WO2017222141A1 - Bacillus subtilis strain for high yield of thrombolytic enzymes - Google Patents

Abstract

The present invention provides a Bacillus subtilis strain (accession number KCTC13020BP) enabling a high yield of thrombolytic enzymes. A Bacillus subtilis strain GDN, according to the present invention, shows notably high thrombolytic enzyme-producing ability compared to existing natto bacteria such as Miyagino bacteria and thus can maintain an equal level of thrombolytic enzyme activities and adjust the ingestion amount of vitamin K2 within the threshold of the recommended daily allowance even when an ingestion amount of natto prepared by means of the Bacillus subtilis strain GDN of the present invention is only one-fifth of an ingestion amount of natto prepared by means of existing natto bacteria. Therefore, the Bacillus subtilis strain GDN can be effectively utilized for preparing various foods for thrombolysis.

Description

Thrombolytic enzyme-producing Bacillus subtilis strains

The present invention relates to a Bacillus subtilis strain that produces high thrombolytic enzymes.

Nattokinase, a thrombolytic enzyme, is a kind of serine protease produced by Bacillus subtilis , a bacterium involved in fermentation of natto, and its soft bacteria. Nattokinase is easily found in soybean fermented foods such as Korea's Cheonggukjang and Japan's Natto, and has strong fibrin-degrading activity. I am getting it.

The Bacillus subtilis is different from the case of culturing soybeans in a medium, but there is a difference, but accumulate thrombolytic enzymes in the medium using various substances as a substrate. Therefore, culturing Bacillus subtilis by various culture methods enables mass production of thrombolytic enzymes. Therefore, efforts have been made to supply nattokinase products at low cost (Japanese Patent Laid-Open No. 2001-299277, Japanese Patent Laid-Open No. 2006-325538).

Recently, research has been conducted on various methods for increasing the thrombolytic enzyme activity of natto, which can be easily consumed as food.

Natto's thrombolytic enzyme activity is known to be influenced by the type of fermentation bacteria, soybean varieties, culture conditions. However, it is possible to use varieties of soybeans that show high activity of thrombolytic enzymes in the manufacture of natto. For example, the busy is known as one of the medium to increase the activity of the thrombolytic enzyme, but mixing the fermented with the bean and soybean fermentation it has a great effect on the texture or appearance of natto is not a preferred method. In addition, the method of changing the culture conditions such as the incubation time, temperature and humidity can improve the activity of the thrombolytic enzyme, but this is a preferable method because it greatly affects the palatability such as taste, aroma and appearance of natto. none.

On the other hand, conventional Natto bacteria such as Miyagi and Takahashi bacteria are known to exhibit different thrombolytic enzyme activity under the same culture conditions. In addition, in the related art, a description of a novel strain having a two-fold increase in the activity of thrombolytic enzyme (Japanese Patent 3564121) compared to commercial natto bacteria has been disclosed. In order to mass-produce thrombolytic enzymes, the development of a strain having high activity is the best. It can be seen that it is an efficient method.

However, when ingesting natto for the purpose of thrombolytic dissolution, side effects due to vitamins such as K2 produced and accumulated in natto by natto bacteria should be considered. Vitamin K2 contained in natto is menaquinone-7 and is known to promote blood coagulation and bone formation. In general, natto contains 600 to 900 μg / 100 g of vitamin K2 and is classified as a high vitamin K2 food. For reference, the recommended daily intake per adult is 75 μg for men, 60-65 μg for women (Journal of Nutritional Science and Vitaminology, 2007, Vol. 53, 464-470) and 120 μg for men in the United States. , Females are 90 μg (J Am Diet Assoc., 1996, vol. 96, 149-154). Thus, when eating a typical natto 1 casing packaged in a volume of 40 to 50 g, a significant amount of vitamin K2 corresponding to arithmetic 300 to 450 μg is consumed. In addition, in the case of taking warfarin, one of the thrombogenic inhibitors, vitamin K2 inhibits the action of warfarin, thereby limiting the intake of natto. As described above, in order to solve the problem of vitamin K2 contained in natto, conventionally, natto is produced by introducing a purification step of removing vitamin K2 contained in natto using an organic solvent such as ethanol (JP 2006-325538 A). , Japanese Patent Application Laid-Open No. 2001-299277), and the purification step as described above has a problem that the manufacturing cost increases due to a complicated process.

