TWI590766B - Thrombotic disease prevention preparation - Google Patents

Description

Thrombotic disease prevention preparation

The present invention relates to a thrombotic disease preventive food which can be easily taken as a health supplement food or the like, and more particularly to a thrombotic disease preventive food containing an extract of a specific microorganism as an active ingredient.

According to the medical treatment fee for injury and illness classification published by the Ministry of Health, Labor and Welfare of Japan, the medical expenses including the diseases of the circulatory system of hypertensive diseases, ischemic diseases and cerebrovascular diseases are the highest, accounting for 21.2% of the total medical expenses. Moreover, these diseases are involved in the abnormal formation of blood clots in blood vessels.

In the blood, there are two kinds of functions of a coagulation system and a fibrinolysis system. If the blood vessel wall is damaged, the platelets are agglomerated, thereby causing a hemostasis, and thereafter thrombin, which is a factor of the coagulation system, acts in the blood fibrinogen, thereby forming fibrin to complete hemostasis. On the other hand, tissue plasminogen activator (tPA, which is secreted from vascular endothelial cells as a fibrinolytic system factor) converts plasminogen, which is an enzyme source, in the blood into fibrinolysis. The enzyme, the plasmin, decomposes the fibrin thus formed in the blood vessel. It is known that the collapse of the balance between the coagulation system and the fibrinolytic system is the cause of thrombotic diseases such as cerebral infarction or myocardial infarction. It is generally believed that the development of new fibrinolytic system hypertonic substances can greatly contribute to the prevention and treatment of such diseases.

The inventors of the present invention have obtained a patent for a method for producing a substance which is reversibly low-molecularized to a stable state by the enzyme or a trace component produced by culturing natto bacteria (Patent Document 1).

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent No. 3902015

It is an object of the present invention to provide a functional food for preventing thrombotic diseases by causing hyperactivity of tPA and causing release of tPA from vascular endothelial cells.

The inventors of the present invention have found that under the subject, a low molecular weight peptide component in which a natto-producing enzyme or a trace component is reversibly reduced to a stable state is added to a vascular endothelial cell, and the tPA in the culture solution is obtained. Further, the activity is hyperactive, and the mice are orally ingested, and as a result, the tPA activity in the blood is increased, thereby completing the present invention.

That is, the present invention provides

[1] A thrombotic disease-preventing food comprising the following components as an active ingredient: a saccharide obtained by hydrolyzing starch with amylase is used as a substrate for a medium, and a yeast extract is added thereto to prepare a medium for fermentation, and The medium is inoculated as Bacillus subtilis AK (registered number: FERM P-18291), and after being fermented and matured, the liquid component formed by fractionation is fractionated;

[2] The thrombotic disease prevention food according to the above [1], wherein the fermentation is carried out at a pH of 4.5 to 6.5 and 28 to 32 ° C for more than 2 months;

[3] The thrombotic disease prevention food according to the above [1] or [2], wherein the ripening is carried out at a pH of 4.0 to 6.0 and 13 to 17 ° C for 4 months or longer;

[4] The thrombotic disease prevention food according to any one of the above [1] to [3] wherein the thrombotic disease is selected from the group consisting of deep venous thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, cloth One or more than one type of venous thrombosis in a group consisting of Jiashi syndrome, sacral-clavicular venous thrombosis, cerebral venous sinus thrombosis, and pulmonary thromboembolism, or selected from cerebral infarction, myocardial infarction, and intestine One or more than one type of arterial thrombosis in a group consisting of membranous artery thrombosis, acute arterial thrombosis of the lower extremity, hepatic artery thrombosis, renal artery thrombosis, splenic arterial thrombosis, and arteriosclerosis of the occlusion;

[5] The thrombotic disease prevention food according to any one of the above [1] to [4], which contains 0.05 to 100% by weight of an active ingredient based on the total weight of the food.

According to the present invention, it is possible to provide a thrombotic disease preventive food which can be easily orally ingested in daily life.

