extraction treatment. The immunostimulant according to (1).
(8) The immunostimulant according to any one of (1) to (7), wherein the filamentous fungus belonging to the genus Mucor is Mucor circinelloides.
(9) The immunostimulant according to any one of (1) to (7), wherein the filamentous fungus belonging to the genus Mortierella is Mortierella isabellina.
(10) A medicament comprising the immunostimulator according to any one of (1) to (9).
(11) An external preparation containing the immunostimulator according to any one of (1) to (9).
(12) A food containing the immunostimulator according to any one of (1) to (9).
(13) A food or drink comprising the immunostimulator according to any one of (1) to (9).

Non-lipid lipids removed from cells belonging to the genus Mucor and Mortierella of the present invention, hot water extracts from defatted cells, etc. are used for tumor cell cytotoxicity tests and cytokine productivity using human lymphocytes. A high immunostimulatory effect is shown in the evaluation. Specifically, the production of IL-12 and TNF-α was remarkably improved by the immunostimulant composed of the nonpolar solvent extraction residue, and the tumor cell damage activity was improved. In addition, immunostimulators consisting of precipitates (polysaccharides, glycoproteins, etc.) obtained by adding ethanol after hot water extraction also significantly improve IL-12 and TNF-α production, and also have tumor cell damage activity Improved.
The genus Mucor and Mortierella according to the present invention can grow at a high concentration in a short period of time under liquid culture conditions, so that the productivity of the cells is very high. Thereby, it is possible to supply a highly active immunostimulant cheaply and in large quantities.
Moreover, the immunostimulant of this invention can be utilized for a pharmaceutical, an external preparation, a foodstuff, food-drinks, etc.

Hereinafter, the present invention will be described in detail.
The immunostimulant derived from the genus Mucor or Mortierella of the present invention can be obtained according to the following procedure (see FIG. 1).

1. Culture of Mucor or Mortierella filamentous fungi Microbes belonging to the genus Mortierella include Mortierella isabellina, Mortierella ramaniana va
r.angrispora), specifically, for example, Mortierella isaverina IFO7824 and Mortierella Ramaniana IFO8187. These strains can be obtained from the Biological Genetic Resource Center (NBRC: 2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture).
Examples of microorganisms belonging to the genus Mucor include Mucor circinelloides and Mucor javanicus, and specific examples include Mucor silcineroides HUT1121 and Mucor Javanics HUT1162. In the present invention, mucor silcineroides HUT1121 is particularly preferred. Mucor Silcineroides HUT1121 was deposited on April 30, 1987 at National Institute of Advanced Industrial Science and Technology Patent Biology Depositary Center (1st, 1st, 1st East, 1st Street, Tsukuba, Ibaraki Prefecture) with accession number FERM P-9359 After that, it was transferred to an international deposit under the Budapest Treaty, and was given a deposit number of FERM BP-3883. On April 30, 1987, Mucor Javanics HUT1162 was registered with the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture). Ferm P-9360 And then transferred to an international deposit based on the Budapest Treaty and assigned a deposit number of FERM BP-3884.

  The medium for culturing the microorganism is not particularly limited as long as the microorganism can grow well, and a medium containing components such as a carbon source, a nitrogen source, inorganic salts and, if necessary, an amino acid suitable for the growth of the microorganism is used. . As the carbon source, saccharides such as glucose, starch and molasses, organic acids, sodium acetate and the like can be used, and saccharides such as glucose are particularly suitable. Examples of the nitrogen source include ammonium salt, yeast extract, corn steep liquor, and peptone. Examples of the inorganic salt include magnesium salt, calcium salt, phosphate, iron salt, and copper salt.

  Other culture conditions, such as temperature and culture time, may be set to preferable conditions in consideration of the culture medium composition and the productivity of the target cells. Usually, 20 to 35 ° C., preferably 25 to 30 ° C., pH is 3 to 7, preferably 3.5 to 6.5, for 72 hours to 240 hours, preferably 96 hours to 168 hours.