As described above, ingestion of natto has the advantage of being able to provide a sufficient amount of thrombolytic enzymes at a cheaper price than purified enzymes while enjoying the original taste and aroma of food, but the content of vitamin K2 is emerging as a problem, Natto bacteria, etc., which are known in the art, have disadvantages of poor palatability when used as a medium, and a study on a method for solving such a problem is required.

[Preceding technical literature]

[Patent Documents]

(Patent Document 1) Japanese Laid-Open Patent 2001-299277 (Published Date: 2001.10.30)

(Patent Document 2) Japanese Patent Laid-Open No. 2006-325538 (published date: 2006.12.07)

(Patent Document 3) Japanese Patent No. 3564121 (Published Date: 2004.08.12)

(Patent Document 4) Korean Patent Publication 10-2014-0123847 (Published Date: 2014.10.23)

The inventors of the present invention have been intensively researched on the production of natto with high activity of thrombolytic enzymes, and as a result, a Bacillus subtilis GDN strain, which is a new strain having higher ability to produce thrombolytic enzymes than the conventionally developed natto strains, has been studied. Was successfully isolated from Bacillus subtilis GDN, and it was confirmed that the thrombolytic enzyme activity was increased more than the soybean fermented product in case of fermented cultivars in the cultivars. It turns out that you can produce with.

Thus, the present invention is to provide a description of the Bacillus subtilis GDN strain that produces a high thrombolytic enzyme nattokinase.

In order to achieve the above technical problem, the present invention provides a Bacillus subtilis GDN strain (Accession No. KCTC13020BP) which produces high thrombolytic enzymes.

In addition, the strain is characterized in that it comprises 16s RNA of SEQ ID NO: 1.

The present invention also provides a culture fermented using the strain described above.

In addition, the culture may be natto, the thrombolytic enzyme contained in the culture may be nattokinase.

In addition, the culture may be a busy fermentation, the busy fermentation is characterized in that the thrombolytic enzyme activity of 3400 to 4300 fibrinolytic unit / g.

The present invention also provides a food composition comprising the culture described above.

The present invention also provides a method for producing a thrombolytic enzyme comprising the step of fermentation using the strain described above.

In addition, the fermentation is characterized in that the fermentation of natto, characterized in that natto kinase is produced by the fermentation of the natto.

In addition, the fermentation is characterized in that the fermentation of raw busy.

The Bacillus subtilis GDN strain according to the present invention has a significantly higher production capacity of thrombolytic enzymes than conventional natto bacteria such as Miyagi, and when ingesting natto prepared with the Bacillus subtilis GDN strain of the present invention, Compared to the intake of natto prepared by conventional natto bacteria, even if the intake of natto is reduced to about one fifth, the activity of the thrombolytic enzyme can be kept the same, and the intake of vitamin K2 is the limit of the recommended daily dose. The Bacillus subtilis GDN strain can be adjusted to be effectively utilized for the production of a variety of food for the purpose of thrombolysis.

In addition, in the fermented product of the Bacillus subtilis GDN strain according to the present invention, the thrombolytic enzyme activity is 1.5 times higher than that of the soybean fermented product, and the thrombolytic enzyme can be efficiently obtained through solid culture without further treatment. It is able to produce chlorophyllase, and can easily purify the thrombolytic enzyme through simple operation from the medium, which not only drastically reduces the production cost of thrombolytic enzyme, but also is eco-friendly in that it can recycle waste paper, industrial waste. .

In addition, the strain produces a large amount of α-glucosidase inhibitor which is effective in lowering blood sugar when fermenting soybeans or raw beef, which is effective in the prevention of adult diseases, as well as in the production of sensory properties such as taste, viscosity, appearance, etc. This excellent natto can be manufactured.

1 is an electron micrograph of the Bacillus subtilis GDN strain.

2 is a schematic showing the taxonomic location of Bacillus subtilis GDN strains.

Hereinafter, the present invention will be described in detail.

The present invention provides a Bacillus subtilis GDN strain (Accession No. KCTC13020BP) which produces high thrombolytic enzymes.