The component contained in the food of the present invention as an active ingredient is a saccharide obtained by hydrolyzing a starch containing corn starch with an amylase as a substrate for a medium, and a yeast extract as a nitrogen source is added thereto to prepare a yeast. The culture medium was used to inoculate Bacillus subtilis AK (registered number: FERM P-18291) as a natto, and the resulting liquid component was fractionated after fermentation and aging.

Here, the saccharide used as the substrate of the fermentation medium may be obtained by using corn starch which has been isolated and purified by amylase hydrolysis from corn seeds, and crude purified corn starch may be used instead of the purified one. Further, in addition to corn starch, hydrolyzed soy flour or rice bran or a mixture of these may be used as a substrate for a medium.

The yeast extract used as a nitrogen source for the fermentation medium can be added as a nitrogen source, usually by drying a water-soluble component obtained by digesting the cells of Saccharomyces cerevisiae Meyen and extracting it.

In the fermentation medium used for the production of the active ingredient of the food of the present invention, in addition to the above-mentioned saccharide and yeast extract, an organic substance such as a protein or an inorganic salt may be formulated as needed. Examples of the organic substance include soybean protein or other vegetable protein, and examples of the inorganic salt include calcium chloride, sodium chloride, and sodium phosphate.

In addition, it is preferable to add sugar to the saccharide which is obtained by hydrolyzing starch with amylase as a substrate for a medium, and examples of such sugars include sucrose (sucrose), glucose, and sucrose.

The yeast strain which was inoculated in the fermentation medium prepared as described above was used as Bacillus subtilis AK of Bacillus natto, and Bacillus subtilis AK was deposited in Japan as "(Register No.) FERM P-18291". Administrative Corporation Industrial Technology Research Institute. Further, as long as it is desired, in addition to Bacillus subtilis AK, Lactobacillus as Lactobacillus or Streptococcus, Saccharomyces cerevisiae or green which are not inhibiting the proliferation of Bacillus subtilis AK may be inoculated. Aspergillus oryzae cells, bacterial extracts or bacterial extracts. Further, in the fermentation medium used for the production of the active ingredient of the food of the present invention, cabbage, cabbage, carrot, medicinal aphid, parsley, and celery which do not inhibit the proliferation of Bacillus subtilis AK can be added as required. Plant extracts such as onions.

Bacillus subtilis AK is obtained by attaching ultraviolet rays, X-ray irradiation, high-low temperature environment (100 ° C, 0 ° C), competition with lactic acid bacteria, and easy production of spores to Bacillus subtilis which is usually natto bacteria [5.0] Various conditions such as weight% of meat extract, 10.0% by weight of peptone, 5.0% by weight of sodium chloride, 15.0% by weight of agar, 65.0% by weight of vegetables (cabbage, carrot, celery, parsley) The drug-resistant bacteria found were repeatedly subcultured and selected.

The Bacillus subtilis AK strain obtained in the above manner has the bacteriological properties as described below.

(a) the nature of morphology

1 cell shape and size

Bacillus 1.0~1.2×3.0~50 μm

2 with or without pleomorphism of cells

no

3 With or without exercise

Yes (circle hairy flagella)

4 with or without spores

The approximate center of the elliptic cell

(b) Culture nature

1 gravy agar plate culture

Round cluster white turbid

2 broth liquid culture

Upper or lower bacterium

(c) Biochemical properties

1 Gram staining positive

2 nitrate reduction positive

3 MR (methyl red) test negative

4 VP (Voges-Proskauer) test positive

5 吲哚 Formation negative

6 Hydrogen sulfide generation negative

7 Utilization of citric acid Positive

8 catalyst positive

9 range of growth

pH 5.5~7.0

Temperature 25 ° C ~ 40 ° C

The Bacillus subtilis AK strain is subjected to the above-mentioned fermentation medium together with other fermenting bacteria containing lactic acid bacteria at a pH of about 4.5 to 6.5 at a temperature of about 28 to 32 ° C for more than 2 months. yeast. It is presumed that the specific natto used in the present invention does not efficiently assimilate the sugar and nitrogen sources in the pH region or the temperature region below the region, even if it is subjected to fermentation for two months or more. The effect obtained by the obtained tPA releasing the entangled substance is obtained. On the other hand, in the pH region or the temperature region higher than the above region, since the fermentation is not sufficient, the specific natto bacteria do not efficiently assimilate the sugar and nitrogen sources, and the obtained tPA is not sufficiently obtained. The effect that the substance is expected to be.

