KR20080066115A - The functional fermented products increased fibrinolytic activity and fermentation method thereof - Google Patents

Abstract

A functional fermented product having an enhanced fibrinolytic activity, and its fermentation method are provided to increase the fibrinolytic activity of fermented soybeans by using Bacillus subtilis NNT-0701(KCCM 10674) and adding medicinal herb extracts having fibrinolytic activities. Functional fermented soybeans having an enhanced fibrinolytic activity are prepared by the steps of: (i) mixing water with red ginseng, mugwort, pine needles, Ganoderma lucidum, Angelica gigas Nakai, and green tea leaves, and soaking soybeans in the mixed water in a weight ratio of 1:2~3 for 3~5hr; (ii) boiling down the soaked soybeans over medium heat for about 2~4hr; (iii) steaming the boiled soybeans at 100~130°C for 30~50min, and inoculating them with 0.5~1.0wt% a culture solution of Bacillus subtilis NNT-0701(KCCM 10674); and (iv) fermenting the inoculated soybeans at 36~45°C for 24~72hr to obtain fermented soybeans. A functional fermented liquid having an enhanced fibrinolytic activity are prepared by the steps of: (a) mixing steamed soybeans or soybeans with water, grinding the mixture, adding 0.3~0.9wt% of each of red ginseng or a red ginseng extract, a mugwort extract, a pine needle extract, a Ganoderma lucidum extract, an Angelica gigas Nakai extract, and a green tea leaf extract to the ground soybean liquid, and sterilizing it at 110~130°C for 10~30min; (b) inoculating the sterilized soybean liquid with 0.5~1.0wt% a culture solution of Bacillus subtilis NNT-0701(KCCM 10674); (c) fermenting the inoculated soybean liquid at 36~45°C for 24~72hr to obtain a fermented soybean/medicinal herb liquid; and (d) lyophilizing the fermented soybean/medicinal herb liquid to obtain a fermented soybean/medicinal herb product.

Description

The functional fermented products increased fibrinolytic activity and fermentation method

Figure 1 shows the artificial thrombosis picture of Cheonggukjang prepared with Bacillus subtilis NNT-0701 (KCCM 10674). (B: boiled soybeans, C: medicinal plant extract-free Cheonggukjang, S: medicinal plant extract Cheonggukjang)

Figure 2 shows the artificial thrombolysis picture of the soybean medicinal plant fermentation solution prepared with Bacillus subtilis NNT-0701 (KCCM 10674). (C: no addition, H: red ginseng extract, SS: mugwort extract, S: pine needle extract Added, D: Angelica extract added, Y: ganoderma lucidum extract, N: green tea extract, T: 6 medicinal plant extracts mixed)

Figure 3 shows the artificial thrombolysis picture when the red ginseng extract was added to the 20% soybean medium and not fermented. (B: blank (not added soybean solution), H-1: 0.5% red ginseng added, H : Inoculate NNT-0701 after adding 0.5% of red ginseng)

Figure 4 shows the artificial thrombosis picture when medicinal plant extract is added to soybean and BN-NUC1 strain is used as a fermentation strain.

5 shows the 16S rDNA partial sequence of Bacillus subtilis NNT-0701.

The present invention uses Bacillus subtilis NNT-0701 (KCCM 10674) as a fermentation strain at the time of fermentation of soybean, extracts of red ginseng, preferably red ginseng, Angelica, mugwort, greens, pine needles, ganoderma, green tea, etc. among medicinal plants. The present invention relates to a thrombolytic-enhanced functional fermented product and a method for preparing the same, which increase the thrombolytic function and prevent adult diseases such as arteriosclerosis, high blood pressure, and stroke due to accumulation of blood clots.

With the modernized diet, cardiovascular and vascular system diseases are increasing, and various medicines for treating cardiovascular diseases, which are the leading cause of mortality in the world, have been developed. However, there is no proper treatment due to side effects and prevention is the best treatment. Doing. Therefore, research is actively conducted on materials that can prevent or improve stroke, myocardial infarction, thrombosis and the like by the intake of functional foods. Traditional fermented foods are one of them, and research is being actively conducted. Nowadays, preference for traditional soybeans is lowered, and recently, intake of miso, soy sauce, cheonggukjang, and red pepper paste is hardly made. Unfortunately, it is pointed out that traditional foods are gradually disappearing from our tables, Western foods are becoming westernized, and adult diseases and cancers are increasing in proportion. In this trend, food nutritionists advise that modern people should be able to eat traditional methods in order to prevent and treat adult diseases and other diseases in modern times. It is due to the result.