The strain can be obtained by separating the bacteria that form heat-resistant spores from rice straw and hay, and selecting strains that produce high-content thrombolytic enzymes during fermentation, such as legumes or raw waste. The selected strains produce thrombolytic enzymes, in particular, nattokinase with high content, and can produce natto having a thrombolytic enzyme activity that is five times higher than in the case of using a general strain in fermentation of natto.

Accordingly, when ingesting natto prepared with the Bacillus subtilis GDN strain of the present invention, compared to the case of ingesting natto prepared by conventional natto bacteria, even if the intake of natto is reduced to about one fifth, thrombi The activity of the lytic enzyme can be kept the same, and the intake of vitamin K2 can be adjusted within the limits of the recommended daily dose, so that the Bacillus subtilis GDN strain can be utilized for the preparation of various foods for the purpose of thrombolysis.

In addition, in the fermented product of the Bacillus subtilis GDN strain according to the present invention, the thrombolytic enzyme activity is 1.5 times higher than that of the soybean fermented product, and the thrombolytic enzyme can be efficiently obtained through solid culture without further treatment. It is able to produce chlorophyllase, and can easily purify the thrombolytic enzyme through simple operation from the medium, which not only drastically reduces the production cost of thrombolytic enzyme, but also is eco-friendly in that it can recycle waste paper, industrial waste. .

 The strain produces a large amount of α-glucosidase inhibitors that are effective in lowering blood sugar when fermenting soybeans or raw beef, which is effective for the prevention of adult diseases, and also has excellent sensory properties such as taste, viscosity, and appearance. Natto can be prepared.

The strain is characterized by having the 16s RNA of SEQ ID NO: 1.

SEQ ID NO 1:

GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGT

In addition, the present invention provides a culture fermented using the Bacillus subtilis GDN strain.

Preferably, the culture may be a fermentation of fermented by inoculating the strain into live busy fermentation, the fermented fermentation may exhibit excellent thrombolytic enzyme activity of 3400 to 4300 fibrinolytic unit / g.

In addition, the culture may be a culture of the beans fermented by inoculating the strain to the beans, preferably, may be natto. Natto fermented by inoculating the strain exhibits thrombolytic enzyme activity of 2300 to 2500 fibrinolytic units / g, compared to natto (400 to 500 fibrinolytic units / g) inoculated with conventional inoculations of natto bacteria such as microorganisms. It has more than 5 times better thrombolytic enzyme activity.

In addition, the present invention provides a food composition comprising the culture.

In addition, the present invention provides a method for preparing a thrombolytic enzyme comprising the step of fermentation using the strain. In the method for preparing thrombolytic enzymes, fermented raw paper can be used to produce fermented bean curds, fermented various soybeans to produce soybean fermented products such as natto, and the soybean fermented product is nattokinase as a thrombolytic enzyme. It may include.

In addition, the busy fermentation product exhibits excellent thrombolytic enzyme activity, and the method for preparing the thrombolytic enzyme can efficiently produce thrombolytic enzymes through solid culture without further treatment by using the industrial waste paper, from the medium. The thrombolytic enzyme can be easily purified through simple manipulation, and thus can be utilized for manufacturing various foods for the purpose of thrombolysis.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The examples presented are merely illustrative of the invention and are not intended to limit the scope of the invention.

< Example >

1. Search for Strains

About 500 rice straws and hay collected from around the world, such as Korea, China, Japan, and the United States, microorganisms were isolated. Each sample was added to a small amount of sterile saline and suspended, followed by heat treatment for 30 minutes in a constant temperature water bath at 80 ° C. LB plate medium containing 2% agar (1% tryptone, 0.2% sugar, 0.5% yeast extract and 0.5%) % NaCl, pH7.0), and incubated in a 37 ° C incubator for 1 day, the cells forming the colonies were isolated.

To examine the production of thrombolytic enzymes, the isolated strains were inoculated in a medium suspended with 5 ml of 5% soy flour and shaken at 37 ° C. for 24 hours. The culture was centrifuged to measure the thrombolytic enzyme activity from the supernatant by the fibrin plate method.

The fibrin plate method described above was performed by the method shown below.

