WO2017222141A1 - Bacillus subtilis strain for high yield of thrombolytic enzymes - Google Patents
Bacillus subtilis strain for high yield of thrombolytic enzymes Download PDFInfo
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- WO2017222141A1 WO2017222141A1 PCT/KR2017/001753 KR2017001753W WO2017222141A1 WO 2017222141 A1 WO2017222141 A1 WO 2017222141A1 KR 2017001753 W KR2017001753 W KR 2017001753W WO 2017222141 A1 WO2017222141 A1 WO 2017222141A1
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- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- the present invention relates to a Bacillus subtilis strain that produces high thrombolytic enzymes.
- Nattokinase a thrombolytic enzyme
- 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).
- Natto's thrombolytic enzyme activity is known to be influenced by the type of fermentation bacteria, soybean varieties, culture conditions.
- soybean varieties that show high activity of thrombolytic enzymes in the manufacture of natto.
- 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.
- 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.
- Vitamin K2 contained in natto is menaquinone-7 and is known to promote blood coagulation and bone formation.
- natto contains 600 to 900 ⁇ g / 100 g of vitamin K2 and is classified as a high vitamin K2 food.
- the recommended daily intake per adult is 75 ⁇ g for men, 60-65 ⁇ g for women (Journal of Nutritional Science and Vitaminology, 2007, Vol. 53, 464-470) and 120 ⁇ g for men in the United States.
- 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).
- JP 2006-325538 A Japanese Patent Application Laid-Open No. 2001-299277
- the purification step as described above has a problem that the manufacturing cost increases due to a complicated process.
- 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)
- 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.
- Bacillus subtilis GDN strain which is a new strain having higher ability to produce thrombolytic enzymes than the conventionally developed natto strains.
- 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.
- the present invention is to provide a description of the Bacillus subtilis GDN strain that produces a high thrombolytic enzyme nattokinase.
- the present invention provides a Bacillus subtilis GDN strain (Accession No. KCTC13020BP) which produces high thrombolytic enzymes.
- 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.
- the culture may be natto
- the thrombolytic enzyme contained in the culture may be nattokinase.
- 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.
- the fermentation is characterized in that the fermentation of natto, characterized in that natto kinase is produced by the fermentation of the natto.
- 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.
- 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 ⁇ -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.
- FIG. 2 is a schematic showing the taxonomic location of Bacillus subtilis GDN strains.
- 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.
- 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 ⁇ -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.
- the present invention provides a culture fermented using the Bacillus subtilis GDN strain.
- 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.
- 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.
- the present invention provides a food composition comprising the culture.
- the present invention provides a method for preparing a thrombolytic enzyme comprising the step of fermentation using the strain.
- 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.
- the busy fermentation product exhibits excellent thrombolytic enzyme activity
- 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.
- 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.
- 0.1 M sodium phosphate buffer pH8.5 in which 2.5 ml of 1.2% fibrinogen solution, 500 ⁇ l 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 ⁇ l 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.
- the isolated strain was Gram-positive, and the cell size was found to be approximately 0.6-0.7 ⁇ 1.4-1.6 ⁇ m.
- Table 1 the taxonomy of the Bacillus subtilis GDN strain The location is shown in FIG. 2.
- 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 °C to prepare a soybean fermented product.
- a commercial Miyagi-no-gyun was used as a control.
- 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.
- 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.
- the daily recommended amount of vitamin K2 is 75 ⁇ g for men in Japan, and 6 to 65 ⁇ g for women, but 300 to 450 ⁇ g 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.
- 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.
- 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.
- 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.
- the thrombolytic enzyme activity of the prepared busy fermentation products (3 types) was measured, and the results are shown in Table 5 below.
- 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.
- 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.
- thrombolytic enzyme activity was measured and the results are shown in Table 6 below with the yields.
- 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.
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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
본 발명은 혈전용해효소를 고생산하는 바실러스 서브틸리스 균주에 관한 것이다.The present invention relates to a Bacillus subtilis strain that produces high thrombolytic enzymes.
혈전용해효소인 낫토키나아제(nattokinase)는 낫토의 발효에 관여하는 균인 바실러스 서브틸리스(Bacillus
subtilis) 및 그 유연균에 의해 생산되는 세린 단백질 가수분해효소(serine protease)의 일종이다. 낫토키나아제는 콩 발효식품인 한국의 청국장, 일본의 낫토 등에서 쉽게 확인할 수 있으며, 강력한 피브린(fibrin) 분해 활성을 가지므로 혈전의 용해에 유효하다고 알려져 청국장과 낫토의 중요한 기능성을 나타내는 성분의 하나로 각광을 받고 있다. 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.
