JP2002255853A - Immunoactivating composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly stable immunoactivating composition comprising an immunoactivating ingredient readily absorbable into the human body. SOLUTION: This immunoactivating composition is characterized by comprising active BT-1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunostimulating composition,
More specifically, the present invention relates to an immunostimulatory composition characterized by containing a low molecular weight immunostimulatory substance BT-1 having excellent immunostimulating action.

[0002]

2. Description of the Related Art Immunostimulants containing lactic acid bacteria as an active ingredient have been known (JP-A-6-80575).
And JP-A No. 1-242532. Also, immunostimulatory components contained in lactic acid bacteria have been studied, and it has been reported that cell wall fractions of lactic acid bacteria and polysaccharides produced by lactic acid bacteria have immunological activity (CI Park et al., M.
ilchwissenschaft, 46 , 87 (19
91); Yasui et al. Dairy Sc
i. , 74 , 1187 (1991); Nagaok
a. Biochem. , 108 , 568 (199
0); Haruki Kitazawa et al., Research on Dairy Chemicals and Foods, 40 , A-
261, (1991)). Also, an immunostimulant containing a cytoplasmic fraction of lactic acid bacteria cells is known (Japanese Patent Laid-Open No. 5-2 / 1993).
No. 52900).

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide an immunostimulating composition containing an immunostimulating component which has high stability and is easily absorbed into the body.

[0004]

Means for Solving the Problems The present inventors conducted further studies on immunostimulatory components contained in lactic acid bacteria and metabolites thereof.
For example, a low molecular weight immunostimulating substance (hereinafter referred to as “BT-1”) contained in a disrupted lactic acid bacterium, a cytoplasmic fraction or cell wall obtained therefrom, or a culture supernatant does not exhibit an immunostimulating effect (not effective). BT-1 exerts an immunostimulatory effect when the lactic acid bacteria-treated product is subjected to the activation treatment of the present invention, for example, by separation using a gel filtration chromatograph according to the difference in molecular weight. An unexpected finding that it is induced into a state (active state) was obtained. In addition, in the conventional immunostimulant using the above-mentioned lactic acid bacteria, since the above-mentioned activation treatment was not performed, even if BT-1 was contained, it was inactive BT-1 itself. It does not contribute to immunostimulatory effects.

[0005] As a demonstration supporting the above description, more specifically, the following experimental results by the present inventors can be mentioned.
That is, when the culture supernatant of lactic acid bacteria confirmed to exhibit immunostimulatory activity was separated by ion-exchange chromatography, there were a fraction exhibiting immunostimulatory activity and a fraction not exhibiting immunostimulatory activity. When fractions showing no immunostimulatory activity were separated by difference in molecular weight using gel filtration chromatography, unexpectedly, immunostimulatory activity was observed in a low molecular weight fraction having a molecular weight of about 10 kDa or less. Further, when the culture supernatant of lactic acid bacteria confirmed to exhibit immunostimulatory activity was separated by molecular weight difference using gel filtration chromatography, a high molecular weight fraction having a molecular weight of 100 kDa or more and a molecular weight of about 10 kDa or less were obtained. The immunostimulatory activity was observed in each of the low molecular weight fractions. The strength of the immunostimulatory activity of the culture supernatant before separation by gel filtration chromatography should be the sum of the strengths of the immunostimulatory activities of the high molecular weight fraction and the low molecular weight fraction. Actually, however, only the strength of the immunostimulatory action exhibited by the high molecular weight fraction was shown. Therefore, it can be said that BT-1 contained in the culture supernatant before separation by gel filtration chromatography is in an inactive state showing no immunostimulating effect.

In addition, when a culture supernatant of lactic acid bacteria, which has been confirmed to exhibit immunological activity, is separated by molecular weight difference using gel filtration chromatography, a high molecular weight fraction exhibiting immunostimulatory activity includes: The intensity of the immune activity decreased over time. That is, it was suggested that such a high molecular weight immunostimulatory component was inferior in stability. On the other hand, in the aforementioned low molecular weight fraction, the intensity of the immunoreactivity did not decrease over time. Therefore, the active BT
It can be said that -1 is excellent in stability. Furthermore, active BT-1 is easily absorbed in the body because of its low molecular weight.
Based on such knowledge, the present inventors have further studied and completed the present invention.