Cheonggukjang is Korea's representative fermented food made from soybean. It is a traditional Korean soy sauce that has been commercialized with soy sauce, soybean paste and red pepper paste. It has more soy protein and fat content and higher digestion absorption than doenjang. In addition, strong fermentation enzymes such as amylase and protease are produced in the fermentation process, so that the digestibility of boiled soybean is 50-70%, while the soybean soup is 90-92%, which is digested and absorbed well, and serves as a digestive agent. Cheonggukjang is a traditional food that decomposes soy protein by fermenting boiled soybean at an appropriate temperature. Microorganisms fermenting Cheonggukjang include Bacillus subtilis and Bacillus licheniformis. In particular, Cheonggukjang is the traditional fermented food with the shortest fermentation period (2 ~ 3 days). Therefore, except for the uniqueness of the flavor, it is recognized as the easiest and most effective way of ingesting soybeans as well as the most easily and easily processed among fermented foods. In particular, in addition to the nutritional function of soybeans, soybeans have various bio-regulatory functions. In addition, traditional Jangjang is said to be effective in the prevention and treatment of heart and vascular diseases such as hypertension, as well as the effect of eliminating poison in the body as described in [Consent Bogam].

The lecithin and proteolytic enzymes contained in Cheonggukjang are excellent for dissolving blood clots and cholesterol that block blood vessels, so they can be the best way to treat and prevent stroke. Natto in Japan, similar to Korea's Cheonggukjang, is a traditional fermented food made by inoculating Bacillus subtilis natto with steamed soybeans. ) Is found to be an enzyme that breaks down fibrin strongly, and it is also expected to be used as a thrombolytic agent. At present, natto is rapidly increasing in sales as a health food (see US Pat. No. 5,750,650).

And herbal medicines with the function of thrombolytic ability and blood circulation improvement, Ganoderma lucidum is the Bulchocho (不 老 草) seen in the jangjang cadet symbolizes health longevity. It is found in Chinese medicine as a medicinal herb, and it has been found that modern medicine contains a large amount of polymer protein polysaccharides that have preventive, healing, and improving effects, and the effects are revealed one after another through experiments. Eliminates blood clots inside the blood vessels and inhibits the formation of lipid peroxide. Accumulation of lipid peroxide causes liver failure, arteriosclerosis, and various thrombosis. The ingredient improves blood circulation and removes harmful substances in the blood to experience the treatment effect of adult diseases or to improve the constitution. Germanium, which is contained in a large amount of Ganoderma lucidum, sends fresh oxygen to the body and activates the cells to maintain health at all times, and strengthens the natural healing power born by humans. It is a good food for high blood pressure, diabetes, stroke, cancer, arteriosclerosis, liver disease, gastric ulcer, duodenal ulcer.

Donkey's water extract has a blood pressure-lowering effect in experimental animals, diuretic action, cerebral sedation, soft water excitement and numbness, and helps protein synthesis and new uterine tissues. In addition, ethanol extract is said to activate uterine movement by activating metabolism and regenerating bone marrow such as increasing glucose absorption. And Angelica extract is said to have a function of lowering intraocular pressure and blood pressure.

Red ginseng has a sedative and arousing effect on the central nervous system, and it acts on the circulatory system to prevent hypertension or atherosclerosis. At the same time, hematopoietic effect (당 作用) and blood sugar levels (血糖 値) lowers, protects the liver, acts on the endocrine system indirectly effective on sexual behavior and reproductive effects, anti-inflammatory And anti-tumor effect (抗 腫 瘍 作用), the protective effect against radiation, protects and softens the skin. The most important of the effects of red ginseng is the adaptogen effect, which increases the ability of the organism to adapt more easily by increasing the defensive ability against the disease, stress, and other harmful effects from the environment. Scientifically proven. It is good for body and mind to improve the fragile constitution and to recover from fatigue. It is a herb that is applied to almost all diseases that makes blood circulation in the body to improve blood circulation and improve the function or development of the body. Dr. Okuda, a professor of biochemistry at the Department of Biochemistry, Ehime University, Japan, is not only recognized for its efficacy in diabetes, but also, Korean red ginseng promotes blood circulation in capillaries, thereby preventing symptoms such as arteriosclerosis, which diabetes patients often suffer from. It is said to be a medicinal benefit.

On the other hand, the patent that used red ginseng for fermentation of Cheonggukjang, Patent Application 2004-0010316 (Diet food composition using Cheonggukjang), Patent Application 2003-0046438 (Registration 10-0519484, Functional Cheonggukjang to reduce the odor and browning phenomenon by adding ginseng and Its manufacturing method), patent application 2004-0115400 (registered 10-0597853, manufacturing method of cheonggukjang using red ginseng extract), patent application 2003-0001238 (functional cheonggukjang), patent application 2004-0110028 (registered 10-0606241, antidiabetic Red ginseng Cheonggukjang production method is enhanced, the patent application 2006-0052823 (registration 10-0659015, functional health food and its manufacturing method) and the like are known. In addition, regarding the anti-thrombotic function of home ginseng, Patent Publication 2004-0060032 (Anti-thrombotic composition), Patent Publication 2001-0059137 (Blood circulatory improving agent using red ginseng complex and a method of manufacturing the same) are known.