First, 0.1 M sodium phosphate buffer (pH8.5) in which 2.5 ml of 1.2% fibrinogen solution, 500 μl of 20 units / ml of thrombin, and 7 ml of 1% agarose solution were dissolved. Fibrin plates were prepared by placing them in a petri dish and solidifying them evenly. The Pasteur pipette was used to form voids at regular intervals in the fibrin plate. 2 μl of the sample prepared by culturing each strain was injected into the pores and cultured at 37 ° C., and the activity of the thrombolytic enzyme was measured from the area of the formed clear zone.

Through the above process, 80 kinds of strains were isolated in the order of the strongest activity, and then selected as thrombolytic enzyme-producing bacteria first, and then the simple identification method (The prokaryotes, A handbook on the biology of bacteria, vol. 2. p. .1668, 1992, Springer-Verlag), two strains with high thrombolytic enzyme activity were selected. Five isolates were identified as Bacillus subtilis through 16s RNA sequencing and confirmed to have the nucleotide sequence of SEQ ID NO: 1. One strain with the highest thrombolytic enzyme activity was named Bacillus subtilis GDN and was deposited to Korea Biotechnology Research Center (KCTC) on May 3, 2016 (Accession Number: KCTC13020BP). .

SEQ ID NO 1:

GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGT

In addition, the isolated strain was observed under a microscope (see Fig. 1), and the morphological characteristics are shown in Table 1 below.

As shown in Table 1, the isolated strain was Gram-positive, and the cell size was found to be approximately 0.6-0.7 × 1.4-1.6 μm.To summarize the above results, the taxonomy of the Bacillus subtilis GDN strain The location is shown in FIG. 2.

In addition, the results of biochemical characterization of the isolated strains are shown in Table 2 below.

2. Using Fibrin Solution Thrombolytic enzyme  Active measurement

100 μl of the diluted sample, 200 μl of 0.1M Tris-HCl buffer (containing 10 mM CaCl2, pH7.8), and 200 μl of 0.4% fibrin solution (pH 7.0) were mixed and reacted at 37 ° C. for 30 minutes. After the reaction, 500 μl of a reaction stop solution (0.22M sodium acetate, 0.11M trichloroacetic acid including 0.33M acetic acid) was added and centrifuged to obtain a supernatant. The absorbance was measured by irradiating light of 275 nm wavelength to the obtained supernatant (wherein 1 unit of thrombolytic enzyme activity was equal to the amount of 1 μg of tyrosine produced at 1 ° C. for 1 minute).

3. Beans Fermented product  Manufacture and Thrombolytic enzyme  Activity analysis

The washed beans were soaked overnight at room temperature, and then autoclaved at 121 ° C. for 40 minutes to make boiled beans. Boiled soybeans were inoculated prior to cooking at a rate of 1 ml of Bacillus subtilis GDN strain spore solution (10 ⁵ spores / ml) per 50 g of boiled soybeans, placed in a foamed styrene container, and incubated at 37 ° C. for 18 hours. Thereafter, aged for 24 hours at 4 ℃ to prepare a soybean fermented product. A commercial Miyagi-no-gyun was used as a control.

The activity of the thrombolytic enzymes of the prepared soybean fermentation was measured, and the results are shown in Table 3 below.

As shown in Table 3, as a result of measuring the activity of thrombolytic enzyme in soybean fermented product, the thrombolytic enzyme activity of soybean fermented with GDN bacterium of Example was increased about 5 times the enzyme activity compared to Miyagi-no, which is a control group. I could confirm it.

That is, the GDN bacterium of the embodiment has a significantly higher production capacity of thrombolytic enzymes compared to the conventional natto bacteria, and thus, natto prepared by the bacterium is reduced to about one fifth of the intake of the conventional natto, even if ingested by conventional natto 1 It was confirmed that the thrombolytic enzyme could be supplied from the case.

For reference, the daily recommended amount of vitamin K2 is 75 μg for men in Japan, and 6 to 65 μg for women, but 300 to 450 μg of vitamin K2 is supplied when a case of natto made of conventional natto bacteria is ingested. It is about five times the recommended daily amount. However, the natto prepared with the GDN strain of Example can maintain the thrombolytic enzyme activity and control the amount of vitamin K within the limit of the recommended daily amount even if the intake of 1/5 tablets of the conventional natto described above. I could confirm that there is.