2. Cell collection, crushing treatment, and dry cell collection can be performed by centrifugation, filtration, or the like. After the collection of the cells, the cells are preferably crushed. This crushing step is effective in improving the removal efficiency of nonpolar lipids by the nonpolar solvent in the next step and improving the efficiency of hot water extraction from defatted cells. Crushing of cells is usually performed mechanically, and can be performed by, for example, a French press, an ultrasonic crusher or the like (see Japanese Patent Application Laid-Open No. 5-17796). After the pulverization treatment, the microbial cells are preferably dried. The cells can be dried by, for example, a freeze dryer, a vacuum dryer, an air dryer, or the like (see JP-A-5-17796).

3. Removal of non-polar lipids from bacterial cells by non-polar solvent extraction The first immunostimulant of the present invention is a defatted product obtained by extracting fungal cells belonging to the genus Mucor or Mortierella with a non-polar solvent. It is an immunostimulant characterized by containing microbial cells (hereinafter referred to as immunostimulant 1).
Examples of the nonpolar solvent used include n-hexane and n-heptane. In particular, n-hexane is preferable from the viewpoint of extraction rate and food application.
Extraction using a solvent is not particularly limited, but it is particularly preferable to perform extraction using a packed column (column). Extraction and recovery using a packed column is performed, for example, by packing dehydrated crushed cells into a cylindrical packed column, allowing the extraction solvent to flow down from the top, collecting the solvent flowing out from the bottom of the packed column, and concentrating. (See JP-A-5-17796). The extraction conditions when using hexane are usually 5 to 60 ° C., preferably 40 to 60 ° C., 0.5 to 12 hours, preferably 1 to 6 hours.
By nonpolar solvent extraction, nonpolar lipids accumulated in the fungus bodies of the genus Mucor and Mortierella can be removed. When this lipid is oxidized by oxygen in the air, it becomes a lipid peroxide exhibiting a specific odor and harmfulness. Therefore, removal of the lipid reduces problems of odor and harmfulness.

4). Extraction of the defatted microbial cells in 3 with a polar solvent The second immunostimulant of the present invention is a defatted microbial cell obtained by subjecting fungal cells belonging to the genus Mucor or Mortierella to extraction with a nonpolar solvent. Is an immunostimulant characterized by further containing a polar solvent extraction residue obtained by extraction with a polar solvent (hereinafter referred to as immunostimulator 2).
Examples of the polar solvent used include chloroform / methanol mixed solution, acetone, methanol, ethanol and the like. In particular, ethanol is desirable from the viewpoint of application to food.
Extraction using a solvent can be performed under ordinary conditions by a method similar to the nonpolar solvent extraction method described above. The extraction conditions when ethanol is used are usually 5 to 60 ° C., preferably 40 to 60 ° C., 0.5 to 12 hours, preferably 1 to 6 hours.
Extraction with polar solvent makes it possible to remove colored components and components having a specific odor contained in the defatted cells (decolorization and deodorization of the defatted cells). Moreover, since polar lipids are also extracted, the oxidation stability is further improved.

5. Extraction of the defatted microbial cell according to 3 and the polar solvent extraction residue according to 4 with hot water The third immunostimulant of the present invention is a nonpolar solvent for microbial cells belonging to the genus Mucor or Mortierella. It is an immunostimulant characterized in that it contains a hot water extract of a polar solvent extraction residue obtained by extracting a defatted microbial cell obtained by extraction with a polar solvent or the defatted microbial cell (hereinafter referred to as immunity). Called activator 3).
Extraction with hot water is performed at 50 to 135 ° C. for 15 minutes to 3 hours, for example. In order to perform in a short time, extraction is performed at 100 ° C. or higher under pressure, for example, at about 120 ° C. under 1 to 2 atm using a pressure cooker for about 30 minutes to about 1 hour (see Japanese Patent Laid-Open No. 9-238697).

6). Drying of the hot water extract obtained in 5 above After adding 2 to 5 times the amount of ethanol to the hot water extract and precipitating it, the precipitate may be collected and then dried by spray drying or freeze drying. Is possible.