After the fermentation, the fungus is incubated on the same medium, and the culturing is carried out for 4 months or more at a pH of about 4.0 to 6.0 at about 13 to 17 °C. It is presumed that in the pH region or the temperature region below the region, even if it is matured for 4 months or more, various active amino acids, lipoproteins, lipopolysaccharides, lipids, and the like are used as fermentation products. Also, the effect of lowering the molecular weight is not sufficiently obtained, and the desired effect of the obtained tPA releasing the entangled substance is not sufficiently obtained. On the other hand, it is presumed that the activity is lowered in the pH region or the temperature region higher than the above region, and the effect obtained by the obtained tPA releasing the entangled substance is not sufficiently obtained as described above.

When the liquid component formed by the fermentation and the ripening stage is fractionated, a well-known separation method such as filtration or centrifugation may be used, and the fractionated food stock solution may be used as the thrombus of the present invention directly or concentrated or diluted. The active ingredient of disease prevention foods.

As an active ingredient of the thrombotic disease prevention food of the present invention, for example, Enzamin stock solution (ENM) manufactured by Enzamin Research Co., Ltd. or Enzamin concentrate (ENM-HL) as a 20-fold concentrated extract thereof can be adjusted.

Therefore, as a constituent component of the active ingredient obtained in the above manner, a substance for facilitating enzyme synthesis in the organic body is contained, and the enzyme obtained by fermentation can be reversely cleaved as a fragment of the active amino acid residue. In addition, it contains useful substances such as adenine, black mites, cytosine, thymine, uracil which can be used in the body. It is generally considered that such a useful substance is a culture filtrate comprising: an antiviral (Antivirus) tissue active factor advocated by Besredka; a life source stimulating hormone described by Filatov, and borrowed These are combined by a biochemical reaction to be treated in a safe and effective manner.

The thrombotic disease preventing food of the present invention containing such an active ingredient can appropriately prevent the balance of the clotting system and the fibrinolytic system by stimulating the life phenomenon of fibrinolysis of blood, thereby preventing the onset of thrombotic diseases.

The thrombotic disease which can be prevented by the thrombotic disease prevention food of the present invention is not particularly limited as long as it is caused by a thrombus, and examples thereof include: selected from deep vein thrombosis, portal vein thrombosis, and renal vein thrombosis. Venous thrombosis in a group consisting of syndrome, jugular vein thrombosis, Budd-Chiari syndrome, sacral-clavicular venous thrombosis, cerebral venous sinus thrombosis, and pulmonary thromboembolism, or from cerebral infarction, myocardial infarction, intestine Arterial thrombosis, a group of arterial thrombosis, lower extremity arterial thrombosis, hepatic artery thrombosis, renal artery thrombosis, splenic arterial thrombosis, and arteriosclerosis obliterans.

The thrombotic disease prevention food of the present invention may be an active ingredient prepared in the above manner and an excipient conventionally used in the food field (for example, starch or dextrin, cellulose, lactose, maltose, reduced lactose, reduced maltose, sorbus) a sugar alcohol, mannitol, erythritol, xylitol, etc.) or an adjuvant (for example, a solvent, a dispersion medium, a coating agent, a stabilizer, a diluent, a preservative, a preservative, a bactericide, an antifungal agent, Isotonic pressure reagent, absorption inhibiting reagent, disintegrating agent, emulsifier, binder, lubricant, pigment, etc.) are mixed and prepared, for example, into tablets, capsules, granules, by a formulation method conventionally used in the food field. The form of powder, extract, solution, slurry, suspension, and emulsion.