However, the use of red ginseng in fermentation of Cheonggukjang has not been greatly improved.

Mugwort is characterized by a high content of minerals and vitamins. In particular, a lot of vitamin A, about 80g to eat the amount you need to be able to supply a day. If you lack vitamin A, the body's resistance to weakened bacteria invaded. Wormwood is also known to be effective for warming the body, especially for women's diseases. It is good for people who are cold and good for colds and diarrhea. Because it warms the blood vessels and the meridians, the uterus and lower abdomen are cold and bleeding during pregnancy, bleeding during pregnancy, hemostasis, nosebleeds, and bleeding when used to hemostatic. The herb is weak and cold, cold and painful in the abdomen, and it is effective for menstrual irregularities, menstrual pain and cramps, and the bronchial smooth muscle ion effect is effective for chronic bronchitis, chronic hepatitis and cirrhosis. In addition, it suppresses the growth of various bacteria, so decoction of bacterial dysentery, eczema, itching skin, etc. is also helpful.

Pine needles are the main components of turpentine, which is a volatile ingredient that is unique to pine needles, and tannin, which tastes astringent.Terbine lowers cholesterol levels and expands peripheral nerves to increase hormone secretion. It is effective for the symptoms that usually occur. It also stabilizes nerves and helps prevent and treat colds. Tannins are a good ingredient for people with nervous constipation, which helps promote appetite, protects the gastric mucosa, and relaxes the intestinal tract and relaxes the intestines.In addition, glycinine, which is good for diabetes, lowers blood sugar levels, and iron, which is good for anemia. It is composed of beneficial ingredients such as rutin to strengthen blood vessels, apiginic acid to remove harmful substances from tobacco, and vitamin C.

Green tea has been shown to have anti-cancer effects by inhibiting the synthesis of N-nitroso compounds in all tea leaves, including green tea, black tea and oolong tea, according to a study by the Chinese Academy of Preventive Medicine. Tea contains a lot of antioxidants to prevent aging, to prevent adult diseases such as high blood pressure, and low calorie beverages with little calories. In addition, green tea is a typical alkaline drink that has a hangover, prevent tooth decay, anti-inflammatory function.

Cheonggukjang as described above is known to have a thrombolytic ability, but the effect is still not expected. The factor that greatly affects the thrombolytic ability in fermentation of Cheonggukjang is the strain used for fermentation. Conventional patents related to Cheonggukjang fermented strains with thrombolytic ability include patent application 2003-0053090 (a new strain of genus Bacillus and antithrombogenic material produced therefrom), and patent application 2004-0031505 (registered 10-0588521, Bacillus subtilis CB1339 strain with excellent immune cell activating function, patent application 2002-0061116 (new strain Bacillus megaterium jabi-42 and new thrombolytic enzyme produced therefrom), patent application 2002-0022551 (registered 10-0511637) , BDR-54 strain of the genus Bacillus (JPCTC 10183PP) and enzyme protein having thrombolytic ability produced from this strain, patent application 2004-0019198 (pharmaceutical composition for thrombolysis containing Cheonggukjang viscous extract isolated from Cheonggukjang), Patent application 2005-0028064 (registered 10-0630272, instant cheoncheonggukjang prepared by fermenting naphtha bacteria and boiled soybeans with excellent thrombolytic ability) is known.

However, Cheonggukjang by these patent applications is the situation that the thrombolytic ability is still not expected. Accordingly, the present applicant has filed a patent application 2006-86676 (Bacillus subtilis NNT-0701 strain and Cheonggukjang produced by the present invention) for strains having excellent thrombolytic ability and Cheonggukjang prepared thereby.

 The present inventors have studied to provide Cheonggukjang that has better thrombolytic ability than Cheonggukjang known to date, and as a result of adding medicinal plant extracts having the above effects to fermentation of Cheonggukjang, fermentation of Cheonggukjang is greatly enhanced. The present invention has been completed.

In particular, when preparing Cheonggukjang fermentation or soybean medicinal plant fermentation solution using Bacillus sutilis NNT-0701 (KCCM 10674) strain previously filed by the applicant, unlike other strains, red ginseng, preferably red ginseng When fermented by adding wormwood, pine needles, ganoderma lucidum, and green tea, the thrombolytic function of soybean fermentation was found to be greatly enhanced to complete the present invention.