That is, the present invention provides (1) active BT-1
(2) an immunostimulatory composition comprising: (2) an active BT-1-containing cell or a processed product of BT-1-containing cells; The immunostimulatory composition according to (2), wherein the (3) cells are cells obtained by culturing under conditions that induce BT-1 production; ,
(5) The immunostimulatory composition according to (3), wherein the cells are cells obtained by culturing in an AMC medium, and (5) the cells are cells. (6) The immunostimulatory composition according to (2), which is a processed product of cells obtained by culturing in an AMC medium, comprising a low molecular weight fraction having a molecular weight of 10 kDa or less. (7) the cells are Bifidobacterium bi
fidum or Bifidobacterium longum; the immunostimulatory composition according to the above (5) or (6), which comprises (8) inactive BT-1;
And extracellular inactive BT-
(1) the immunostimulatory composition, wherein (1) is in an activated state, (9) the Peyer's patch cell activating composition,
To (8), (10) Inactive BT
-1 containing cells or a processed product of the cells is separated into a low molecular weight fraction having a molecular weight of 10 kDa or less and a high molecular weight fraction having a molecular weight of 10 kDa or more, and a fraction containing a low molecular weight fraction having a molecular weight of 10 kDa or less is collected. (11) The method for producing an immunostimulatory composition according to the above (1), (11) converting an inactive BT-1 contained in a cell containing the inactive BT-1 or a processed material of the cell into an active BT. And activating the converted active BT-1 on a living body.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, "active BT-1" is used.
The term "immunostimulating component" refers to an immunostimulatory component having a molecular weight of about 10 kDa or less contained in cells. Here, the immunostimulatory component is a component that exerts an immunostimulatory effect. Specifically, when the immune activity is measured by the method described in Examples, the value (stimulation rate IS) is 3 or more. Are preferred. Also,
"Inactive BT-1" refers to BT-1 in a state where the active BT-1 cannot exert an immunostimulatory action due to the presence of an inhibitor or the like, that is, in an inactive state.

[0009] The present invention provides an immunostimulatory composition containing the above-mentioned active BT-1. Active BT-1 contained in the immunostimulatory composition has the advantage of being excellent in stability and being easily absorbed into the body because of its low molecular weight. One embodiment of the immunostimulatory composition according to the present invention includes, for example, an immunostimulatory composition containing active BT-1-containing cells or a treated product of BT-1-containing cells.

The treated BT-1 containing cells according to the present invention include, for example, (a) cells containing active BT-1 (hereinafter sometimes abbreviated as "active BT-1 containing cells"). ), Or (b) cells containing inactive BT-1 (hereinafter sometimes abbreviated as “inactive BT-1 containing cells”), or further processed cells. A treated product of an inactive BT-1 containing cell in which BT-1 is in an active state, that is, in a state capable of exerting an immunostimulatory effect by removing the BT-1 inhibitor by treatment or the like, may be mentioned.

The above-mentioned (a) treated BT-1 containing cells are those obtained by treating active BT-1 containing cells, and the treatment is not particularly limited. Specific examples of the treated product include a crushed liquid of the cells by ultrasonic waves or the like, a culture liquid or a culture supernatant of the cells, and a solid residue obtained by separating them by a solid-liquid separation means such as filtration or centrifugation. In addition, a treatment solution in which cell walls are removed by an enzyme or mechanical means, a protein complex (protein, lipoprotein, glycoprotein, etc.) or a peptide complex (peptide, glycopeptide, etc.) obtained by trichloroacetic acid treatment or salting out treatment, etc. ) And the like are also mentioned as the treated product. Furthermore, these concentrates, their dilutions, or their dried products are also included in the processed product. Further, those obtained by further treating the disrupted solution of the cells by ultrasonic waves or the like, the culture solution or the culture supernatant of the cells, such as fractions separated by various chromatography, etc., are also treated in the present invention. Included in things.