The thrombotic disease-preventing food of the present invention may contain about 0.05 to 100% by weight, preferably about 0.1 to 90% by weight, more preferably about 1 to 85% by weight, and more preferably about 1 to 85% by weight, based on the total weight of the food. It is preferably about 5 to 80% by weight, preferably about 10 to 50% by weight, of a liquid component formed by bacteria as an active ingredient.

Further, as another aspect of the present invention, a saccharide obtained by hydrolyzing starch containing corn starch by amylase is used as a substrate for a medium for producing the above-mentioned thrombotic disease preventive food, and a nitrogen source is added thereto as a nitrogen source. The yeast extract is used to prepare a fermentation medium, and the medium is inoculated as Bacillus subtilis AK (registered number: FERM P-18291), and after being fermented and matured, the liquid component is fractionated. Use of the ingredient; a use of Bacillus subtilis AK for the manufacture of a thrombotic disease prevention food; characterized by the prevention or treatment of a thrombotic disease in the prevention of food for thrombotic diseases.

Hereinafter, the present invention will be described in more detail based on the examples, but the present invention is not limited by the examples.

[Example 1]

50 kg of purified water was added to 2.3 kg of yellow corn starch, 0.5 kg of soy peptone, 0.5 kg of rice bran juice, 80 g of calcium chloride, and 150 g of salt, and heated and dissolved. It was then allowed to cool and 50 g of amylase was added to fully saccharify it. After the saccharification, add 1.5 kg of sugar, 1.5 kg of glucose, 450 g of yeast extract (Japanese pharmaceutical (stock)), 1.5 kg of rice bran, 80 g of sodium phosphate, 5 kg of vegetable crush Juice (cabbage, carrots, celery, parsley) and purified water to a total weight of 150 kg.

Further, sodium hydroxide was added to adjust the pH to a range of 7.2 to 7.6, which was placed in a culture tank, and autoclaved at 120 ° C for 20 minutes. After cooling, the Bacillus subtilis AK strain was inoculated and fermented in a constant temperature room at a temperature of 30±2 ° C for 60 days at a pH of 4.5 to 6.5, followed by a thermostatic chamber at a temperature of 15 ± 2 ° C. It is aged for 120 days under the conditions of pH 4.0~6.0. The supernatant was sterilized at 110 ° C for 20 minutes, and allowed to stand naturally to cool the culture solution. After filtering with a filter paper, the pH was adjusted to 3.5-3.7 by citric acid, and after sterilization at 95 ° C, it was placed in a 5 gallon tank at a temperature of 90 ° C or higher. In the above manner, 125 liters of liquid food stock solution (ENM) was obtained.

The general analysis results in 100 g of the obtained food stock solution (ENM) are shown in Table 1 below.

Further, regarding the above-mentioned food stock solution, a column (TSKgel G2500PWXL) manufactured by Tosoh Corporation was used, and a liquid high-speed chromatogram of a 55:45:0.1 mixture of water, acetonitrile and trifluoroacetic acid was used (Shodex Corporation) Manufacture: GPC SYSTEM-21) and measure the size exclusion chromatography (SEC), and compare the detector sensitivity (ultraviolet spectrophotometer: mV) with the dissolution time of the known molecular weight. The molecular weight distribution is shown in Fig. 1, and the ratio (percentage) of the area of the molecular weight fraction in the figure to the whole is shown in Table 1.

Then, whether or not the low molecular substance contained in the obtained stock solution for food (ENM) is modified or decomposed by heat is evaluated.

The molecular weight distribution of the ENM heated at 121 ° C for 30 minutes was measured by size exclusion chromatography using a TSK gel G2500 PWXL column (manufactured by Tosoh Co., Ltd.). Also, the molecular weight distribution of the same batch of ENM (Control ENM) not placed in a high temperature was measured in the same manner. The results of these are shown in Table 2.