상기한 바실러스 서브틸리스는 콩을 배지로 배양한 경우 이외에도, 차이는 있으나, 다양한 물질을 기질로 하여 배지 중에 혈전용해효소를 축적한다. 따라서, 바실러스 서브틸리스를 다양한 배양 방법으로 배양하면 혈전용해효소의 대량 생산이 가능하므로, 낫토키나아제 제품을 염가로 공급하기 위하여 노력을 하고 있다(일본특개 2001-299277, 일본특개 2006-325538).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).
최근, 식품으로 간편하게 섭취할 수 있는 낫토의 혈전용해효소 활성을 높이기 위한 다양한 방법에 관한 연구가 진행되고 있다.Recently, research has been conducted on various methods for increasing the thrombolytic enzyme activity of natto, which can be easily consumed as food.
낫토의 혈전용해효소 활성은 발효균의 종류, 콩의 품종, 배양 조건 등에 의해 영향을 받는 것으로 알려져 있다. 하지만, 낫토 제조시 혈전용해효소의 활성이 높게 나타나는 콩의 품종을 사용하는 것은 가능하나, 콩의 품종을 제한하는 방법으로 동일한 품질의 낫토를 대량 생산한다는 측면에서는 제한성이 있다. 일례로, 비지는 혈전용해효소의 활성을 높게 해주는 배지의 하나로 알려져 있으나 비지를 콩과 함께 섞어 발효시키는 것은 낫토의 식감이나 외형에 큰 영향을 미치게 되므로 바람직한 방법은 되지 않는다. 또한, 배양시간, 온도 및 습도 등의 배양 조건을 변화시키는 방법은 혈전용해효소의 활성을 제고할 수 있으나 이는 낫토의 맛, 향, 외관과 같은 기호성에 지대한 영향을 미치게 되므로 바람직한 방법이라고는 할 수 없다.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.
한편, 기존의 미야기노균 또는 타카하시균 등의 시판 낫토균은 같은 배양 조건에서 서로 다른 혈전용해효소 활성을 나타내는 것으로 알려져 있다. 또한, 종래에는 시판 낫토균에 비하여 혈전용해효소(일본특허 3564121) 활성이 2배 증가한 신규 균주에 관한 기술 내용이 개시된 바 있어, 혈전용해효소를 대량 생산하기 위하여서는 활성이 높은 균주의 개발이 가장 효율적인 방법임을 알 수 있다. 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.
그러나, 혈전 용해를 목적으로 낫토를 섭취하는 경우에는, 낫토균에 의하여 낫토에 생성 및 축적되는 K2 등의 비타민에 의한 부작용을 고려하여야만 한다. 낫토에 함유된 비타민 K2는 메나퀴논-7(menaquinone-7)으로 혈액응고를 촉진하며 골 형성을 촉진하는 인자로서 알려져 있다. 일반적으로, 낫토에는 600 내지 900μg/100g의 비타민 K2가 함유되어 비타민 K2 고함량 식품으로 분류되어 있다. 참고로, 성인 1인당 1일 권장 섭취량은 일본의 경우 남성은 75μg이고, 여성은 60 내지 65μg이며(Journal of Nutritional Science and Vitaminology, 2007, Vol. 53, 464-470), 미국의 경우 남성은 120μg, 여성은 90μg(J Am Diet Assoc., 1996, vol.96, 149-154)이다. 따라서, 40 내지 50g의 내용량으로 포장되어 시판되는 통상적인 낫토 1 캐이스(case)를 취식하는 경우, 산술적으로 300 내지 450μg에 해당하는 상당한 양의 비타민 K2가 동반 섭취되게 된다. 또한, 혈전형성억제제의 하나인 와파린을 복용하는 환자의 경우에 비타민 K2가 와파린의 작용을 억제하므로 낫토의 섭취를 제한하고 있다. 상기한 바와 같이 낫토에 포함된 비타민 K2의 문제점을 해소하기 위하여 종래에는 에탄올 등의 유기용매를 이용하여 낫토에 포함된 비타민 K2를 제거하는 정제 단계를 도입하여 낫토를 생산하고 있으나(특개 2006-325538, 특개 2001-299277), 상기와 같은 정제 단계는 공정이 복잡하여 제조비용이 증가하는 문제가 있다.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.
