JPH10265396A - Thrombolytic agent, functional food and their production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a highly safe thrombolytic agent useful for preventing and treating diseases caused by thrombosis by including a fermentation product of soybean curd refuses with a specific bacterium. SOLUTION: This thrombolytic agent contains the fermentation product of soybean curd refuses with Bacillus natto. The fermentation product is obtained e.g. by adjusting the water content of soybean curd refuses by-produced in a process for producing soy bean curds, etc., to about 70-80 wt.%, sterilizing the soybean curd refuses at a temperature of 105-130 deg.C at a pressure of 1.2-3 kg/cm<2> , etc., inoculating a Bacillus natto such a Takahasi bacterium in the sterilized soybean curd refuses in an amount of 10<3> -10<6> cells/g (converted into the weight of dry soybean curd refuses), fermenting the refuses at a temperature of 20-40 deg.C for 10 hr or longer, and further fermenting the refuses at a temperature of 20-25 deg.C for 15-20 hr. The fermentation product is mixed with other additives. prepared in the form of powder, etc., and subsequently orally administered. The fermentation product is also prepared in the form of functional foods such as semi-raw type sticks, biscuits, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thrombolytic agent, a functional food having a thrombolytic effect, and a method for producing the same.

[0002]

BACKGROUND ART Brain diseases such as cerebral infarction and transient ischemic attack,
Angina, heart disease such as myocardial infarction, or chronic arterial occlusion,
Intermittent claudication, numbness of limbs, peripheral diseases such as pain, as a method of preventing or treating diseases involving circulatory insufficiency or obstruction of blood vessels as a major cause, preventing or forming thrombus It has been conventional to administer a drug that dissolves thrombus.

As the above-mentioned drugs, antiplatelet agents such as aspirin and ticlopidine, anticoagulants such as warfarin, thrombolytic agents such as prourokinase, urokinase, t-PA, streptokinase and chemically modified streptokinase have been conventionally used. Have been. However, these drugs have drawbacks such as high side effects and heavy burden on patients, and the necessity of adjusting the dose so as not to cause side effects, requiring administration by a physician for administration. These drawbacks are particularly serious when a drug is continuously administered to prevent the onset of the various diseases.

[0004] By the way, okara, which is generated as squeezed lees during the production of tofu and the like, is usually discarded without being used because of its spoilage. It has been proposed to ferment okara with Bacillus natto in order to obtain a good food (Japanese Patent Laid-Open No. Hei 4-21339).
No.). However, it has not been conventionally known that fermentation of okara with Bacillus natto produces a substance having a thrombolytic action that can be used in oral fibrinolytic therapy.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a thrombolytic agent which can be used for the prevention or treatment of a disease caused by a thrombus, and particularly to a thrombolytic agent which is highly safe and which prevents the onset of the disease or is chronically effective. It is an object of the present invention to provide a thrombolytic agent suitable for continuous administration for alleviating the symptoms of the disease.

[0006] It is a further object of the present invention to provide a functional food suitable for daily ingestion of the thrombolytic agent.

Another object of the present invention is to provide a method for producing the thrombolytic agent with high efficiency.

[0008]

According to the present invention, there is provided a thrombolytic agent containing a fermented product of Okara natto.

Further, according to the present invention, there is provided a functional food containing the thrombolytic agent.

[0010] The present invention further provides a method for producing a thrombolytic agent, which comprises a step of fermenting okara with Bacillus natto.

[0011]

DETAILED DESCRIPTION OF THE INVENTION The thrombolytic agent of the present invention comprises a fermented product of Okara natto.

[0012] The okara is not particularly limited, and may be any okara such as tofu, soymilk, frozen tofu, soybean oil, soybean protein, and the like generated during the production process, or a mixture thereof.

The Bacillus natto is not particularly limited, and may be a commercially available Bacillus natto, such as Takahashi, Naruse, Miyagi, or a mixture thereof.

The fermented product is obtained by fermenting the okara with the Bacillus natto and, if necessary, drying and purifying. The fermentation method is not particularly limited. For example, a fermentation product having high activity as a thrombolytic agent can be easily obtained by a preferable fermentation method described below.

The purification method is not particularly limited,
Since a protein separated from the fermented product according to a general enzyme separation method exhibits thrombolytic activity, a purification method containing this fraction is preferred.

As a purification method for obtaining the protein fraction, specifically, for example, the fermented product is extracted with physiological saline, and the extract is treated with 50 to 70% ethanol to remove mucilage. And a method of subjecting to ion exchange gel chromatography.

The thrombolytic agent of the present invention contains the above-mentioned fermented products, and further includes additives for formulating glucose, lactose, powdered starch syrup, sucrose, dextrin, vitamins, organic acids, etc. May be included.

The administration route of the thrombolytic agent of the present invention is not particularly limited, but when used for a long term for prevention of disease, alleviation of chronic disease symptoms, etc., an oral administration route with a small burden on the patient. Is particularly preferred.