 According to an embodiment of the present invention, in the fermented product prepared by fermenting soybean, red ginseng is added to the soybean and Bacillus sutilis NNT-0701 (KCCM 10674) strain is used as a fermentation strain. A thrombolytic enhancing functional fermented product is provided. The fermented product may be in various forms, such as fermented soybean medicinal plant produced by lyophilizing soybean medicinal plant fermentation solution obtained by fermenting soybean oil (fermented soybean oil).

In addition, according to another embodiment of the present invention there is provided a method for producing a thrombolytic enhanced functional fermentation product obtained by solid state fermentation and liquid fermentation. In the case of solid phase fermentation, fermentation method of Cheonggukjang is generally used, but there is no big difference from the conventionally known method. Only medicinal plants such as red ginseng are added during fermentation. For example, as an example of the solid phase fermentation according to the present invention, the step of immersion by adding water mixed with red ginseng to soybean; Boiled soybeans for heating; Cook boiled soybeans and inoculate them with Bacillus sutilis NNT-0701 (KCCM 10674); The fermented soybean is fermented to obtain Chunggukjang. Specifically, red ginseng, mugwort, pine needles, ganoderma lucidum, Angelica, and water mixed with green tea 2 to 3 times by weight of soybeans are added for 3 to 5 hours soaking; Boiled soybeans on medium heat for about 2-4 hours and boiled until there is little water (which facilitates soybean penetration of medicinal plants); Life is 30-50 minutes steaming and soybean germs soybeans from 100 ℃ ~ 130 ℃ Bacillus subtilis (Bacillus sutilis) NNT-0701 (KCCM 10674), the culture medium 0.5 to 1.0 of boiled soybean weight (approximately 10 ⁸ cell / ml or less) Inoculating weight percent; The inoculated soybeans are fermented at 36 ° C. to 45 ° C. for 24 to 72 hours to obtain Cheonggukjang.

Liquid fermentation is different from solid fermentation in that it uses soybean liquid instead of solid soybeans. For example, preparing a soybean solution of cooked soybean added red ginseng; Inoculating the soy solution with Bacillus sutilis NNT-0701 (KCCM 10674); Shaking fermentation of the inoculated soy solution; The fermented soybean medicinal plant fermentation broth is lyophilized to obtain a soybean medicinal plant fermentation. Specifically, add red ginseng or red ginseng, mugwort, pine needles, ganoderma lucidum and green tea extract to 0.3-9.0% by weight (relative to soy solution) to the ground soybean juice mixed with cooked soybean or soybean and water. Sterilizing for 10-30 minutes at ℃ ~ 130 ℃; Inoculating Bacillus subtilis NNT-0701 (KCCM 10674) culture solution (approximately 10 ⁸ cells / ml) to 0.5-1.0 wt% of soybean solution to which medicinal plants are added; Shaking fermentation at 36 ° C. to 45 ° C. for 24 to 72 hours to obtain a soybean plant fermentation broth with thrombolytic ability; The soybean medicinal plant fermentation broth is lyophilized to obtain a soybean medicinal plant fermentation.

The medicinal plants used in the present invention may be used only with red ginseng, but it is more preferable to ferment by adding mugwort, pine needles, ganoderma lucidum, and Angelica. These red ginsengs, mugwort, pine needles, ganoderma lucidum, Angelica and green tea are both extract phases extracted from raw materials or powdered powders pulverized. Raw materials are more preferable. The extract phase can be produced directly by generally known means, but is currently commercially available. The powder phase needs to be added a lot for the purpose of thrombolytic activity, in which case it has a disadvantage in that the original function, such as Cheonggukjang.

In the case of extract phase, the addition amount is preferably 0.1 to 3.0% by weight based on soybeans, and in the case of powder form, the addition amount is preferably 1.0 to 10.0% by weight based on soybean. If the amount of the extract is too small, the thrombolytic ability is lowered, and if the amount is too high, there is a risk of inhibiting the growth of fermented strains.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these examples are only for illustrating the present invention in detail, and the scope of the present invention is not limited to these examples.

Bacillus subtilis NNT-0701 used in the present invention is similar to the morphological and biochemical properties of the general production of Cheonggukjang, but is characterized by excellent thrombolytic ability and excellent protease activity to degrade soy protein. In addition, it is possible to produce a Chungkukjang excellent in the ability to produce a viscous substance is also excellent strain thrombolytic ability.