As a result of the measurement of the molecular weight distribution, ENM was not found to change in the molecular weight distribution of the low molecular composition even under high temperature conditions, and thus it was found that ENM is resistant to heat.

Further, in the case where the low molecular substance contained in the obtained stock solution for food (ENM) is subjected to a strong acid condition, whether or not the modification or decomposition is caused is evaluated.

While maintaining the ENM (pH 3.7) at 37 ° C, stirring was carried out, and hydrochloric acid was added to adjust the pH to 1.2. After leaving for 15 minutes, it was reduced to the original pH of 3.7 with sodium hydroxide, and the molecular weight distribution of the sample was determined by size exclusion chromatography using a TSK gel G2500PWXL column (manufactured by Tosoh Co., Ltd.). Also, the molecular weight distribution of the same batch of ENM (Control ENM) not placed in a strong acid was measured in the same manner. The results of these are shown in Table 3.

As a result of the measurement of the molecular weight distribution, ENM was hardly found to change in the molecular weight distribution of the low molecular composition even under strong acid conditions, and thus ENM was also resistant to strong acid, and was also resistant to gastric acid when orally administered.

Based on the above results, it was confirmed that the ENM is a raw material which is stable to high temperature or strong acidic conditions, and it is found that it can be subjected to various processing or utilization without being affected by high temperature or acidic conditions even in the case of food processing or oral ingestion.

Then, the prepared ENM is charged into a concentrating device, concentrated at a temperature of 40 ° C or lower, and then concentrated at a vacuum of 20-60 cmHg, and sterilized at 90 ° C for 30 minutes, and then refilled into a concentrating device. The mixture was concentrated at a temperature of 40 ° C or lower at a vacuum of 20-60 cmHg to obtain an ENM-HL having a concentration ratio adjusted to 20 times.

Study on the effect of ENM-HL on the plasmin activity of synthetic substrate S-2251

Then, the present inventors have used plasmin in order to directly study the effect of ENM-HL (manufactured by Enzamin Research Co., Ltd.) on the plasmin activity of tissue plasminogen activator (tPA). The synthetic substrate S-2251 of the specific cleavage site was studied. A dilution (100 to 0.13 vol%) of ENM-HL with physiological saline was used as a sample.

Add 10 μl of this sample to 90 μl of tPA (10 IU/ml), 20 μl of Glu-plasminogen (200 μg/ml) and 100 μl of S-2251 (1 mM) for 2.5 minutes. The absorbance at 450 nm was measured for 2 hours, and the degree of increase (ΔA450 nm) was calculated to evaluate the tPA activity.

As a result, significant increase in tPA activity using ENM-HL was found (Fig. 2).

[Embodiment 2]

Study on the effect of ENM-HL on the effect of tPA activity using fibrin plate

Then, the present inventors conducted a study on the effect of ENM-HL on tPA activity using a fibrin plate method. A dilution (100 to 0.35 vol%) of ENM-HL with physiological saline was used as a sample. To 10 μl of the sample, 10 μl of tPA (25 IU/ml) was added to a fibrin culture dish and allowed to react for 24 hours to evaluate the tPA activity.

As a result, even in the fibrin plate method, the effect of the tPA activity of ENM-HL was confirmed (Fig. 3).

[Example 3]

Effect of ENM-HL on the activity and production of tPA in vascular endothelial cells

Then, the present inventors conducted studies on the effects of ENM-HL on tPA activity and tPA production ability in vascular endothelial cells. Bend.3 cells derived from mouse cerebral vascular endothelium were used as vascular endothelial cells. The BEnd.3 cells were seeded at 2 × 10 ⁵ cells/well on a 24-well plate to achieve fusion, and after 2 days, ENM-HL was added to the serum-free medium at a concentration of 0.001 to 0.1% by volume. Cultured in hours, 12 hours, and 24 hours. The tPA activity in the culture solution was evaluated by fibrin zymography.