The form of the thrombolytic agent of the present invention is not particularly limited, but the fermented product which is not subjected to drying, purification, etc., as it is, is dried by freeze-drying or the like to obtain a fine powder, or the fermented product is further processed. If necessary, other additives may be added to make a powder form such as powder, granule, tablet or the like, or a capsule such as an enteric capsule filled into a capsule to form a capsule.

The dose of the thrombolytic agent of the present invention is, for example, in the case of oral administration to humans, 5 to 70 g, preferably 10 to 50 g, in terms of the above-mentioned dried fermentation product without purification.
It is desirable that

The indication of the thrombolytic agent of the present invention is not particularly limited as long as it is a disease associated with circulatory insufficiency or occlusion of blood vessels due to thrombus. Heart disease, heart disease such as myocardial infarction, or chronic arterial occlusion, intermittent claudication, numbness of the extremities, hearing loss, and other peripheral diseases such as pain. It can also be used to prevent or alleviate symptoms that are thought to be involved as one of the causes of circulatory failure.

Since the fermented product is a fermented product of natto of okara, which is soybean meal, it is a fermented product substantially similar to natto,
The safety of the thrombolytic agent of the present invention is considered to be high because its safety is sufficiently ensured by long-term dietary experience.

The functional food of the present invention contains the thrombolytic agent.

The form of the functional food of the present invention is not particularly limited. In addition to the various forms exemplified as the form of the thrombolytic agent of the present invention, the form of semi-life stick, biscuit, etc. And a drink prepared by dissolving the purified fermented product and adding a sweetener, a flavor and the like.

The dosage of the functional food of the present invention is desirably 5 to 70 g, preferably 10 to 50 g, in terms of the dry fermented product which has been simply dried without purification.

By ingesting the functional food of the present invention, the various diseases exemplified as indications of the thrombolytic agent of the present invention can be treated or prevented, or the symptoms can be alleviated. In particular, since the functional food of the present invention can be taken on a daily basis with little burden on the user, it is particularly suitable for taking for the prevention of the above-mentioned various diseases.

The method for producing a thrombolytic agent of the present invention includes a step of fermenting okara with Bacillus natto.

The okara is not particularly limited, and various okara exemplified above can be used. A fermented material having a high activity is obtained when the seed coat portion is mixed at 2 to 10% by weight. It is particularly preferred because it can be used. Also,
The water content of okara when subjected to fermentation is preferably about 70 to 90% by weight. Therefore, when the water content of okara is out of this range during fermentation, it is preferable to appropriately adjust the water content to a preferable range by dehydration with a roller press or addition of water.

The Bacillus natto is not particularly limited, and various commercially available Bacillus natto, etc. exemplified above can be used. Particularly, Takahashi is capable of producing a highly active thrombolytic agent. preferable.

The specific operation of the fermentation is not particularly limited as long as the okara is fermented with the Bacillus natto, but it is preferable to follow, for example, the following method.

First, the water content of okara used for fermentation is adjusted to a range of about 70 to 90% by weight, if necessary, and then heat-treated. The heat treatment is preferably performed under high pressure using a pressure cooker or the like. As the heating conditions, a heating temperature of 10
It is preferable that the temperature is 5 to 130 ° C., the pressure is 1.2 to 3 kg / cm ² , and the heating time is 20 to 50 minutes. By the heat treatment, sterilization, sterilization, and the like of various bacteria that hinder fermentation can be performed.

Subsequently, the natto bacteria are inoculated into the heat-treated okara. The inoculation amount of the natto bacteria is 10 ³ ~
It is preferably 10 ⁶ / g (weight in terms of dry okara).

Subsequently, the okara inoculated with the natto is fermented. In the fermentation, it is preferable to supply oxygen sufficiently. Specifically, it is preferable that the okara is placed in a layer of about 1 cm on a net-like support such as a stainless steel wire net and subjected to fermentation.

The fermentation temperature during the fermentation is preferably 20 to 40 ° C. The fermentation time is usually 10 hours or more, since a substance showing a thrombolytic action is generated in the fermented product from 10 hours after the start of fermentation.
More preferably, it is 4 to 30 hours.

More preferably, at 35 to 37 ° C.
After the fermentation for 15 hours, the fermentation is further performed for 15 to 20 hours at a temperature of 20 to 25 ° C., whereby a fermented product having low viscosity and low ammonia smell can be obtained.

In the case of following the above-mentioned method, it is possible to add a cultivation aid such as glucose, which helps cultivation of the bacterium, without adding any auxiliaries.
Fermentation can be performed.

The obtained fermented product can be used as a thrombolytic agent as it is or by further drying, purifying, adding additives, and other various preparations as necessary.

[0038]

EFFECTS OF THE INVENTION The thrombolytic agent of the present invention has a thrombolytic effect and can be used for the prevention or treatment of diseases caused by thrombus. Is useful as a thrombolytic agent that can be continuously administered to prevent the onset of the disease or to alleviate the chronic symptoms of the disease.

Since the functional food of the present invention contains the above-mentioned thrombolytic agent, it is useful as a functional food that can be taken daily to prevent or treat diseases caused by thrombus.