상기한 바와 같이, 낫토의 섭취는 식품 본래의 맛과 향을 즐기면서도, 정제한 효소보다는 충분한 양의 혈전용해효소를 염가로 제공받을 수 있는 장점이 있으나 비타민 K2의 함량이 문제점으로 대두되고 있고, 종래에 알려진 낫토균 등은 비지를 배지로 활용할 경우 기호성이 떨어지는 단점이 있어, 이와 같은 문제점을 해결할 수 있는 방법에 대한 연구가 필요하다.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.
[선행기술문헌][Preceding technical literature]
[특허문헌][Patent Documents]
(특허문헌 1) 일본공개특허 2001-299277 (공개일 : 2001.10.30)(Patent Document 1) Japanese Laid-Open Patent 2001-299277 (Published Date: 2001.10.30)
(특허문헌 2) 일본공개특허 2006-325538 (공개일 : 2006.12.07)(Patent Document 2) Japanese Patent Laid-Open No. 2006-325538 (published date: 2006.12.07)
(특허문헌 3) 일본등록특허 3564121 (공개일 : 2004.08.12)(Patent Document 3) Japanese Patent No. 3564121 (Published Date: 2004.08.12)
(특허문헌 4) 한국공개특허 10-2014-0123847 (공개일 : 2014.10.23)(Patent Document 4) Korean Patent Publication 10-2014-0123847 (Published Date: 2014.10.23)
본 발명의 발명자들은 혈전용해효소의 활성이 높은 낫토의 제조를 목표로 예의 연구해 온 결과, 종래에 개발되었던 낫토 균주보다 혈전용해효소를 고생산하는 능력을 갖는 새로운 균주인 바실러스 서브틸리스 GDN 균주(Bacillus
subtilis GDN)를 분리하는데 성공하였으며, 해당 균주를 비지에서 배양한 비지 발효물의 경우, 혈전용해효소 활성이 콩 발효물에 비해 더욱 증가한다는 사실을 확인하여 고체배양을 통하여도 혈전용해효소를 효율적으로 생산할 수 있음을 알게 되었다.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.
이에, 본 발명에서는 혈전용해효소인 낫토키나아제를 고생산하는 바실러스 서브틸리스 GDN 균주에 대한 기술 내용을 제공하고자 한다.Thus, the present invention is to provide a description of the Bacillus subtilis GDN strain that produces a high thrombolytic enzyme nattokinase.
상기한 바와 같은 기술적 과제를 달성하기 위해서 본 발명은, 혈전용해효소를 고생산하는 바실러스 서브틸리스 GDN 균주(Bacillus
subtilis GDN, 수탁번호KCTC13020BP)를 제공한다.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.
또한, 상기 균주가 서열번호 1의 16s RNA를 포함하는 것을 특징으로 한다.In addition, the strain is characterized in that it comprises 16s RNA of SEQ ID NO: 1.
또한, 본 발명은 상기에 기재된 균주를 이용하여 발효시킨 배양물을 제공한다.The present invention also provides a culture fermented using the strain described above.
또한, 상기 배양물은 낫토일 수 있으며, 상기 배양물에 포함된 혈전용해효소가 낫토키나아제일 수 있다.In addition, the culture may be natto, the thrombolytic enzyme contained in the culture may be nattokinase.
또한, 상기 배양물은 비지 발효물일 수 있으며, 상기 비지 발효물은 3400 내지 4300 fibrinolytic unit/g의 혈전용해효소 활성을 나타내는 것을 특징으로 한다.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.
또한, 본 발명은 상기에 기재된 배양물을 포함하는 식품 조성물을 제공한다.The present invention also provides a food composition comprising the culture described above.
또한, 본 발명은 상기에 기재된 균주를 이용하여 발효시키는 단계를 포함하는 혈전용해효소의 제조방법을 제공한다.The present invention also provides a method for producing a thrombolytic enzyme comprising the step of fermentation using the strain described above.
또한, 상기 발효가 낫토의 발효인 것을 특징으로 하며, 상기 낫토의 발효에 의해 낫토키나아제가 제조되는 것을 특징으로 한다.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.
또한, 상기 발효가 생비지의 발효인 것을 특징으로 한다.In addition, the fermentation is characterized in that the fermentation of raw busy.