< Example  1> Bacillus Subtilis ( Bacillus sutilis ) NNT -0701 ( KCCM  10674) Cheonggukjang Fermentation Using Strains

The soybeans were soaked with 3 times the weight of water and immersed for 18 to 20 hours, and then cooked at 115 ° C. for 40 minutes. The fermented Bacillus subtilis NNT-0701 (KCCM 10674) strain (10 ⁸ cell) / ml) was inoculated to 0.5% by weight of the cooked soybean fermented at 40 ℃ 48 hours to prepare a cheonggukjang.

< Example  2> Cheonggukjang Fermentation with Natural Medicinal Plant Extracts

The Cheonggukjang fermented bacterium Bacillus subtilis (Bacillus sutilis) NNT-0701 (KCCM 10674) strain can be prepared according to the following diagram to improve the thrombolytic ability Cheonggukjang. Red ginseng, mugwort, pine needles, Angelica, Ganoderma lucidum, green tea extract (available from Basan Co., Ltd.), each 0.5% by weight of the added soybeans, 3 times by weight of the weight of the soybeans added, for 1-3 hours After immersion, it is heated and heated until it disappears about 3 ~ 5 hours at medium heat and then it is increased for 40 minutes at 115 ℃. Bacillus sutilis NNT-0701 (KCCM 10674) strain culture solution (10 ⁸ / ml) was inoculated to 0.5% by weight of soybeans and fermented at 40 ° C for 48 hours to increase thrombolytic ability. Prepared cheongukjang.

       Scheme 1. Manufacturing method of Cheonggukjang added with medicinal plant extracts with enhanced thrombolytic ability

           Soybeans: Red ginseng, mugwort, pine needles, Angelica, ganoderma lucidum, green tea extract 0.5% by weight sap = 1: 3

               ↓

             Immersion: 1-3 hours

               ↓

             Heating: 3-5 hours. Until there is no moisture

               ↓

             Increase: 115 ℃, 40 minutes

               ↓

             Spawn: 0.5% of soy

               ↓

             Fermentation: 40 ℃, 48 hours

               ↓

      Acquired Cheonggukjang with enhanced thrombolytic ability

( Experimental Example  1) preparation of fermented extract of Cheonggukjang and Thrombolytic ability  Comparative measurement

The thrombolytic activity test was performed using Fibrin Plate method to confirm the thrombolytic activity of Fibrin solubility in the gel fibrin plate formed by Thrombin and fibrinogen treatment. )]. Fibrin plate was dissolved in 0.067M sodium phosphate buffer (pH 7.4) in 0.5% fibrinogen and 10ml was added to a petri dish 9cm in diameter. 0.1 ml of thrombin (100 NIH / mL) dissolved in 0.067 M sodium phosphate buffer (pH 7.4) was added thereto, mixed quickly, and a uniform plate was prepared. After standing at room temperature for 30 minutes, the fibrin plate was completely solidified. To Cheonggukjang fermented by the method of Example 1 and Example 2, 0.1M sodium phosphate buffer (pH 7.4) was added twice, shaken for about 1 hour, and the filtrate was filtered through a 0.45um filter. After incubating at 20 μL and incubating at 37 ° C. (10 hours incubation), the area of thrombolytic ring was measured to compare the thrombolytic activity. The results are shown in Table 1 and FIG. The resolution of artificial blood clot was nearly doubled in the Cheonggukjang fermented with the medicinal plant extract than the Cheonggukjang without the medicinal plant extract.

Bacillus Subtilis NNT -0701 ( KCCM  Cheonggukjang made with 10674 Thrombolytic ability  result  Sample Name  Diameter of melt ring (mm) Area of dissolution ring (r ² × π)  ratio  Increased soybeans (B)  0  0  0  Cheonggukjang without medicinal plant extract (C)  25.0  490.6  100  Cheonggukjang added with medicinal plant extract (S)  34.5  934.4  190

< Example  3> Natural medicinal plants Extract  Added liquid Soybean Fermentation  Produce

Red ginseng, mugwort, pine needles, ganoderma lucidum, and green tea extract (commercially available from Basan Co., Ltd.) Extracts) to each 0.5% by weight, each or all of the addition and sterilization at 121 ℃, 20 minutes, followed by the culture solution of Bacillus sutilis NNT-0701 (KCCM 10674) (10 ⁸ cell / ml) 0.5 wt% was inoculated. Then, shake fermentation at 40 ° C. for 48 hours to obtain a soybean medicinal water fermentation solution with enhanced thrombolytic ability.

( Experimental Example  2) Soybean Medication Of water fermentation liquid  Pharmacy and Thrombolytic ability  Comparative measurement

The thrombolytic ability of the soybean medicinal plant fermentation broth prepared in Example 3 was measured in the same manner as in Experimental Example 1 and compared with the soybean fermentation broth without adding the medicinal plant extract. The results are shown in Table 2 and FIG.