In the above (b), examples of the treated product of the inactive BT-1 containing cells include the treated products obtained by treating the inactive BT-1 containing cells as described above. Further, the treatment for bringing the inactive BT-1 containing cells or the inactive BT-1 contained in the treated product into an active state includes:
A cell containing inactive BT-1 or a processed product of the cell is treated with a low molecular weight fraction having a molecular weight of 10 kDa or less and a molecular weight of 10 kDa or less.
Separated into a high molecular weight fraction of at least Da and a molecular weight of 10 kDa
It is preferable to collect the fraction containing the following low molecular weight fraction. More specifically, a low molecular weight fraction, preferably from a processed product of inactive BT-1 containing cells, for example, a lysate obtained by sonication, a culture solution or a culture supernatant of the cells, is subjected to gel filtration chromatography. It is more preferable to collect a fraction of about 10 kDa or less or a substance containing the fraction.

The cells in the "active BT-1 containing cells" or "inactive BT-1 containing cells" of the present invention include, for example, microorganisms, insect cells, plant cells or animal cells. The microorganism may be a prokaryote such as a lactic acid bacterium or a eukaryote such as a yeast. Among them, the cells are preferably bacterial cells, and more preferably enterobacteria. Intestinal bacteria are a general term for bacteria living in the intestine, and specifically, for example, Lactobacillus acidophilus, L. casei, L. gasser
i, L. plantalum, L. delbrueckii subsp bulgaricus,
Lactobacillus genus such as L. delbrueckii subsp lactis (also called lactobacilli); for example, Leuconostoc mecenter
oides, Streptococcus (Enterococcus) faecalis, Strep
tococcus (Enterococcus) faecium, Streptococcus (Ent
erococcus) hirae, Lactococcus lactis, Streptococc
Lactococci such as us thermophilus; Bifidobacterium bifid
um, B. longum, B. breve, B. adrecentis, B. infant
genus of Bifidobacterium (also referred to as Bifidobacterium) such as is, B. pseudolongum and B. thermophirum; genus of Bacteroides such as fragilis or melaninogenicus; Genus Eubacterium; for example, Peptstreptococcus (Peptstr.
genus Enterococcus, for example, E. faecalis; Enterobacter, for example, aerogenes; Propioniba, for example, Acnes
cterium); for example, Streptococcus genus such as mutans; for example, Mitsuokella such as multi-acidida; sarsina (for example, Bentriculi); Ruminococcu
s) genus; for example, Veillon (Veillon)
ella) genus; for example, microorganisms belonging to the genus Megasphaera, such as Elsdeni bacteria.

Further, as the cell, Bifidobacterium
bifidum, B. longum, B. breve, Bifidobacterium genus such as B. infantis, Lactobacillus acidophilus, L.
Lactobacillus such as casei, L. gasseri, L. bulgaricus, or Enterococcus faecalis, Enterococcus faec
ium, Streptococcus lactis, Streptococcus thermoph
Lactococci such as ilus are more preferably used.
Most preferably, Bifidobacterium bifidum or B. longum is used. Further, among the strains of Bifidobacterium bifidum, E-139 is more preferably used. The cells can be easily obtained from an organization such as the ATCC or IFO, or the Japan Bifidobacterium Center. Also, commercially available ones can be used as appropriate.

In the present invention, the above-mentioned cells may be purified or those having a purity of a roughly purified level. Also, regardless of the state of the cells, they may be alive or dead, or may be freeze-dried. Furthermore, the above-mentioned cells may be used alone or as a mixture of two or more.

The active BT-1 containing cell according to the present invention may be any of the above-mentioned cells containing active BT-1, and among them, a cell capable of releasing active BT-1 out of the cell is preferable. . The cells that can release the active BT-1 outside the cell may be those that constantly release BT-1 outside the cell, or those that release BT-1 outside the cell under certain specific conditions (environment). You may. Thus, active BT-
In the production of the immunostimulatory composition according to the present invention, the active BT-1 is extracted from the active BT-1 containing cells or the processed product thereof in order to obtain a desired immunostimulatory effect by releasing 1 outside the cells. Since such a step is not required, an inexpensive product can be supplied. Furthermore, by ingesting or administering the cells in a living state, and culturing and growing the cells in the ingested or administered organism, active BT-
1 can also be taken or administered continuously.