본 발명에 따른 바실러스 서브틸리스 GDN 균주는, 미야기노균 등과 같은 종래의 낫토균에 비해 혈전용해효소의 생산 능력이 현저히 높아, 본 발명의 바실러스 서브틸리스 GDN 균주로 제조한 낫토를 섭취할 경우, 종래의 낫토균에 의해 제조한 낫토를 섭취하는 경우에 비해, 낫토의 섭취량을 5분의 1정도로 줄이더라도, 혈전용해효소의 활성은 동일하게 유지할 수 있고, 비타민 K2의 섭취량은 1일 권장량의 한계치 내로 조절할 수 있어 상기 바실러스 서브틸리스 GDN 균주는 혈전용해를 목적으로 한 다양한 식품의 제조를 위해 효과적으로 활용이 가능하다. 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.
또한, 본 발명에 따른 바실러스 서브틸리스 GDN 균주를 배양한 비지 발효물의 경우, 콩 발효물에 비해 혈전용해효소 활성이 1.5배 이상 높은 특성을 보여, 추가적인 처리없이 고체배양을 통해 효율적으로 혈전용해효소를 생산할 수 있고, 배지로부터 간단한 조작을 통해 혈전용해효소를 손쉽게 정제할 수 있어, 혈전용해효소의 생산비용을 획기적으로 절감할 수 있을 뿐만 아니라, 산업 폐기물인 비지를 재활용할 수 있다는 점에서 친환경적이다. 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. .
아울러, 상기 균주는 콩류 또는 생비지 등을 발효시 혈당강하에 효과가 있는 α-글루코시다아제 저해제를 다량 생성하여 성인병 예방에도 효과적인 식품 제조가 가능할 뿐만 아니라, 맛, 점질물 강도, 외형 등의 관능 특성이 우수한 낫토를 제조할 수 있다.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.
도 1은 바실러스 서브틸리스 GDN 균주의 전자현미경 사진이다.1 is an electron micrograph of the Bacillus subtilis GDN strain.
도 2는 바실러스 서브틸리스 GDN 균주의 분류학상 위치를 나타낸 개략도이다.2 is a schematic showing the taxonomic location of Bacillus subtilis GDN strains.
이하, 본 발명을 상세히 설명하도록 한다.Hereinafter, the present invention will be described in detail.
본 발명은, 혈전용해효소를 고생산하는 바실러스 서브틸리스 GDN 균주(Bacillus subtilis GDN, 수탁번호KCTC13020BP)를 제공한다.The present invention provides a Bacillus subtilis GDN strain (Accession No. KCTC13020BP) which produces high thrombolytic enzymes.
상기 균주는 볏짚 및 건초로부터 내열성 포자를 형성하는 균을 분리하고, 콩류 또는 생비지 등의 발효시 고함유의 혈전용해효소를 생산하는 균주를 선별함으로써 얻을 수 있다. 선별된 상기 균주는 혈전용해효소, 특히, 낫토키나아제를 고함유로 생산하며, 낫토의 발효에 있어서 일반 균주를 사용한 경우에 비하여 5배 이상으로 높은 혈전용해효소 활성을 갖는 낫토를 생산할 수 있다.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.
이에 따라, 본 발명의 바실러스 서브틸리스 GDN 균주로 제조한 낫토를 섭취할 경우, 종래의 낫토균에 의해 제조한 낫토를 섭취하는 경우에 비해, 낫토의 섭취량을 5분의 1정도로 줄이더라도, 혈전용해효소의 활성은 동일하게 유지할 수 있고, 비타민 K2의 섭취량은 1일 권장량의 한계치 내로 조절할 수 있어 상기 바실러스 서브틸리스 GDN 균주는 혈전용해를 목적으로 한 다양한 식품의 제조를 위해 활용이 가능하다.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.
또한, 본 발명에 따른 바실러스 서브틸리스 GDN 균주를 배양한 비지 발효물의 경우, 콩 발효물에 비해 혈전용해효소 활성이 1.5배 이상 높은 특성을 보여, 추가적인 처리없이 고체배양을 통해 효율적으로 혈전용해효소를 생산할 수 있고, 배지로부터 간단한 조작을 통해 혈전용해효소를 손쉽게 정제할 수 있어, 혈전용해효소의 생산비용을 획기적으로 절감할 수 있을 뿐만 아니라, 산업 폐기물인 비지를 재활용할 수 있다는 점에서 친환경적이다. 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. .