Further, cDNA (ribonucleic acid, ribonucleic acid) was extracted from bEnd.3 cells cultured for 24 hours to synthesize cDNA (complementary deoxyribonucleic acid), and polymerase chain reaction (PCR) was used. The method was used to study the amount of tPA mRNA expression. The amount of expression of GAPDH mRNA as an endogenous control was measured and corrected.

The tPA activity in the culture supernatant was found to be hyperactive after 6 hours, 12 hours, and 24 hours after the addition of ENM-HL (Fig. 4A). Further, no increase in the expression amount of tPA mRNA due to ENM-HL was observed, and thus it was suggested that the production of tPA of vascular endothelial cells was not affected (Fig. 4B).

[Example 4]

Study on the binding of ENM-HL to tPA using an intermolecular interaction device (IASYS)

The inventors of the present invention considered the binding of ENM-HL to tPA by using an organic intermolecular interaction analysis device (IASYS) in which ENM-HL contains a substance directly acting on tPA. The tPA was solid-phased in a colorimetric tube, and ENM-HL (1 to 20% by volume) was added to evaluate the binding property.

As a result, it was found that the concentration-dependent binding of ENM-HL was strong to tPA (Fig. 5). According to this situation, it is suggested that ENM-HL contains a substance which strongly binds to tPA.

[Example 5]

Effect of ENM-HL on the activity of tPA in the blood of mice

Then, the inventors of the present invention conducted research using C57BL6/J mice in order to study the effect of ENM-HL in the organism. 0.3 ml of ENM-HL (0.008-25% by volume) was orally administered to male C57BL6/J mice (12 weeks old, body weight 25 ± 3 g, 3 in each group), and blood was collected 2 hours later to take plasma. And separate from the euglobulin component. The tPA activity of the fraction was evaluated by fibrin zymography.

As a result, an increase in tPA activity was observed at a concentration of ENM-HL 1% by volume and 0.1% by volume (Fig. 6).

Study on the sustained effect of ENM-HL on the tPA activity in the blood of mice

Then, in order to study the sustained effect of the hyperactivity of tPA activity in the organism, ENM-HL, which was found to have a 1% concentration of tPA activity, was orally administered to C57BL6/J mice, and was performed 1, 2, 3, and 4 hours later. Blood was collected and the euglobulin component was isolated to evaluate the tPA activity by fibrin zymography.

As a result, it was found that the hyperactivity of tPA activity was the highest value after 2 hours and was maintained for 4 hours or more (Fig. 7).

According to the results of the above experiments, it is suggested that ENM-HL promotes the activity of tPA in the blood and maintains its effect for a relatively long time.

[Embodiment 6]

Study on the effect of medium added components in the blood of mice on tPA activity

(1) Lactic acid bacteria extract

Then, the inventors of the present invention conducted studies on the effects of other bacterial fermentation extracts in the organism. 300 μl of lactic acid bacteria extract (stock solution, 1 to 50% by volume) diluted with physiological saline was orally administered to male C57BL6/J mice (12 weeks old), and the plasma was separated from 2 hours later. Globulin component. The activity of tPA and uPA (urokinase-type plasminogen) of this component was evaluated by fibrin zymography.

As a result, in the lactic acid bacteria extract, no effect of enhancing the activity of tPA and uPA was observed (Fig. 8).

(2) natto fermentation extract

Then, the inventors of the present invention conducted studies on the effects of other medium-added components in the organism. 300 μl of S. cerevisiae extract (20 times concentrated solution, 0.2-25% by volume) diluted with physiological saline was orally administered to male C57BL6/J mice (12 weeks old), taken from 2 hours later. A single euglobulin component in plasma. The activity of tPA and uPA of this component was evaluated by fibrin zymography.

As a result, no effect of enhancing the activity of tPA and uPA was observed in the natto extract extract (Fig. 9).