When the medicinal plant extract-free soybean fermentation had a thrombolytic ability of 100, the thrombolytic ability of the red ginseng extract-added soybean fermentation solution was 166%, and the soybean fermentation solution containing six extracts was added. 183% was similar to the results of the medicinal plants added Cheonggukjang of Experimental Example 1. When the medicinal plant extract was added as a single, the thrombolytic ability was enhanced when mixed.

Bacillus Subtilis NNT -0701 ( KCCM  10674) Soybean Medicinal Plant Fermentation Solution Thrombolytic ability  result Sample Name diameter( mm ) area( r ² × π) ratio C (no addition) 11.25 99.35 100  H (red ginseng extract) 14.50 165.05 166.13 SS  ( Mugwort extract ) 9.75 74.62 75.11 S (pine needle extract) 9.75 74.62 75.11  D (Dongsu extract) 12.15 115.88 116.64  Y (gear extract) 10.50 86.55 87.12  N (Green Tea Extract) 7.5 44.16 44.45 T (6 extracts mixed) 15.25 182.56 183.75

( Comparative example  1) of red ginseng itself Thrombolytic ability  Measure

The thrombolytic ability (18 hours incubation) was measured in the same manner as in Experimental Example 1 without adding the red ginseng extract extract to soybean liquid medium and fermented with the culture, and compared with the soybean fermentation solution containing the red ginseng extract extract. The results are shown in Table 3 and FIG. 3. When red ginseng extract extract was added to soybean liquid medium and fermented with bacteria (prepared in the same manner as in Example 3 except that 0.5% red ginseng extract was added to 20% soybean), thrombolysis occurred. It can be seen that thrombolysis does not occur.

Soybean 20 %in Red Ginseng Extract  According to addition (0.5%) Thrombolytic ability Sample Name Diameter 1 (mm) Diameter 2 (mm) Radius (r) Area (mm ² )  B 0 0 0 0  H-1 0 0 0 0  H 13.0 13.0 6.5 132.7

( Comparative example  2) Other strains  Used Thrombolytic Measure

Inoculated using BN-NUC1 strain (a strain isolated from Natto as Bacillus subtilis) as a fermentation strain, and cultured for 68 hr to produce liquid soybean fermentation broth with natural medicinal plant extract in the same manner as in Example 3 Soybean fermentation broth was prepared, and the thrombolytic ability was measured in the same manner as in Experimental Example 1. The results are shown in Table 4 and FIG. In contrast to the case of using the Bacillus subtilis (Bacillus sutilis) NNT-0701 (KCCM 10674) strain according to the present invention it can be seen that in the case of BN-NUC1 strain increased the thrombolytic ability of pine needles, Angelica, and red ginseng If the thrombolytic ability is not very high.

Other strains  Used Thrombolytic Measure Sample Name Diameter 1 mm ) Diameter 2 mm ) mean (r) area( r ² × π) control ratio%  C ( control ) 14.8 14 7.20 162.78 -  H (red ginseng) 16.2 16 8.05 203.48 125.00 SS  (Wormwood) 14 13.2 6.80 145.19 89.20  S (pine leaf) 16 15.5 7.88 194.73 119.63  D (Dangi) 17 16.2 8.30 216.31 132.89  Y (manor) 14 15 7.25 165.05 101.39  N (green tea) 15 15.2 7.55 178.99 109.96  T (mixed) 16 15.0 7.75 188.60 115.86

On the other hand, Bacillus as a fermentation strain Although licheniformis BNT11 strain was used and the thrombolytic activity was tested in 20% soybean medium, the thrombolytic activity was increased in mugwort, Angelica, ganoderma lucidum, pine mushroom and pine needles. Not high.

 Reference Example 1: Search for strains and identification of strains

The discovery and identification of Bacillus subtilis NNT-0701, a novel strain excellent in thrombolytic ability, mucus component formation, and protease activity and low in odor of Cheonggukjang, is as follows.

Rice straw grown in agrochemical and organic forms in Korea was obtained from Gyeongju, Pohang, Yeongdeok, Uljin, Yangsan, Sangju, Yeoju, Suwon, Namwon, and Wonju, soaked for 15 hours, and sterilized soybeans for 30 minutes at 121 ℃. Cheonggukjang was used to isolate strains with high lytic activity with large diameters of lysis zones formed by fibrinogen reacting with artificial blood clots formed by the reaction of thrombin.

Bacillus subtilis NNT-0701 used in the present invention is similar to the morphological and biochemical properties of the general production of Cheonggukjang, but is characterized by excellent thrombolytic ability and excellent protease activity to degrade soy protein.

Table 5 is a chart showing high activity of artificial thrombi of Bacillus subtilis NNT-0701 used in the present invention.