상기 균주는 콩류 또는 생비지 등을 발효시 혈당강하에 효과가 있는 α-글루코시다아제 저해제를 다량 생성하여 성인병 예방에도 효과적인 식품 제조가 가능할 뿐만 아니라, 맛, 점질물 강도, 외형 등의 관능 특성이 우수한 낫토를 제조할 수 있다. 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.
상기 균주는 하기 서열번호 1의 16s RNA를 갖는 것을 특징으로 한다.The strain is characterized by having the 16s RNA of SEQ ID NO: 1.
서열번호 1:SEQ ID NO 1:
GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGT
또한, 본 발명은 상기 바실러스 서브틸리스 GDN 균주를 이용하여 발효시킨 배양물을 제공한다. In addition, the present invention provides a culture fermented using the Bacillus subtilis GDN strain.
바람직하게는, 상기 배양물은 생비지에 상기 균주를 접종하여 발효시킨 비지 발효물일 수 있으며, 상기 비지 발효물은 3400 내지 4300 fibrinolytic unit/g의 우수한 혈전용해효소 활성을 나타낼 수 있다.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.
또한, 상기 배양물은 콩류에 상기 균주를 접종하여 발효시킨 콩류의 배양물일 수 있으며, 바람직하게는, 낫토일 수 있다. 상기 균주를 접종하여 발효시킨 낫토는 2300 내지 2500 fibrinolytic unit/g의 혈전용해효소 활성을 나타내, 미아기노균 등과 같은 종래의 낫토균을 접종하여 발효시킨 낫토(400 내지 500 fibrinolytic unit/g)에 비해 혈전용해효소 활성이 5배 이상 우수하다.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.
또한, 본 발명은 상기 배양물을 포함하는 식품 조성물을 제공한다.In addition, the present invention provides a food composition comprising the culture.
또한, 본 발명은 상기 균주를 이용하여 발효시키는 단계를 포함하는 혈전용해효소의 제조방법을 제공한다. 상기 혈전용해효소의 제조방법에서는, 생비지를 발효시켜 비지 발효물을 제조할 수 있으며, 각종 콩류를 발효시켜 낫토 등의 콩 발효물을 제조할 수 있으며, 상기 콩 발효물은 혈전용해효소로 낫토키나아제를 포함할 수 있다.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.
또한, 상기 비지 발효물은 우수한 혈전용해효소 활성을 나타내며, 상기 혈전용해효소의 제조방법은, 산업 폐기물인 비지를 이용하여 추가적인 처리없이 고체배양을 통해 효율적으로 혈전용해효소를 생산할 수 있고, 배지로부터 간단한 조작을 통해 혈전용해효소를 손쉽게 정제할 수 있어 혈전용해를 목적으로 하는 다양한 식품 제조를 위해 활용가능하다.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.
이하, 본 발명을 실시예를 들어 더욱 상세히 설명하도록 한다.Hereinafter, the present invention will be described in more detail with reference to Examples.
제시된 실시예는 본 발명의 구체적인 예시일 뿐이며, 본 발명의 범위를 제한하기 위한 것은 아니다.The examples presented are merely illustrative of the invention and are not intended to limit the scope of the invention.
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실시예Example
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1. 균주의 탐색1. Search for Strains
한국, 중국, 일본, 미국 등 세계 각지에서 수집한 약 500여점의 볏짚 및 건초를 시료로 하여 미생물을 분리하였다. 각 시료를 멸균 식염수에 소량 가하여 현탁시킨 다음, 80℃ 항온 수조에서 30분간 열처리하여 얻은 포자액을 2% 한천을 함유하는 LB 평판배지(1% 트립톤, 0.2% 설탕, 0.5% 효모추출물 및 0.5% NaCl, pH7.0)에 도말하고, 37 ℃의 배양기에서 1일간 배양한 후, 균 집락을 형성하는 균체를 분리하였다. 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.
혈전용해효소의 생산량을 조사하기 위하여, 분리된 균주를 5 ml의 5% 콩가루를 현탁한 배지에 접종하고, 37℃에서 24시간 동안 진탕 배양하였다. 배양액을 원심분리하여 상등액으로부터 혈전용해효소 활성을 피브린 평판법(fibrin plate method)로 측정하였다. 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.
상기한 피브린 평판법은 하기에 나타낸 방법으로 수행하였다.The fibrin plate method described above was performed by the method shown below.
