KR102141341B1 - Cheonggukjang Fibrinolytic Enzyme for Improving Blood Circulation and Manufacturing Method - Google Patents
Cheonggukjang Fibrinolytic Enzyme for Improving Blood Circulation and Manufacturing Method Download PDFInfo
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- KR102141341B1 KR102141341B1 KR1020180093752A KR20180093752A KR102141341B1 KR 102141341 B1 KR102141341 B1 KR 102141341B1 KR 1020180093752 A KR1020180093752 A KR 1020180093752A KR 20180093752 A KR20180093752 A KR 20180093752A KR 102141341 B1 KR102141341 B1 KR 102141341B1
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- thrombolytic enzyme
- thrombolytic
- cheonggukjang
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- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
본 발명은 혈행개선 증진용 혈전용해효소의 제조방법에 관한 것으로, 청국장을 발효하여 혈전용해능이 우수한 혈전용해효소를 제조하는 단계와, 청국장 발효액으로부터 청국장효소인 혈전용해효소를 추출 및 농축하는 단계, 상기의 혈전용해효소를 제형화하는 단계로 구성된다.
본 발명의 혈전용해효소는 혈행개선 효과가 뛰어나고, 청국장의 이취나 거부감이 없는 청국장 유래 혈전용해효소를 제공할 수 있다.The present invention relates to a method of manufacturing a thrombolytic enzyme for improving blood circulation, producing a thrombolytic enzyme having excellent thrombolytic ability by fermenting cheonggukjang, and extracting and concentrating thrombolytic enzyme, cheonggukjang enzyme, from the fermentation broth of cheonggukjang, It consists of the step of formulating the thrombolytic enzyme.
The thrombolytic enzyme of the present invention has excellent circulatory effect, and can provide a thrombolytic enzyme derived from cheonggukjang without odor or rejection.
Description
본 발명은 혈행개선 증진용 혈전용해효소 및 그 제조방법에 관한 것이다. 보다 상세하게는 청국장을 배지에서 발효하여 혈전용해능이 우수한 혈전용해효소를 제조하는 단계와, 청국장 발효액으로부터 혈전용해효소를 추출 및 농축하는 단계, 상기의 혈전용해효소를 비드(bead)나 젤리 또는 캡슐 등으로 제형화하는 단계로 구성된다.The present invention relates to a thrombolytic enzyme for improving blood circulation and a method for manufacturing the same. More specifically, the step of preparing a thrombolytic enzyme having excellent thrombolytic ability by fermenting cheonggukjang in a medium, and extracting and concentrating thrombolytic enzyme from the chunggukjang fermentation broth, beads or jelly or capsules of the thrombolytic enzyme. And the like.
본 발명의 혈전용해효소는 혈행개선 효과가 뛰어나고, 청국장의 이취나 거부감이 없는 혈전용해효소를 제공할 수 있다.The thrombolytic enzyme of the present invention has excellent blood circulation improving effect, and can provide a thrombolytic enzyme without odor or rejection of Cheonggukjang.
콩은 우리나라, 중국, 일본에서 전통식품인 장류, 두부 및 콩가공식품의 원료로서 콩의 영양 및 건강기능성에 대한 인식이 확산되면서 그 수요도 꾸준히 늘어나고 있다. 우리나라도 고령화인구의 증가로 노인성 치매환자의 60%가 혈전에서 기인하므로 순환계 질환에 대한 대책은 매우 중요한 문제이다. 우리나라에서 청국장은 볏짚 유래의 고초균(Bacillus Subtilis sp .)을 이용하는 것으로, 삶은 콩을 볏짚 위에 깔고 비교적 고온으로 보온해주며 약 일주일 정도 발효시키는 전통적인 방법으로 만들어 왔다. 근래에는 순수 분리한 고초균을 사용하여 발효탱크에서 40∼45℃로 2∼3일간 발효시켜 상업적으로 제조하고 있다. 일본에서는 대두발효식품인 낫토로부터 분리된 효소인 나토키나제(nattokinase)를 경구 투여 시 생체내의 혈전용해능을 높일 수 있다고 보고된 후, 수많은 나토키나제 제품이 상업화되어 유통되고 있다. 우리나라에서도 대두발효 식품인 청국장, 된장, 간장 등에서 항산화효과, 혈전용해효과, 혈압강화효과 등 각종 생리활성 물질이 밝혀진 바 있다. 특히 청국장을 섭취할 때 생체내의 혈전용해능이 알려져 관심이 높아지고 있는데, 청국장 발효에 관여하는 혈전용해능이 우수한 균주를 선발하여 이들이 생산하는 효소를 고도로 정제하여 기능성 식품소재로 이용할 필요가 있다. 한국특허등록번호(10-0849833) 바실루스 낫토(Bacillus subtilis natto)가 혈전 용해효소인 나토키나제를 다량으로 함유하는 식품, 예를 들면, 바실루스 낫토 배양 추출물을 분말 또는 캡슐화시킨 상품이 건강식품으로서 판매되고 있다. 