[Industrial availability]

It is generally considered that tPA is also used as a therapeutic drug for thrombotic diseases such as cerebral infarction, and its hyperactivity (enhanced activity) is effective for the treatment and prevention of such thrombotic diseases. This time, it is clear that the component derived from natto as a low molecular peptide enhances the activity of tPA in an organism. Since this ingredient has been prepared into a nutraceutical food, it is expected to prevent thrombotic diseases by ingesting food.

The thrombotic disease prevention food of the present invention belongs to the fields of nutritional functional foods, nutritional supplement foods, health supplement foods, specific health foods, and particularly health supplement foods. The thrombotic disease-preventing food of the present invention is an active ingredient from a microorganism culture which has been previously used for foods and the like and which has been confirmed to be safe, and can be easily taken. The thrombotic disease-preventing food of the present invention can effectively prevent the onset of a thrombotic disease and reduce the huge medical expenses associated with a thrombotic disease by simple and daily ingestion.

Fig. 1 is a graph showing the molecular weight distribution of the active ingredient of the present invention.

Fig. 2 is a graph showing the effect of the hyperactivity of the tPA activity of the active ingredient of the present invention measured using a synthetic substrate S-2251 having a cleavage site specific for plasmin.

Fig. 3 is a photograph (A) and a graph (B) showing the effect of the use of the tPA activity of the active ingredient of the present invention measured using a fibrin plate.

Fig. 4 is a graph (B) showing the tPA activity in the culture solution measured by the mouse brain vascular endothelium cell bEnd. 3 and the gene expression amount of the intracellular tPA (B).

Fig. 5 is a graph showing the bondability of the active ingredient of the present invention to tPA measured using an organic intermolecular interaction analysis apparatus (IASYS).

Fig. 6 is a photograph and graph showing the tPA activity in the blood of the blood collection after the mice were ingested with the active ingredient of the present invention 2 hours later.

Fig. 7 is a photograph and graph showing the effect of the hyperactivity of tPA activity in the blood of mice when the mouse is ingested with the active ingredient of the present invention.

Fig. 8 is a photograph showing the effect of the ingestion of the lactic acid bacteria extract in mice, and the effect of the tPA and uPA activities in the blood collected after 2 hours.

Fig. 9 is a photograph showing the effect of the ingestion of tPA and uPA activity in the blood of the blood collected by the mouse after the natto bacterium fermentation extract was taken.

Claims (7)

The use of a stock solution for the preparation of a thrombotic disease prevention preparation, the stock solution comprising: a saccharide obtained by hydrolyzing starch with an amylase as a substrate for a medium, a yeast extract is added thereto to prepare a fermentation medium, and The medium is inoculated with Bacillus subtilis AK (registered number: FERM BP-11451), which is fermented and matured, and then fractionated to obtain the liquid component; and 80% of the stock solution (peak area) The percentage) has a molecular weight of 500 or less. For the purpose of claim 1, the fermentation is carried out for 2 months or more at a pH of 4.5 to 6.5 and 28 to 32 °C. For the purpose of claim 1, the culturing is carried out for 4 months or more at a pH of 4.0 to 6.0 and 13 to 17 °C. For the purpose of claim 2, the ripening is carried out for 4 months or more at a pH of 4.0 to 6.0 and 13 to 17 °C. The use according to any one of claims 1 to 4, wherein the thrombotic disease is selected from the group consisting of deep venous thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Budd-Chiari Syndrome, One or more than one type of venous thrombosis of sputum-clavicular venous thrombosis, cerebral venous sinus thrombosis, and pulmonary thromboembolism, or selected from cerebral infarction, myocardial infarction, One or more than one type of arterial thrombosis of a group consisting of intestinal mesenteric artery thrombosis, acute arterial thrombosis of lower extremity, hepatic artery thrombosis, renal artery thrombosis, splenic artery thrombosis, and arteriosclerosis of the occlusion. The use of any one of claims 1 to 4, wherein the whole is relative to the preparation The weight contains 0.05 to 100% by weight of the above stock solution. The use of claim 5, wherein the raw material is contained in an amount of 0.03 to 100% by weight based on the total weight of the preparation.