7). Drying of Hot Water Extraction Residue After Hot Water Extraction The fourth immunostimulant of the present invention comprises a defatted microbial cell obtained by extraction of a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent. Furthermore, it is an immunostimulant characterized in that it contains a hot water extraction residue obtained by hot water extraction treatment (hereinafter referred to as immunostimulator 4).
The fifth immunostimulant of the present invention is obtained by subjecting a defatted microbial cell or a defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent to a hot water extraction treatment. It is an immunostimulant characterized by including a polar solvent extract of the obtained hot water extract (hereinafter referred to as immunostimulant 5). Although nonpolar lipid is removed by the said process 3, the immunostimulant 5 which is a polar solvent extract contains glycolipid and another polar lipid other than phospholipid.
The sixth immunostimulant of the present invention is obtained by subjecting a defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent to a polar solvent and hot water extraction treatment. It is an immunostimulant characterized by containing an extraction residue (hereinafter referred to as immunostimulant 6).
The conditions such as extraction with a polar solvent and extraction with hot water are the same as in Steps 4 and 5 above.
Drying of the hot water extraction residue can be performed by, for example, a freeze dryer, a vacuum dryer, an air dryer, or the like (see JP-A-5-17796).

8). Measurement of immunostimulatory activity The immunostimulatory activity can be measured, for example, according to the method described in Japanese Patent No. 3565741.
The following items are listed as immunostimulation measurement items.
(1) Examination using lymphocytes in human blood ・ Measurement of tumor cell dysfunction ・ Measurement of cytokine (I): IL-12
・ Cytokine measurement (II): TNF-α
(2) Examination using mouse-derived macrophage-like cell line ・ Cytokine measurement (I): IL-12
・ Cytokine measurement (II): TNF-α

  The immunostimulant of the present invention obtained according to the above procedure has autologous tumor cytotoxicity (ATK) activity and the ability to produce IL-12 and TNF-α, as shown in the following examples. IL-12 is a cytokine that has an action of activating natural killer cells, has an action of proliferating and activating cytotoxic T cells (killer T cells), and an interferon that promotes activation of killer T cells. It is also known to enhance gamma production.

  Therefore, the immunostimulant of the present invention is effective for the treatment or prevention of infections caused by bacteria or viruses, diseases such as leukemia, diseases in which immunity is reduced, tumors and the like. Inflammatory cytokines typified by TNF-α are reported to be released mainly from macrophages and exhibit antitumor effects and the like. Therefore, the immunostimulant of the present invention can be used as an immunostimulant, an antitumor agent, an IL-12 or TNF-α production promoter and the like for the purpose of treating or preventing these diseases. Moreover, the immunostimulant of this invention can be administered to a patient for the purpose of the treatment or prevention of these diseases.

  The immunostimulant of the present invention is useful in immunotherapy. Specifically, after collecting blood from a patient, the immunostimulant of the present invention is used to activate lymphocytes separated from blood, Can be returned.

The medicament containing the immunostimulant of the present invention may be administered as it is or in a mixture with a pharmacologically acceptable carrier according to known means generally used in the production method of pharmaceutical preparations. Can do.
Examples of pharmacologically acceptable carriers include excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, buffers in liquid formulations. And soothing agents.
For the formulation of the medicament of the present invention, various components usually used for formulation are optionally used. Examples thereof include starch, dextrin, lactose, corn starch, inorganic salts and the like.

Examples of the pharmaceutical dosage form containing the immunostimulant of the present invention include ampoules, tablets, capsules, granules, fine granules, powders, infusions, drinks, etc. It is not limited to.
The preferable content of the immunostimulant in the preparation is 0.1 to 80% by weight, more preferably 1 to 5% by weight, based on the total amount of the preparation.

  The administration method of the immunostimulant of the present invention is not particularly limited, and examples thereof include oral administration and intravenous administration. The preferred dose varies depending on the administration subject, target organ, symptom, administration method and the like, and is not particularly limited. For example, for a patient (weight 60 kg), about 0.1 to 100 mg, preferably about 1. It is 0 to 50 mg, more preferably about 1.0 to 20 mg.