먼저, 1.2%의 피브리노겐 용액(fibrinogen solution) 2.5ml, 20unit/ml의 트롬빈(thrombin) 500 μl, 1%의 아가로스 용액(agarose solution) 7ml이 용해된 0.1M 인산 나트륨 완충액(pH8.5)을 페트리디시(petri dish)에 넣고 고르게 펴진 상태로 고화시켜 피브린 평판(fibrin plate)을 제조하였다. 파스퇴르 피펫을 이용하여 피브린 평판에 일정 간격으로 공극을 형성시켰다. 각각의 균주를 배양해서 만든 시료 2 μl을 공극에 주입하여 37 ℃에서 배양하고, 형성된 클리어존(clear zone)의 면적으로부터 혈전용해효소의 활성을 측정하였다. 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.
상기의 과정을 통해, 활성이 강한 순으로 80종의 균주를 분리하여 혈전용해효소 생산균으로 1차 선발한 후, 간이 동정법(The prokaryotes, A handbook on the biology of bacteria, vol.2. p.1668, 1992, Springer-Verlag)으로 혈전용해효소 활성이 높은 5종의 균주를 2차 선발하였다. 분리한 5종의 균주는 16s RNA 염기 서열 분석을 통하여 바실러스 서브틸리스로 동정이 되었고, 서열번호 1의 염기서열을 갖는다는 사실을 확인하였다. 혈전용해효소 활성이 가장 높은 1종의 균주를 바실러스 서브틸리스 GDN(Bacillus
subtilis GDN)으로 명명하고 한국생명공학 연구원 생물자원센터(KCTC)에 2016.05.03.일자로 기탁하였다(기탁번호: KCTC13020BP).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). .
서열번호 1:SEQ ID NO 1:
GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGT
또한, 분리한 균주를 현미경으로 관찰하였으며(도 1 참조), 형태학적 특성을 분석하여 하기의 표 1에 나타내었다.In addition, the isolated strain was observed under a microscope (see Fig. 1), and the morphological characteristics are shown in Table 1 below.
표 1에 나타낸 바와 같이, 분리한 균주는 그람 양성이었고, 세포의 크기는 대략 0.6 내지 0.7 × 1.4 내지 1.6 μm인 것을 확인할 수 있었으며, 상기한 결과를 종합하여, 바실러스 서브틸리스 GDN 균주의 분류학상 위치를 도 2에 나타내었다.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.
또한, 분리한 균주의 생화학적 특성 분석결과를 하기의 표 2에 나타내었다.In addition, the results of biochemical characterization of the isolated strains are shown in Table 2 below.
2. Fibrin 용액을 이용한 2. Using Fibrin Solution
혈전용해효소Thrombolytic enzyme
활성 측정 Active measurement
희석한 시료 100 μl와 0.1M Tris-HCl 완충액 200 μl(10mM CaCl₂ 포함, pH7.8), 0.4% 피브린(fibrin) 용액(pH 7.0) 200 μl를 혼합한 후, 37℃에서 30분간 반응시켰다. 반응이 끝난 후 반응정지액(0.22M sodium acetate, 0.33M 아세트산 포함 0.11M 트리클로로 아세트산) 500 μl를 첨가하고 원심분리하여 상등액을 수득하였다. 수득한 상등액에 275nm 파장의 빛을 조사하여 흡광도를 측정하였다(단, 혈전용해효소 활성 1 unit은 37℃에서 반응하여 1분간 만들어내는 1 μg의 tyrosine의 양과 같음).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. 콩 3. Beans
발효물의Fermented product
제조 및 Manufacture and
혈전용해효소Thrombolytic enzyme
활성 분석 Activity analysis
세척한 콩을 실온에서 하룻밤 침지한 후, 121 ℃에서 40분간 고압증기멸균(autoclave)하여 삶은 콩을 만들었다. 삶은 콩 50g당 바실러스 서브틸리스 GDN 균주 포자액(105포자/ml) 1 ml의 비율로 삶은 콩이 식기 전에 접종하여 발포스티렌 용기에 넣고 피막을 하여 37℃에서 18시간 배양하였다. 그 후, 4℃에서 24시간 숙성하여 콩 발효물을 제조하였다. 대조군으로는 시판의 미야기노균(宮城野菌)을 사용하였다. 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.
제조한 콩 발효물의 혈전용해 효소의 활성을 측정하여 하기의 표 3에 그 결과를 나타내었다.The activity of the thrombolytic enzymes of the prepared soybean fermentation was measured, and the results are shown in Table 3 below.