The inactive BT-1 containing cell according to the present invention may be any cell containing inactive BT-1 among the above-mentioned cells. Among them, BT-1 can be released outside the cell. Are preferred. Furthermore, the released BT-1
It is more preferable that the compound be activated outside the cell, such as in a living body to which the cell is administered or ingested, for the reasons described above. However, the released BT-1 may be in an inactive state. This is because, for example, an inactive BT-1 can be activated by, for example, subjecting an extracellular solution such as a culture solution to gel filtration chromatography to extract only a low molecular weight fraction. Examples of such inactive BT-1 containing cells that release BT-1 in an inactive state include, for example, the above-mentioned Bifidobacterium such as Bifidobacterium bifidum or B. longum, Lactobacillus or Lactococci and the like.

The active BT-1-containing cells according to the present invention are not limited to cells containing BT-1 in an active state at all times, but may be used under conditions that induce BT-1 production or when BT-1 production is induced. A cell that produces BT-1 only when cultured under conditions that induce production and that contains the BT-1 in an active state may be used. In addition, inactive BT-
1-containing cells are not limited to cells that always contain BT-1 in an inactive state, and were cultured under conditions that induce BT-1 production or under conditions that induce BT-1 production. Only in such cases, the cell may produce BT-1 and contain the BT-1 in an inactive state.
More specifically, the cells, preferably enterobacteria, more preferably Bifidobacterium bifidum or B. in AMC medium.
When culturing the above-mentioned Bifidobacterium such as longum, Lactobacillus or Lactococcus, cells containing BT-1 in an inactive state and capable of releasing BT-1 out of the cell in an inactive state are obtained. can get. Here, the AMC medium contains 0.5% by weight of casamino acid, 0.5% by weight of bactitryptone, inorganic salts, amino acids, vitamins, nucleic acids, and Tween80,
pH 6.8 medium. More preferably, the AMC medium described in Examples is mentioned. Since the culture conditions vary depending on the medium and the cultured cells, they cannot be unconditionally determined. For example, when culturing Bifidobacterium bifidum using the above-mentioned AMC medium as a condition for inducing BT-1 production, about 3
It is preferable to perform stationary culture at about 7 ° C. for about 2 days.

In a more preferred embodiment of the present invention, Bifi
dobacterium bifidum, B. longum, B. breve, B. infan
Bifidobacterium such as tis, Lactobacillus acido
Lactobacillus such as philus, L. casei, L. gasseri, L. bulgaricus, or Enterococcus faecalis, Enter
ococcus faecium, Streptococcus lactis, Streptococ
lactococci such as C. thermophilus, preferably Bifidobact
erium bifidum or B. longum is cultured in an AMC medium, preferably the AMC medium described in the Examples. The culture supernatant or the crushed microbial cells obtained by sonication are subjected to gel filtration chromatography to obtain a low molecular fraction, preferably about 10 kD.
a. An embodiment in which the following fraction is collected and used as an immunostimulatory composition according to the present invention. As the separation conditions for gel filtration chromatography, those described in Examples are preferable.

The immune composition according to the present invention comprises (a) active B
Activated BT-1 contained in a T-1 containing cell or a processed product of the cell, or (b) Inactive BT-1 contained in an inactive BT-1 containing cell or a processed product of the cell, was activated B
The active BT-1 converted into T-1 is allowed to act on a living body. Here, as a method of converting inactive BT-1 to active BT-1, inactive BT-1
And a treated product of the cells containing
A method is preferable in which a low molecular weight fraction having a molecular weight of 10 kDa or less is separated into a low molecular weight fraction having a molecular weight of 10 kDa or more, and a fraction containing a low molecular weight fraction having a molecular weight of 10 kDa or less is collected. More specifically, a low-molecular-weight fraction, preferably a low-molecular-weight fraction, is obtained by gel filtration chromatography from a processed product of an inactive BT-1-containing cell, for example, a lysate obtained by sonication, a culture solution or a culture supernatant of the cell, or the like. A more preferred method is to collect a fraction of about 10 kDa or less or a substance containing the fraction.