The immunostimulant of the present invention is a health food having high immunostimulatory activity, a dietary supplement, and a nutritional composition by being blended with raw materials generally used as foods, such as proteins, lipids, carbohydrates, vitamins and the like. Can also be used.
A food can be produced by mixing the immunostimulant of the present invention with a raw material usually used for food.
The amount of the immunostimulant contained in the food of the present invention is not particularly limited and may be appropriately selected. For example, it is 0.1 to 50% by mass, preferably 1 to 10% by mass in the food. Good.
Moreover, the immunostimulant of this invention can be used also as food and drink, animal feed, pet food, health food for pets, etc.
The immunostimulant of this invention can be contained in skin external preparations, such as cosmetics, a pharmaceutical, and a quasi-drug. Cosmetics include emulsion, soap, facial cleanser, bath preparation, cream, emulsion, lotion, eau de cologne, shave cream, shave lotion, cosmetic oil, sunscreen / sunscreen lotion, funny powder, foundation, perfume, pack , Nail cream, enamel, enamel remover, eyebrow, blusher, eye cream, eye shadow, mascara, eyeliner, lipstick, lip balm, shampoo, rinse, hair dye, dispersion, detergent and the like. Examples of pharmaceuticals or quasi drugs include pharmaceuticals such as ointments, creams, and external liquids.
In addition, the external preparation for skin of the present invention contains ingredients, oils, higher alcohols, fatty acids, ingredients blended in external preparations for skin such as cosmetics and quasi-drugs in addition to the above-mentioned physiologically active composition. Ultraviolet absorbers, powders, pigments, surfactants, polyhydric alcohols / sugars, polymers, physiologically active substances, solvents, antioxidants, fragrances, preservatives, and the like can be blended.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, it goes without saying that the present invention is not limited to these examples.

Example 1: Mucor filamentous fungus culture and production of defatted dry cells 18 L of a medium having the following composition was placed in a 30 liter jar fermenter, and this medium was inoculated with Mucor cilcineroides HUT1121 strain (FERM BP-3883). The culture was aerated and stirred at 30 ° C. for 4 days.

Glucose 100g / L
Ammonium sulfate 6.6g / L
Monopotassium phosphate 4.5g / L
Magnesium sulfate heptahydrate 0.5g / L
Yeast extract 0.3g / L
Trace metals, ferrous sulfate, heptahydrate 20 mg / L
・ Calcium chloride ・ Dihydrate 2.4 mg / L
・ Copper sulfate ・ pentahydrate 0.4 mg / L
・ Zinc sulfate ・ 7 water salt 2.0 mg / L
Manganese chloride tetrahydrate 2.0 mg / L

After the main culture, the culture solution was filtered to recover the cells. The collected cells are uniformly dispersed and suspended in water to a concentration of 10%, and then, using a crusher (Microfluidizer M110Y type, manufactured by Microfluidizer), 700 kg / cm ² It was crushed by pressure. Thereafter, the cells were lyophilized to obtain dry cells.
10 g of this dried cell was packed in a glass column having an inner diameter of 20 mm, and 100 ml of hexane was allowed to flow from the top. Thereafter, the cells were collected and 6.6 g of defatted and dried cells from which hexane was removed by drying under reduced pressure was obtained.

Example 2: Polar solvent extract 50 ml of ethanol was added to 5 g of the defatted and dried cells obtained in Example 1 above, followed by extraction at 40 ° C for 1 hour. After the extract was filtered, 50 ml of ethanol was added again to the residue and extraction was performed in the same manner. This was filtered, and the obtained extract filtrate was combined with the first extract filtrate. By removing ethanol from the filtrate by a vacuum drying method, 0.9 g of an ethanol extract (polar solvent extract) was obtained.

Example 3: Polar solvent extraction residue The residue after ethanol extraction obtained in Example 2 was dried under reduced pressure to obtain 4.0 g of ethanol extraction residue (polar solvent extraction residue).

Example 4: Hot water extract 50 ml of deionized water was added to 5 g of the defatted and dried cells obtained in Example 1 above, and an extraction treatment was performed at 60 ° C for 1 hour. After the extract was filtered, 50 ml of water was added again to the residue, and extraction was performed in the same manner. This was filtered, and the obtained extract filtrate was combined with the first extract filtrate. To this solution was added 4 volumes of ethanol (400 ml) and allowed to stand at 20 ° C. for 1 hour. Thereafter, the precipitate was separated by centrifugal separation and freeze-dried to obtain 0.8 g of a hot water extract.

Example 5: Hot water extraction residue The residue obtained by hot water extraction in Example 4 was freeze-dried to obtain 4 g of hot water extraction residue.

Example 6: Extraction residue using polar solvent and hot water The polar solvent extraction residue obtained in Example 3 was subjected to hot water extraction in the same manner as in Example 4, and the resulting residue was lyophilized. As a result, 3.0 g of an extraction residue using a polar solvent and hot water was obtained.