표 3에 나타낸 바와 같이, 콩 발효물에서 혈전용해효소의 활성 측정결과, 실시예의 GDN균으로 제조한 콩 발효물의 혈전용해효소 활성은 대조군인 미야기노균에 비하여 약 5배의 효소 활성이 증가된 것을 확인할 수 있었다. 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.
즉, 실시예의 GDN균은 종래의 낫토균에 비하여 혈전용해효소의 생산 능력이 현저하게 높으므로, 해당 균으로 제조한 낫토는 일반적인 낫토의 섭취량의 5분의 1정도로 줄여서 섭취하여도 종래의 낫토 1케이스로부터 기대할 수 있는 혈전용해 효소를 공급받을 수 있음을 확인할 수 있었다. 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.
참고로, 비타민 K2의 1일 권장량은 일본의 경우 남성이 75μg이고, 여성이60 내지 65μg이나, 종래의 낫토균으로 제조한 낫토 1케이스를 섭취할 경우, 300 내지 450 μg의 비타민 K2가 공급되므로 1일 권장량을 약 5배 초과하게 된다. 하지만, 실시예의 GDN 균주로 제조한 낫토는 상기한 종래의 낫토의 1/5정로의 양을 섭취하여도, 혈전용해효소 활성은 그대로 유지하고, 비타민 K의 양을 1일 권장량의 한계치 내로 조절할 수 있다는 사실을 확인할 수 있었다. 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. 관능평가 3. Sensory evaluation
상기의 방법으로 얻어진 콩 발효물에 대하여 관능평가를 수행하였다. 관능평가는 5단계 평가법으로 수행하였으며, 5점은 매우 좋음, 4점은 좋음, 3점은 보통, 2점은 나쁨, 1점은 매우 나쁨으로 하여 하기의 5항목에 대하여 평가하였으며 그 결과를 하기의 표 4에 나타내었다.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.
표 4에 나타낸 바와 같이, 관능평가를 수행한 결과, GDN 균주로 제조한 콩 발효물의 관능 평가는 3.6 내지 4.8점의 높은 점수가 나와 낫토와 같은 콩 발효물로 개발하여도 훌륭한 제품이 될 수 있음을 확인할 수 있었다.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. 비지 4. Busy
발효물의Fermented product
제조 Produce
당일 제조된 생비지(3종)를 그대로 121℃에서 40분간 고압증기멸균하고, 멸균한 비지 50g당 105포자 /ml의 포자액을 1 ml의 비율로 식기 전에 접종하여 37℃에서 24시간 배양하였다. 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.
제조한 비지 발효물(3종)의 혈전용해효소 활성을 측정하여 하기의 표 5에 그 결과를 나타내었다.The thrombolytic enzyme activity of the prepared busy fermentation products (3 types) was measured, and the results are shown in Table 5 below.
표 3에 나타낸 바와 같이, 서로 다른 3개의 공장에서 제조한 비지를 발효하여 생성된 비지 발효물의 혈전용해효소 활성을 측정한 결과, 비지 발효물이 콩 발효물보다 효소생산량이 대략 1.5배 높은 것으로 확인되었다. 이를 통해, 산업 폐기물인 비지를 이용하여 추가적인 처리없이 고체배양을 통하여 효율적으로 혈전용해효소의 생산에 이용될 수 있음을 확인할 수 있었다.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. 비지 5. Busy
발효물에서In fermentation
혈전용해효소의Thrombolytic enzyme
정제 refine
비지를 30cm × 24cm × 6cm 크기의 스테인리스 트레이(stainless tray)에 500 g씩 넣고 121℃에서 40분간 고압증기멸균하였다. 멸균한 비지 1kg당 105포자/ml의 포자액 20 ml를 식기 전에 가하여 접종하고, 비닐 피막을 하여 37℃에서 24시간 동안 배양하여 비지 발효물을 제조하였다. 제조한 비지 발효물에 3배량의 물을 가하고 잘 교반하여 혈전용해효소를 추출하였다. 여기에 CaCl2의 최종 농도가 0.1M이 되도록 CaCl2 분말을 가하고 30분간 정치한 후, 원심분리하여 침전을 제거하고 상등액인 비지 추출물을 제조하였다. 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.
또한, 상등액에 2배량의 에탄올을 가하고 원심분리하고 침전을 동결건조하여 혈전용해효소 분말을 제조하였으며, 혈전용해효소 활성을 측정하여 그 결과를 수율과 함께 하기의 표 6에 나타내었다.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.