또한 낫토 배양 추출물에는 다량 함유되어 있는 비타민 K2를 제거하기 위해 키토산을 첨가하여 배양액으로부터 99% 이상 제거시킨 나토키나제를 제공한다.Soybeans are a raw material for traditional foods such as soybean, tofu and soybean processed foods in Korea, China, and Japan. In Korea, as the aging population increases, 60% of senile dementia patients are from blood clots, so countermeasures against circulatory diseases are very important. In Korea, Cheonggukjang is Bacillus derived from rice straw. Subtilis sp . ), the boiled beans are laid on rice straw, kept at a relatively high temperature, and traditionally fermented for about a week. Recently, it is commercially produced by fermenting the fermentation tank in a fermentation tank at 40 to 45°C for 2 to 3 days using purely isolated archaea. In Japan, after oral administration of nattokinase , an enzyme isolated from soybean fermented food natto, has been reported to increase the thrombolytic potential in vivo, numerous nattokinase products have been commercialized and distributed. In Korea, various physiologically active substances, such as antioxidant effects, thrombolytic effects, and blood pressure enhancing effects, have been identified in soybean fermented foods such as Cheonggukjang, miso, and soy sauce. In particular, when ingesting Cheonggukjang, interest in thrombolytic in vivo is known, and interest is increasing. It is necessary to select strains with excellent thrombolytic ability to participate in the fermentation of Cheonggukjang and highly purify the enzymes produced by them to use it as a functional food material. Korea Patent Registration number (10-0849833) Bacillus natto (Bacillus subtilis natto ) is a food containing a large amount of thrombolytic enzyme nattokinase , for example, a product obtained by powdering or encapsulating Bacillus natto culture extract as a health food. Also natto culture extract is added to the chitosan in order to remove the K2 vitamin, which is contained a large amount provides NATO kinase was removed more than 99% from the culture broth.
한국특허등록번호 10-1680640(청국장에서 분리한 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소활성이 있는 바실러스 서브틸리스 2RL2-1 균주 및 이의 용도)는 유해 미생물인 바실러스 세레우스를 생산하지 않는 균주를 이용하여 미생물제제 또는 발효식품을 제공한다. 그밖에 일본의 노구찌나토키나제 혈전용해효소(fibrinolytic enzyme) 개발(1987), 혈전용해능이 강화된 기능성 청국장의 제조 및 나토키나제 생산을 위한 액체발효 기술개발(Bacillus subtilis와 99% 상동성을 나타내는 균주를 Bacillus subtilis DGCK2 개발)동국대학교 경주캠퍼스 산학협력단(2009), 미세캡슐화된 청국장 유래 혈전용해 조효소 농축물 분말”의 상용화를 위한 제품화에 대한 연구(고려대학교 2007)등이 있으나, 이들 종래기술은 본 발명과 기술적구성이 다른 것이다.Korean Patent Registration No. 10-1680640 (Bacillus subtilis 2RL2-1 strain having thrombolytic and enzymatic activity while having a biogenic amine resolution isolated from Cheonggukjang and its use) is a strain that does not produce Bacillus cereus, a harmful microorganism To provide a microbial preparation or fermented food. In addition, Japan's Noguchinatokinase fibrinolytic enzyme was developed (1987), the production of functional Chungkookjang with enhanced thrombolytic ability and liquid fermentation technology for the production of nattokinase ( Bacillus Strains representing the 99% homology to Bacillus subtilis and subtilis DGCK2 development) Dongguk University Gyeongju Campus Industry-University Cooperation Group (2009), research on commercialization of microencapsulated Cheonggukjang-derived thrombolytic coenzyme concentrate powder (Korea University 2007), etc. The composition is different.