The immunostimulatory effect of the composition of the present invention is not limited as long as it acts on and enhances the immune function. Specifically, the immunostimulatory effect includes, for example, enhancement of immune response of lymph node cells, activation of spleen cells and / or thymocytes, activation of bone marrow-derived B cells, activation of Peyer's patch cells (eg, IgA antibody Such as enhancement of intestinal endocrine antibody production)
And the like. Among them, the immunostimulatory composition according to the present invention preferably has an activity of activating Peyer's patch cells, which is the key to intestinal immunity.

The immunostimulating composition according to the present invention can be used as a medicine. The immunostimulating composition as a medicament of the present invention comprises the above-mentioned (a) active BT-1, (b) active BT
Treated cells containing -1 or BT-1, or (c) containing inactive BT-1 and inactive B
Cells that release T-1 extracellularly (hereinafter collectively referred to as “the immunostimulatory component of the present invention”) may be directly administered. However, in general, it is desirable to administer in the form of a pharmaceutical composition containing the immunostimulatory component according to the present invention, which is an active ingredient, and one or more pharmaceutical additives. Such a pharmaceutical composition can be manufactured according to a method known or commonly used in the field of pharmaceuticals itself.

For the preparation of liquid preparations suitable for oral administration, for example, sugars such as water, sucrose, sorbitol, fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil;
-Pharmaceutical additives such as preservatives such as -hydroxybenzoic acid esters can be used. Also, capsules,
For the production of solid preparations such as tablets, powders or granules, for example, excipients such as lactose, glucose, sucrose, mannitol; disintegrants such as starch and sodium alginate; lubricating agents such as magnesium stearate and talc Agents; binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin; surfactants such as fatty acid esters; plasticizers such as glycerin.

Among the preparations suitable for parenteral administration, preparations for intravascular administration such as injections and drops can be prepared preferably using an aqueous medium isotonic with body fluids. For example, an injection can be prepared as a solution, suspension or dispersion using an aqueous medium selected from a salt solution, a glucose solution or a mixture of a salt solution and a glucose solution together with a suitable auxiliary according to a conventional method. Suppositories for enteral administration can be prepared using carriers such as, for example, cocoa butter, hydrogenated fats or hydrogenated carboxylic acids. Sprays can be prepared using a carrier that does not irritate the oral cavity and respiratory tract mucosa, and that can promote absorption by dispersing the active ingredient of the present invention as fine particles as fine particles. As such a carrier, for example, lactose or glycerin can be used. Depending on the properties of the immunostimulatory component and the carrier used according to the present invention, it can be prepared as a preparation in the form of an aerosol or dry powder. For the preparation of a parenteral preparation, for example, one or more selected from diluents, flavors, preservatives, excipients, disintegrants, lubricants, binders, surfactants, plasticizers and the like Pharmaceutical additives can be used. The form of the medicament of the present invention and the method for producing the same are not limited to those specifically described above.

The route of administration, dosage and frequency of administration of the immunostimulatory composition as a medicament of the present invention are not particularly limited, and the type of immunostimulatory component according to the present invention, the type of pathological condition to be treated,
It can be appropriately selected according to various conditions such as the age and weight of the patient, the condition, and the severity of the disease.
Oral administration is preferred as the administration route. Also, for example,
When the immunostimulatory composition containing Bifidobacterium bifidum or B. longum as the immunostimulatory component of the present invention is orally administered systemically, the dose is about 0.1 to 1500 mg / kg / day for an adult. It is preferably about day. When the composition is administered intravenously or intramuscularly, the dose is about 0.01 to 1200.
It is preferably about mg / kg / day. However, the dosage is not limited to this particular example.

The immunostimulatory composition according to the present invention may be used not only as a medicine but also as a food or drink such as a nutritional food, a functional food, a food for specified health use, a powdered milk for childcare or a drink. When used as food, granules,
It can be provided in the form of tablets, gums, candy, jellies, beverages and the like. When the immunostimulating composition according to the present invention is used as a food, the content of the immunostimulating component according to the present invention can be appropriately selected according to the type of the component and the like. For example, when Bifidobacterium bifidum or B. longum is contained as the immunostimulatory component of the present invention, about 1.0%
× 10 ⁵ / g or more, preferably about 1.0 × 10 ⁶ to
It is preferable that about 1.0 × 10 ¹² cells / g of the cells are contained in the immunostimulatory composition according to the present invention as food.