Example 7: Study on improvement of tumor cytotoxicity using human lymphocytes
1) Details of the test In order to test the immunostimulatory effect of the defatted dry cells, hot water extract, and hot water extraction residue, ATK-inducing activity was measured as an immunoassay in 5 subjects. In addition, ATK-inducing activity was also measured for picibanil, which is known to have an immunostimulatory effect.
2) Inspection method Various samples were dissolved in the culture medium, and then diluted and adjusted to the following concentrations. All samples including the control contained 0.5% DMSO at the time of culture. The concentration of picibanil was 0.05 KE / ml.
Here, Pisibanil 1KE means that it corresponds to 2.8 mg of Streptococcus pyogenes (A group 3 type) Su strain penicillin-treated lyophilized powder and 0.1 mg as dried cells. The freeze-dried powder contains magnesium sulfate, DL-methionine, maltose, and benzylpenicillin potassium as additives.

Delipidated and dried cells of Example 1: 10 μg / ml, 100 μg / ml, 250 μg / ml
Polar solvent extract of Example 2: 10 μg / ml, 100 μg / ml, 250 μg / ml
Polar solvent extraction residue of Example 3: 10 μg / ml, 100 μg / ml, 1000 μg / ml
Hot water extract of Example 4: 10 μg / ml, 100 μg / ml, 250 μg / ml
Hot water extraction residue of Example 5: 10 μg / ml, 100 μg / ml, 1000 μg / ml
Extraction residue using polar solvent and hot water of Example 6: 10 μg / ml, 100 μg / ml, 1000 μg / ml

Blood was collected from the subjects and lymphocytes were separated. After adding the above sample to lymphocytes, C
Incubate overnight in O ₂ incubator.
On the next day, the cultured lymphocytes and cancer cells K562 labeled with a labeling substance (radiochrome 51) that elutes when injured are in a lymphocyte-to-cancer cell ratio of 40: 1, 20: 1, 10: 1, 5 respectively. 1 was mixed to determine how much cancer cells were damaged by the elution amount of the labeling substance (see Japanese Patent No. 3655741).

The degree of injury activity was calculated according to the formula and was defined as ATK-inducing activity.
ATK-inducing activity is determined by the following formula. The official maximum release amount indicates the amount of labeling substance released when all cancer cells are artificially destroyed, and the spontaneous release amount indicates the amount of background labeling substance released when lymphocytes are not added.
ATK-inducing activity (%) = (labeled substance released from damaged cancer cells-spontaneous release)
/ (Maximum release amount-spontaneous release amount) x 100

In addition, ATK-inducing activity of only lymphocytes to which no health food was added was measured and used as a control when evaluating the immunostimulatory effect.
The results are shown in Table 1.

Example 8: Examination of improvement of cytokine production ability in vitro using human lymphocytes
1) Examination content In order to examine the immunostimulatory effect of the following 6 types of samples, cytokine production ability was measured in 5 subjects. In addition, cytokine production ability was also measured for picibanil, which is known to have an immunostimulatory effect.
2) Inspection method Various samples were dissolved in the culture medium, and then diluted and adjusted to the following concentrations. All samples including the control contained 0.5% DMSO at the time of culture. The concentration of picibanil was 0.05 KE / ml.

Delipidated and dried cells of Example 1: 10 μg / ml, 100 μg / ml, 250 μg / ml
Polar solvent extract of Example 2: 10 μg / ml, 100 μg / ml, 250 μg / ml
Polar solvent extraction residue of Example 3: 10 μg / ml, 100 μg / ml, 1000 μg / ml
Hot water extract of Example 4: 10 μg / ml, 100 μg / ml, 250 μg / ml
Hot water extraction residue of Example 5: 10 μg / ml, 100 μg / ml, 1000 μg / ml
Extraction residue using polar solvent and hot water of Example 6: 10 μg / ml, 100 μg / ml, 1000 μg / ml