The method for producing a thrombolytic agent of the present invention is useful as a simple and inexpensive method for producing the thrombolytic agent, since the thrombolytic agent can be easily produced from the normally discarded okara as a raw material. It is.

[0041]

The present invention will be described in more detail with reference to the following examples.
The present invention is not limited to these.

[0042]

Example 1 Okara (moisture content: 80% by weight, seed coat mixed ratio: 6% by weight) generated during the production of tofu was heated in a pressure cooker at 121 ° C. and 1.3 kg / cm ^{2 for} 20 minutes. did.

After treatment, 10 ⁵ natto bacteria (species) was added to Okara.
/ G (in terms of dry okara), inoculated on a stainless steel wire mesh as a layer having a thickness of about 1 cm,
Fermentation was carried out for 20 hours, then the temperature was adjusted to 20 ° C., and the fermentation was further carried out for 15 hours to obtain a fermented product.

[0044]

Example 2 1.0 kg of the fermented product obtained in Example 1 was added to phosphate buffered saline (0.1 M phosphate buffer (pH
To 7.5), 5.0 l of NaCl added to give a concentration of 0.85% by weight) was added, homogenized (5,000 rpm, 5 minutes) with a homogenizer, centrifuged, and the supernatant was collected. Ethanol was added to this supernatant to a concentration of 25 to 50% by weight, and the supernatant was collected and freeze-dried. Next, this was fractionated by Sephadex G-100 gel chromatography. The same operation as in Example 4 described later was performed except that each fraction of the eluate was used as a sample, and fractions having strong thrombolytic activity were collected and used as samples. The molecular weight of this sample measured by SDS-PAGE was 30,000 ± 5,000,
The isoelectric point was 8.5 ± 0.5.

[0045]

Example 3 HD-Val-Leu-, a specific substrate of plasmin
Lys-pNA was added to 0.1 M N to a final concentration of 0.5 mM.
It was dissolved in a 0.1 M phosphate buffer (pH 7.4) containing aCl. 200 μl of the sample (200 μg / ml phosphate buffer) obtained in Example 2 was added to 1.8 ml of the substrate solution, and the reaction was carried out at 37 ° C. for 30 minutes. After the reaction, 4
The absorbance at 05 nm was measured, and the amount of released pNA was calculated as the molar extinction coefficient ε405 = 9620M. As a result, the degree of degradation of the substrate by this sample was 4
1.3 nmol / min / ml, confirming that the sample exhibited the same substrate specificity as plasmin. The same operation as above was performed except that the temperature and pH were variously changed, and the optimum temperature and pH of this reaction were determined to be 45 ° C. and pH 8.5, respectively.

[0046]

Example 4 The sample obtained in Example 2 was used for Ploug
And Kjeldgoapy's method (Biochem. Biophys. Acta, 24 , 27
8, 1957), and its thrombolytic activity was tested by a fibrin plate dissolution method using a plasminogen-free plate. That is, 10 ml of 0.4% by weight fibrinogen dissolved in 0.05 M boric acid-physiological saline buffer (pH 7.8) was placed in a glass petri dish, and bovine thrombin (50 U / ml physiological saline) was added. 5 ml was added, and the mixture was stirred and solidified. On this, 10 μl of the sample (corresponding to 0.2 mg of wet fermented product) was dropped, incubated at 37 ° C., and the area (mm ² ) of the fibrin dissolution window formed after 4 hours was measured. The same measurement was performed a total of five times. Table 1 shows the results. In addition, a standard curve for the relationship between the logarithm of the concentration and the area of the lysis window was obtained using human plasmin as a standard, and this was compared with the above results to determine the thrombolytic activity. Further, the amount of protein in the sample was determined by Lowry.
According to the method (J. Biol. Chem., 193 , 265, 1951), bovine serum albumin was measured as a standard sample.
48 mg protein / ml. From the above,
The specific activity of the sample was found to be 6.25 mg protein / ml. From these results, the enzyme activity per 1 g of the wet fermented product was about 150 CU in terms of plasmin.
It can be seen that this corresponds to the unit.

[0047]

[Table 1]

[0048]

Example 5 50.0 g of the fermented product obtained in Example 1 dried by a freeze-drying method was orally ingested by 10 healthy men and women (5 men and 5 women). 2,
Blood was collected 4, 8, 12, and 24 hours later, and the area of the fibrin lysis window was measured by operating the plasma euglobulin fraction as a sample in the same manner as in Example 4. Table 2 shows the results.

[0049]

[Table 2]

From the results in Table 2, it can be seen that the fibrin degradability in blood increased after ingestion of the fermented product, and the increased value was maintained for a long time. This indicates that the oral intake of the fermented product can increase the fibrinolytic activity in blood over a long period of time.

Claims (3)

[Claims] 1. A thrombolytic agent comprising a fermented product of Okara natto. 2. A functional food containing the thrombolytic agent according to claim 1. 3. A method for producing a thrombolytic agent, comprising a step of fermenting okara with Bacillus natto.