The immunostimulatory composition according to the present invention includes, for example,
Antigenic substances, such as food allergies and resident bacterial flora,
Alternatively, it can be used for imparting a biological defense capability to a living body against infectious microorganisms such as viruses or pathogenic bacteria. When the immunostimulatory composition according to the present invention is incorporated into infant formula, it is possible to prevent various diseases such as artificial nutrition caused by the antigenic substance or the infectious microorganism. However, the use of the immunostimulatory composition is not limited to this. In addition, the immunostimulatory composition according to the present invention can be used not only for humans but also for mammals (eg, mice, rats, rabbits, cats, dogs, cows, horses, monkeys, humans, etc.).
It can also be applied to

[0028]

[Example 1] [Acquisition of culture supernatant] A stock strain of Bifidobacterium bifidum stock (E-139) anaerobically cultured at 37 ° C was used as a seed bacterium. 1/30 ml of this inoculum was added to 30 mL of a VF medium having the following composition.
100 inoculation was performed and anaerobic culture was performed at 37 ° C. After culture, 36
Centrifugation at 00 rpm for 10 minutes to remove the supernatant,
The obtained cells were suspended in 3 L of an AMC medium having the following composition and cultured anaerobically at 37 ° C. for 48 hours. The obtained culture was diluted with 36
Centrifuge at 00 rpm for 10 minutes to separate the supernatant,
This was used as a culture supernatant, that is, a sample specimen.

Composition of VF culture solution Beef / liver exudate 1000 mL Casein peptone 10 g Glucose 10 g Polysorbate 80 1 g L-cysteine 0.5 g These were mixed, adjusted to pH 6.9 ± 0.1, and then subjected to high pressure steam sterilization (121 For 15 minutes).

[0030]

[Table 1] The above components are dissolved in distilled water to adjust the pH to 6.8,
Perform high pressure steam sterilization (115 ° C. for 20 minutes).

[Gel Filtration Column Chromatography] 1 mL of the culture supernatant (sample specimen) obtained as described above was separated by liquid chromatography under the following conditions. Detector: UV absorption spectrophotometer (measuring wavelength: 210 nm) Column: Superose 12 (1.6 × 47.5 cm Amersham Pha)
Column temperature: room temperature Mobile phase: phosphate buffered saline (PBS) Flow rate: 1.0 mL / min Fractionation: 5 mL / tube

[Method for Measuring Immunostimulatory Activity] Fractions of 10 KDa or less separated by liquid chromatography (Fracti
on) (hereinafter referred to as BT-1) was examined for immunostimulatory activity as follows. C57BL / 6 mouse (male 6
Spleen, thymus and Peyer's patch tissues were aseptically removed from BALB / c mice (male, 6 weeks old) or BALB / c mice (male, 6 weeks old), and crushed with a slide glass to prepare a cell suspension. Culture solution 5% FCS-
4.4 × 10 ⁶ each using RPMI1640 (manufactured by Nissui Corporation)
cells / mL and add 1 to a 96-well microplate.
BT-1 was added in an amount of 90 μl per well.
Was added to make a total volume of 100 μl. 5% C at 37 ° C
After culturing for 68 hours under O ₂ , the MTT reagent (5 mg / mL sig
(manufactured by ma) was added to each well in an amount of 10 μl, and after culturing for further 4 hours, the supernatant was removed by centrifugation. 200 μl of dimethyl sulfoxide (DMSO) was added to the residue, stirred, and the absorbance at 570 nm was measured with a microplate reader. The control was PBS instead of BT-1,
Positive controls were ConA (1 μg / mL) and LPS (5 mg
/ ML) was added.

The results are shown in the following table (with the absorbance of the control taken as 1 and expressed as the stimulation rate SI).