3. Sensory evaluation

The sensory evaluation was performed on the soybean fermented product obtained by the above method. The sensory evaluation was performed by a five-step evaluation method. Five points were very good, four points were good, three points were normal, two points were bad, and one point was very bad, and the following five items were evaluated. It is shown in Table 4.

As shown in Table 4, as a result of performing the sensory evaluation, the sensory evaluation of the soybean fermentation produced by the GDN strain can be a good product even if developed as a soybean fermentation such as natto with a high score of 3.6 to 4.8 points Could confirm.

4. Busy Fermented product  Produce

Autoclave sterilized fresh raw paper (3 types) on the same day at 121 ℃ for 40 minutes, inoculate 10 ⁵ spores / ml spore solution per 50 g of sterilized busy paper at a rate of 1 ml and incubate at 37 ℃ for 24 hours. It was.

The thrombolytic enzyme activity of the prepared busy fermentation products (3 types) was measured, and the results are shown in Table 5 below.

As shown in Table 3, as a result of measuring the thrombolytic enzyme activity of the sebaceous fermentation produced by fermenting the sewage produced at three different factories, it was confirmed that the fermented fermented product was approximately 1.5 times higher than the soybean fermented product. It became. Through this, it was confirmed that it can be efficiently used for the production of thrombolytic enzymes through solid culture without additional treatment using the industrial waste paper.

5. Busy In fermentation Thrombolytic enzyme  refine

The paper was placed in a stainless steel tray of 30 cm x 24 cm x 6 cm in 500 g each and autoclaved at 121 ° C. for 40 minutes. 20 ml of spore solution of 10 ⁵ spores / ml per 1 kg of sterilized bean curd was added before inoculation, inoculated with vinyl film, and cultured at 37 ° C. for 24 hours to prepare a fermented bean curd product. Three times the amount of water was added to the prepared fermented fermented product, and the mixture was stirred well to extract thrombolytic enzymes. Here it was then added and the CaCl ₂ powder for 30 minutes to have a final concentration of CaCl ₂ to a 0.1M, centrifuged to remove the precipitate and the supernatant liquid produced in the busy extract.

In addition, twice the amount of ethanol was added to the supernatant, centrifuged, and the precipitate was lyophilized to prepare a thrombolytic enzyme powder. The thrombolytic enzyme activity was measured and the results are shown in Table 6 below with the yields.

As shown in Table 6, it was confirmed that the calcium (CaCl ₂₎ serum enzyme activity of the soluble extract a busy treatment very high. Through this, homogenize three times the weight of water to the fermented product of GDN strain by homogenization, add CaCl ₂ powder to final concentration of 0.1M, and leave it for 30 minutes to solidify from the bean and then easily remove by precipitation. The thrombolytic enzyme remained in the clear supernatant, so the purification of the thrombolytic enzyme can be confirmed by a high yield even by a simple operation.

Bacillus subtilis GDN strain according to the present invention is significantly high production capacity of thrombolytic enzymes, can be utilized in the production of various foods for the purpose of thrombolysis.

[Accession number]

Name of institution: Korea Biotechnology Research Institute Korea Bioresource Center (KCTC)

Accession number: KCTC13020BP

Deposit date: 20160503

[Deposit]

[Revision 10.04.2017 under Rule 26]
deleted

Claims (12)

1. Bacillus subtilis GDN strain (KCTC13020BP) producing high thrombolytic enzymes.
2. The strain according to claim 1, wherein the strain comprises 16s RNA of SEQ ID NO: 1.
3. A culture fermented using the strain of any one of claims 1 to 2.
4. The culture according to claim 3, wherein the culture is natto.
5. The culture according to claim 4, wherein the thrombolytic enzyme contained in the culture is nattokinase.
6. The culture according to claim 3, wherein the culture is busy fermentation.
7. The culture according to claim 6, wherein the busy fermentation product exhibits thrombolytic activity of 3400 to 4300 fibrinolytic units / g.
8. A food composition comprising the culture of claim 3.
9. A method for preparing a thrombolytic enzyme comprising the step of fermenting using the strain of any one of claims 1 to 2.
10. 10. The method of claim 9, wherein the fermentation is fermentation of natto.
11. The method for producing thrombolytic enzymes according to claim 10, wherein nattokinase is produced by fermentation of the natto.
12. 10. The method of claim 9, wherein the fermentation is fermentation of live busy.

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