표 6에 나타낸 바와 같이, 칼슘(CaCl2) 처리한 비지 추출물의 혈청용해효소 활성이 매우 높은 것으로 확인되었다. 이를 통해, GDN 균주의 비지 발효물에 중량대비 3배량의 물을 가하여 균질화하고 거기에 최종농도가 0.1M이 되도록 CaCl2 분말을 가하고 30분간 정치하면 비지로부터 유래한 고형분이 응집하여 쉽게 침전으로 제거되고 혈전용해효소는 맑은 상등액에 남으므로 간단한 조작에 의하여서도 혈전용해효소의 정제가 고수율로 이루어진다는 사실을 확인할 수 있었다.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.
본 발명에 따른 바실러스 서브틸리스 GDN 균주는 혈전용해효소의 생산 능력이 현저히 높아, 혈전용해를 목적으로 한 다양한 식품 제조에 활용될 수 있다. 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.
[수탁번호][Accession number]
기탁기관명 : 한국생명공학연구원 한국생물자원센터(KCTC)Name of institution: Korea Biotechnology Research Institute Korea Bioresource Center (KCTC)
수탁번호 : KCTC13020BPAccession number: KCTC13020BP
수탁일자 : 20160503Deposit date: 20160503
[기탁증][Deposit]
[규칙 제26조에 의한 보정 10.04.2017]
deleted[Revision 10.04.2017 under Rule 26]
deleted
deleted[Revision 10.04.2017 under Rule 26]
deleted
Claims (12)
- 혈전용해효소를 고생산하는 바실러스 서브틸리스 GDN 균주(Bacillus subtilis GDN, 수탁번호KCTC13020BP). Bacillus subtilis GDN strain (KCTC13020BP) producing high thrombolytic enzymes.
- 제1항에 있어서, 상기 균주가 서열번호 1의 16s RNA를 포함하는 것을 특징으로 하는 균주.The strain according to claim 1, wherein the strain comprises 16s RNA of SEQ ID NO: 1.
- 제1항 내지 제2항 중 어느 한 항의 균주를 이용하여 발효시킨 배양물.A culture fermented using the strain of any one of claims 1 to 2.
- 제3항에 있어서, 상기 배양물이 낫토인 것을 특징으로 하는 배양물.The culture according to claim 3, wherein the culture is natto.
- 제4항에 있어서, 상기 배양물에 포함된 혈전용해효소가 낫토키나아제인 것을 특징으로 하는 배양물.The culture according to claim 4, wherein the thrombolytic enzyme contained in the culture is nattokinase.
- 제3항에 있어서, 상기 배양물이 비지 발효물인 것을 특징으로 하는 배양물.The culture according to claim 3, wherein the culture is busy fermentation.
- 제6항에 있어서, 상기 비지 발효물이 3400 내지 4300 fibrinolytic unit/g의 혈전용해효소 활성을 나타내는 것을 특징으로 하는 배양물.The culture according to claim 6, wherein the busy fermentation product exhibits thrombolytic activity of 3400 to 4300 fibrinolytic units / g.
- 제3항의 배양물을 포함하는 식품 조성물.A food composition comprising the culture of claim 3.
- 제1항 내지 제2항 중 어느 한 항의 균주를 이용하여 발효시키는 단계를 포함하는 혈전용해효소의 제조방법.A method for preparing a thrombolytic enzyme comprising the step of fermenting using the strain of any one of claims 1 to 2.
- 제9항에 있어서, 상기 발효가 낫토의 발효인 것을 특징으로 하는 혈전용해효소의 제조방법.10. The method of claim 9, wherein the fermentation is fermentation of natto.
- 제10항에 있어서, 상기 낫토의 발효에 의해 낫토키나아제가 제조되는 것을 특징으로 하는 혈전용해효소의 제조방법.The method for producing thrombolytic enzymes according to claim 10, wherein nattokinase is produced by fermentation of the natto.
- 제9항에 있어서, 상기 발효가 생비지의 발효인 것을 특징으로 하는 혈전용해효소의 제조방법.10. The method of claim 9, wherein the fermentation is fermentation of live busy.
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CN113755404A (en) * | 2021-10-13 | 2021-12-07 | 华北制药股份有限公司 | High-yield strain of vitamin K2(MK-7), screening method thereof and method for producing vitamin K2(MK-7) |
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