[Table 2]

Example 2 A stock strain of Bifidobacterium bifidum (E-139) was cultured at 37 ° C. in 3 L of AMC medium.
Anaerobic culture was performed for 2 days, and the supernatant was collected by centrifugation (3600 rpm for 10 minutes). Anion exchange column chromatography (DEAE) for 3 mL of culture supernatant
-Tyopa 1650 1.0 × 30.5 cm Tosoh, mobile phase solution; PBS, eluate; PB with 1 M NaCl
S, measurement wavelength: 210 nm, flow rate: 1 ml / min), fractionation into non-adsorption and adsorption was performed, and the immunostimulatory activity was measured in exactly the same manner as in Example 1. The activity was present in the adsorbed fraction and was not observed in the non-adsorbed fraction. When each fraction was separated by gel filtration column chromatography (the conditions were exactly the same as in Example 1), a high molecular weight (100 kDa or more) fraction was obtained in the adsorbed fraction, and a low molecular weight (10 kDa) was obtained in the non-adsorbed fraction. The following) Immunostimulatory activity was observed in the fraction. The immunostimulatory activity (stimulation rate SI with the absorbance of the control as 1) is shown below.

[0035]

[Table 3]

Example 3 1 mL of the culture supernatant of Bifidobacterium bifidum obtained in exactly the same manner as in Example 2 was subjected to high molecular weight immunostimulating activity fraction (100 Da or more) and low molecular weight immunostimulating activity fraction by gel filtration column chromatography. (BT-1) and change with time (stored at 4 ° C ± 1)
The effect of the activity was investigated. The activity (residual activity%) is shown below. The residual activity is calculated from the following formula,
Each stimulus rate is a value obtained by measuring the immunostimulatory activity in exactly the same manner as in Example 1 and setting the absorbance of the control to 1.

## EQU1 ## Residual activity (%) = (stimulation rate after elapse of predetermined period /
Stimulation rate at start) x 100

[0037]

[Table 4]

[0038]

The active BT-1 according to the present invention has excellent stability and is easily absorbed into the body because of its low molecular weight. Therefore, the immunostimulatory effect when the composition according to the present invention is administered or ingested. The effect is great.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 (C12N 1/20 E C12R 1:01) C12R 1:01) (72) Invention Person Hiroshi Ohno 7-3-4 Ibukidai Higashi-cho, Nishi-ku, Kobe City, Hyogo Prefecture Biofermin Pharmaceutical Co., Ltd. Kobe Factory (72) Inventor Hideki Yamamura 7-3-4, Ibukidai Higashi-cho, Nishi-ku, Nishi-ku, Kobe City, Hyogo Prefecture F-term in Kobe Factory, Biofermin Pharmaceutical Co., Ltd. (Reference) 4B065 AA21X AC14 BA22 BD14 BD18 CA44 4C084 AA17 NA05 NA14 ZB092 4C087 AA01 AA02 AA05 BC30 BC59 BC60 NA05 NA14 ZB09

Claims (10)

[Claims] 1. An immunostimulatory composition comprising active BT-1. 2. A cell or BT containing active BT-1
The immunostimulatory composition according to claim 1, comprising a processed product of cells containing -1. 3. The immunostimulatory composition according to claim 2, wherein the cells are obtained by culturing under conditions that induce BT-1 production. 4. The immunostimulatory composition according to claim 3, wherein the cells are cells obtained by culturing in an AMC medium. 5. The immunostimulatory composition according to claim 2, wherein the cells are bacterial cells. 6. The immunostimulatory composition according to claim 5, wherein the cells are Bifidobacterium bifidum or Bifidobacterium longum. 7. An inactive BT comprising inactive BT-1
-1 which contains cells that release extracellular -1
An immunostimulatory composition in which T-1 is activated. 8. The immunostimulatory composition according to claim 1, which is a Peyer's patch cell activating composition. 9. Separating a cell containing inactive BT-1 or a processed product of the cell into a low molecular weight fraction having a molecular weight of 10 kDa or less and a high molecular weight fraction having a molecular weight of 10 kDa or more,
The method for producing an immunostimulatory composition according to claim 1, wherein a fraction containing a low molecular weight fraction having a molecular weight of 10 kDa or less is collected. 10. An inactive BT-1 which is contained in a cell containing inactive BT-1 or a processed product of the cell.
A method for activating a living body, the method comprising converting the active BT-1 into -1 and causing the active BT-1 to act on the living body.