Blood collected from the subject is placed in a centrifuge tube, diluted twice with phosphate buffer solution (PBS), transferred to a plurality of lymphoprep tubes for blood separation, centrifuged at room temperature, and the lymphocyte layer is centrifuged. Collect in a tube, wash with PBS, centrifuge and discard the supernatant. After repeating this operation three times, the plate was further washed twice with PBS at a low temperature, the RPMI of the culture solution was added, the concentration was measured, and the concentration was adjusted to 4 × 10 ⁶ lymphocytes / ml. This concentration measurement was performed using a hemocytometer and was measured as accurately as possible to obtain a lymphocyte culture stock solution. This stock solution was diluted as necessary. Dispensing 0.1 ml each of these adjusted liquids into a plurality of tubes, adding an equal amount of RPMI as a blank, standing on a tube stand, storing in a CO ₂ incubator, and culturing overnight (see Japanese Patent No. 3656551) .
The supernatant after 24 hours of culture was collected, and the amount of cytokine (IL-12 and TNF-α) produced was measured using a cytokine measurement kit.
IL-12 and TNF-α were measured using a human IL-12 ELISA kit and a human TNF-α ELISA kit (both from Biosource International).
The results are shown in Tables 2 and 3.

Example 9: Examination of cytokine production improvement using a mouse-derived macrophage-like cell line
1) Examination contents Cytokines (IL-12 and TNF-α) were measured in order to examine the macrophage activation ability of six types of samples.
2) Inspection method Various samples were dissolved in the culture medium, and then diluted and adjusted to the following concentrations. All samples contained 0.5% DMSO during culture.

Delipidated and dried cells of Example 1: 10 μg / ml, 100 μg / ml, 250 μg / ml
Polar solvent extract of Example 2: 10 μg / ml, 100 μg / ml, 250 μg / ml
Polar solvent extraction residue of Example 3: 10 μg / ml, 100 μg / ml, 1000 μg / ml
Hot water extract of Example 4: 10 μg / ml, 100 μg / ml, 250 μg / ml
Hot water extraction residue of Example 5: 10 μg / ml, 100 μg / ml, 1000 μg / ml
Extraction residue using polar solvent and hot water of Example 6: 10 μg / ml, 100 μg / ml, 1000 μg / ml

Samples were added to the mouse-derived macrophage-like cell line J774.1 (1.5 × 10 ⁵ cells / well) and allowed to react for a certain period of time.
Cytokine (IL-12 and TNF-α) production was measured by collecting the supernatant after 24 hours of culture and using a cytokine measurement kit.
The results are shown in Tables 4 and 5.

  According to the present invention, a highly active immunostimulator can be provided. Therefore, the present invention relates to an immunostimulatory food, health food, food and drink, medicine, medical treatment (immunotherapy: lymphocytes are activated and returned to the body after blood collection), pet food, pet health food, pet medicine. It can be applied to animal feeds, external preparations including cosmetics, and the like.

The outline of the preparation procedure of the immunostimulant of this invention is shown.

Claims (13)

An immunostimulant comprising a defatted microbial cell of a filamentous fungus belonging to the genus Mucor or Mortierella or an extraction residue or extract thereof. 2. The immunostimulant according to claim 1, comprising a defatted microbial cell obtained by extraction of a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent. It contains a polar solvent extraction residue obtained by further extracting a defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent, and further extracting with a polar solvent. The immunostimulant according to claim 1. Extraction of filamentous fungi belonging to the genus Mucor or Mortierella with a nonpolar solvent or hot water extraction of polar solvent extraction residues obtained by extracting the defatted cells with a polar solvent The immunostimulant according to claim 1, comprising a product. A defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent, further comprising a hot water extraction residue obtained by hot water extraction treatment, The immunostimulant according to claim 1. Polarized solvent extract of defatted cells obtained by extracting fungal cells belonging to the genus Mucor or Mortierella with a nonpolar solvent or a hot water extract obtained by extracting the defatted cells with hot water The immunostimulant according to claim 1, comprising: It comprises an extraction residue obtained by subjecting a defatted microbial cell obtained by extracting a fungal cell belonging to the genus Mucor or Mortierella with a nonpolar solvent to a polar solvent and hot water extraction, Item 12. The immunostimulator according to Item 1. The immunostimulant according to any one of claims 1 to 7, wherein the filamentous fungus belonging to the genus Mucor is Mucor circinelloides. The immunostimulant according to any one of claims 1 to 7, wherein the filamentous fungus belonging to the genus Mortierella is Mortierella isabellina. The pharmaceutical containing the immunostimulant in any one of Claims 1-9. The external preparation containing the immunostimulant in any one of Claims 1-9. The foodstuff containing the immunostimulant in any one of Claims 1-9. Food / drink containing the immunostimulant in any one of Claims 1-9.

Images