The measurement of fibrinolytic activity uses the usual method. For example, 6 ml of a 0.15% (w / v) fibrinogen solution is mixed well with a 0.2 ml thrombin (20 NIH / ml) solution and placed on a petri dish and left for 30 minutes to form a fibrin plate. 10 μl each of a 2 mg / ml viscous solution was added dropwise to the plate, and the resultant was left at 37 ° C. for 5 hours to compare the diameters of the dissolved fibrin areas formed. Meanwhile, the standard plasmin solution was prepared at 0.075, 0.15, 0.30, 0.45, 0.1 unit / ml, and the diameter of the lysis region formed by dropwise adding 10 µl to the fibrin plate was measured and compared with the activity of the viscous substance.

Fibrin lyzed activity of the isolate strain Bacillus subtilis NNT-0701 Experiment list Fibrin dissolution activity ( unit ) Experimental zone (Separated strain B. subtilis NNT-0701 culture solution) 0.541 Experimental zone (Cheonggukjang using isolated strain B. subtilis NNT-0701) 0.537 Imported Sickle A 0.438 Imported sickle B 0.356 Commercial Cheonggukjang A 0.163 Commercial Cheonggukjang B 0.149 B. subtilis KCTC culture 0.412 B. subtilis KCTC culture 0.394

Imported Natto A (Mitotado ^™ , Importer Monomart, Vendor E-mart)

Imported Sickle B (Organic Sickle ^™ , Importer Mono Mart, Vendor E-Mart)

Commercial Cheonggukjang A (Indigenous Cheonggukjang ^™ , manufacturer's native food)

Commercial Cheonggukjang B (Ojang Jant ^™ , Manufacturer Pulmuone)

Table 6 is a table showing the high extracellular protease activity of Bacillus subtilis NNT-0701 used in the present invention. As a method for measuring extracellular protease activity, the azocasein method proposed by Braun, V. and G. Schmitz was used.

Protease Activity of Bacillus subtilis NNT-0701 Experiment list Extracellular  Protease activity Experimental zone (Separated strain B. subtilis NNT-0701) 0.114 unit / mg protein B. subtilis KCTC 0.071 unit / mg protein B. subtilis KCTC 0.067 unit / mg protein

Reference Example 2 Identification of Strains

Physiological and culture characteristics of the isolated strain were investigated according to the Burgy's manual (Bergey's manual of systematic bacteriology, Palleroni, 1984) using YMG medium as a basic medium. Identification of the isolated strain was performed using rDNA sequence requested by KCCM, Yonsei University Biotechnology Research Institute.

The physiological and morphological characteristics of the strain of the present invention isolated by the above method are shown in Table 7.

Physiological and Morphological Characteristics of Bacterial Bacillus subtilis NNT-0701 List Bacillus subtilis Bacillus subtilis NNT -0701 shape Stick Forming Spores Stick Forming Spores Gram Dyeing positivity positivity Anaerobic growth + or- - Catalase + + Oxidase + + Glucose availability + + Arabinos + + Xylose + + Mannitol + + Growth at 2% NaCl + + 5% + + 7% + + 10% + + Growth at 5-10 ℃ - - 30-55 ℃ + + 65 ℃ - -

When the strain of the present invention is cultured in a YMG agar plate medium, it forms a milky white colony, it is possible to grow in a wide temperature range of 20 ~ 55 ℃, it is possible to grow in a wide pH range up to pH 4-11. The optimum culture conditions were 37 ~ 43 ℃ for temperature, and showed the best growth between 5.5 ~ 6 for pH. As a result of analyzing 16s rDNA sequence, homology between Bacillus subtilis and 16S rDNA sequence The sex was 99.0% (595 bp of 600 bp was the same). The 16S rDNA partial sequence of Bacillus subtilis NNT-0701 is shown in FIG. 5.

The strain Bacillus subtilis NNT-0701 of the present invention was identified as a novel strain of the conventional Bacillus subtilis through the physiological and culture characteristics of the strain, dated August 10, 2005, to the Korea Microorganism Conservation Center. Deposited with KCCM 10674P.

The effect of the present invention is red ginseng, as seen in the above embodiment, red ginseng, mugwort, pine needles, ganoderma lucidum, soybean and soybean juice with added green tea extract, when fermented with blue soybean bacilli, Bacillus subtilis NNT-0701 ( When inoculated and fermented with a culture medium of KCCM 10674P) it can be seen that a functional fermentation product with greatly enhanced thrombolytic ability is produced compared to other strains.

Claims (22)

A fermentation product prepared by fermenting soybeans, wherein the red ginseng is added to the soybeans, and the thrombolytic enhancing functional fermentation product is characterized by using Bacillus sutilis NNT-0701 (KCCM 10674) strain as a fermentation strain. The thrombolytic enhancing functional fermentation according to claim 1, which is fermented by further adding mugwort, pine needles, ganoderma lucidum, donkey and green tea. The thrombolytic enhancing functional fermentation according to claim 2, wherein the red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea are extract phases extracted from raw materials. The method of claim 3, wherein the red ginseng, mugwort, pine needles, ganoderma lucidum, Angelica and green tea extract is added in the amount of 0.1 ~ 3.0% by weight based on soybean thrombolytic enhanced functional fermentation. The thrombolytic enhancing functional fermentation according to claim 2, wherein the red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea are in powder form of crushed raw materials. The method of claim 5, wherein the amount of red ginseng, mugwort, pine needles, ganoderma lucidum, Angelica and green tea powder added amount is 1.0 ~ 10.0% by weight based on soybean thrombolytic enhanced functional fermentation. The thrombolytic enhancing functional fermentation according to any one of claims 1 to 6, wherein the fermentation product is Cheonggukjang. The fermentation product according to any one of claims 1 to 6, wherein the fermentation product is a soybean plant fermentation product prepared by freeze-drying the soybean plant fermentation broth. Immersing by adding water mixed with red ginseng; Heating soybeans soaked; Cooking soybeans boiled and inoculated with Bacillus sutilis NNT-0701 (KCCM 10674); A method for producing a thrombolytic-enhanced functional fermented product, comprising the step of fermenting the inoculated soybean to obtain Chungkookjang. 10. The method of claim 9, wherein in addition to the red ginseng in the immersion step, wormwood, pine needles, ganoderma lucidum, donkey and green tea are added to the method for producing a thrombolytic enhancing functional fermentation. The method according to claim 10, wherein the red ginseng, mugwort, pine needles, ganoderma lucidum, Angelica and green tea are extract phases extracted from raw materials. The method according to claim 11, wherein the amount of red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea extract is 0.1 to 3.0% by weight based on soybean. The method of claim 10, wherein the red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea are powdered crushed raw materials. The method according to claim 13, wherein the amount of red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea powder is 1.0 to 10.0% by weight based on soybean. 15. The method according to any one of claims 9 to 14, wherein red ginseng, mugwort, pine needles, ganoderma lucidum, and green tea are mixed with water soaked for 3 to 5 hours by adding 2 to 3 times by weight of soybean; Boiled soybeans on medium heat for about 2-4 hours and boiled until little moisture; Boiled soybeans at 100 ° C. to 130 ° C. for 30 to 50 minutes, and inoculating 0.5 to 1.0% by weight of a culture solution of Bacillus sutilis NNT-0701 (KCCM 10674); Fermented soybean inoculated at 36 ℃ ~ 45 ℃ 24 ~ 72 hours to obtain a cheonggukjang method for producing a thrombolytic enhanced functional fermentation, characterized in that. Preparing soybean solution of cooked soybean containing red ginseng; Inoculating the soy solution with Bacillus sutilis NNT-0701 (KCCM 10674); Shaking fermentation of the inoculated soy solution; A method for producing a thrombolytic enhanced functional fermented product, comprising the step of obtaining the fermented soybean medicinal plant fermentation broth by lyophilization. 17. The method of claim 16, wherein in the preparing of the soybean solution, mugwort, pine needles, ganoderma lucidum, donkey and green tea are further added to the red ginseng. The method according to claim 17, wherein the red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea are extract phases extracted from raw materials. The method according to claim 18, wherein the amount of red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea extract is 0.1 to 3.0% by weight based on soybean. 18. The method of claim 17, wherein the red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea are powdered crushed raw materials. 21. The method of claim 20, wherein the amount of red ginseng, mugwort, pine needles, ganoderma lucidum, donkey and green tea powder is 1.0 to 10.0 wt% based on soybean. The method according to any one of claims 16 to 21, Add red ginseng or red ginseng, wormwood, pine needles, pine needles, Angelica and green tea extract to 0.3 ~ 9.0 wt%, respectively, and sterilize at 110 ℃ ~ 130 ℃ for 10 ~ 30 minutes. Making; Inoculating 0.5 to 1.0% by weight of a culture solution of Bacillus sutilis NNT-0701 (KCCM 10674); Shaking fermentation at 36 ° C. to 45 ° C. for 24 to 72 hours to obtain a soybean plant fermentation broth with thrombolytic ability; Method for producing a thrombolytic enhanced functional fermentation, characterized in that the step of obtaining a soybean medicinal plant fermentation broth freeze-dried to obtain as a soybean medicinal plant fermentation.

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