증자한 콩에 개방형태로 청국장을 발효하면 청국장균 뿐만 아니라 야생에 존재하는 많은 잡균이 증식하여 원하지 않는 성분이 생성되어 이취가 나고 불순물이 혼합되는 등 청국장의 품질을 떨어뜨리고, 또한 효소 활성도 낮으므로 원하는 고 순도 및 고 농도의 혈전용해효소 활성을 얻기가 어렵다. 또한 볏짚 유래의 고초균을 분리하여 얻은 청국장 균주를 순수 배양하더라도 이취가 나고 혈전용해효소의 농도가 낮은 단점이 있다. 본 발명은 한국특허등록번호 10-1680640에서 제공하는 바실러스 스브틸리스 2RL2-1 의 청국장균으로부터 분리한 혈행개선 증진용 혈전용해효소(나토키나제)를 장용성 bead화 또는 캡슐화하여 고기능성 혈행개선 식품을 개발하는 데 있다.When fermenting Cheonggukjang in an open form to the increased soybeans, not only Cheonggukjang fungi, but also many germs present in the wild multiply, and unwanted components are generated, resulting in deodorization and impurity mixing. It is difficult to obtain the desired high purity and high concentration of thrombolytic enzyme activity. In addition, even if the strains of Cheonggukjang obtained by separating archaea bacteria derived from rice straw are purely cultured, there is a disadvantage of low odor and low concentration of thrombolytic enzyme. The present invention provides an enteric bead or encapsulated thrombolytic enzyme (natokinase) for enhancing blood circulation, which is isolated from Bacillus subtilis 2RL2-1, provided by Korean Patent Registration No. 10-1680640, to provide highly functional blood circulation improvement foods. To develop.
본 발명은 혈행개선 증진용 혈전용해효소 및 그 제조방법에 관한 것으로, 청국장을 배지에서 발효하여 혈전용해능이 우수한 혈전용해효소를 제조하는 단계와, 청국장 발효액으로부터 혈전용해효소를 추출 및 농축하는 단계, 상기의 혈전용해효소를 비드(bead)나 젤리 또는 캡슐 등으로 제형화하는 단계로 구성된다.The present invention relates to a thrombolytic enzyme for improving blood circulation and a method for manufacturing the same, producing a thrombolytic enzyme having excellent thrombolytic ability by fermenting cheonggukjang in a medium, and extracting and concentrating thrombolytic enzyme from the cheonggukjang fermentation broth, It consists of the step of formulating the thrombolytic enzyme into beads, jelly or capsules.
본 발명은 대한민국 농촌진흥청이 보유하고 있는 한국특허등록번호 제1680640호(청국장에서 분리한 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소활성이 있는 바실러스 서브틸리스 2RL2-1 균주 및 이의 용도)를 기술이전 받아 상기 균주의 배양액을 유효성분으로 포함하는 혈전용해효소 및 상기 효소를 포함하는 발효식품을 제공한다.The present invention describes the Korean Patent Registration No. 1680640 (Bacillus subtilis 2RL2-1 strain having thrombolytic and enzymatic activity and having biolytic amine resolution isolated from Cheonggukjang and its use) possessed by the Rural Development Administration of the Republic of Korea It provides a thrombolytic enzyme containing the culture medium of the strain as an active ingredient and a fermented food containing the enzyme.
종래의 습윤상태의 청국장은 저장기간이 짧아서 보관과 유통이 어렵고 혈전용해 효소활성이 낮은 단점이 있다. 청국장의 유효성분인 혈전용해효소를 분리하여 캡슐화(capsule)하면 이용의 편리를 도모하여 손쉽게 섭취할 수 있으나, 소화기관인 위에서 실활될 우려가 있으나 이를 비드화(bead)하면 장관에 이르기까지 혈전용해 효소를 전달시킬 수 있으므로 혈행 개선 효과가 증대될 수 있다. 본 발명은 청국장에서 고순도 혈전용해효소가 함유된 청국장을 재래 청국장보다 품질이 우수하고 혈행 개선 능력이 있는 신개념 혈전용해효소를 제공할 수 있다. 본 발명의 최종 목표는 혈전용해능의 혈전용해효소 제조공정 확립 및 품질기준을 설정하여 농축액을 제조하고, 소비자의 기호에 맞는 고농도 혈전용해효소를 비드(bead)나 젤리 또는 캡슐 등으로 제형화하여 제품을 개발하는 데 있다.Conventional wet state Cheonggukjang has a short storage period, which makes storage and distribution difficult, and has low thrombolytic enzyme activity. Separating the thrombolytic enzyme, an active ingredient of Cheonggukjang, and encapsulating it for convenience of use, it can be consumed easily, but there is a concern that it may be inactivated in the digestive tract, but beading this leads to the thrombolytic enzyme to the intestinal tract Because it can deliver the blood circulation improving effect can be increased. The present invention can provide a new concept thrombolytic enzyme having a higher quality and improved blood circulation ability than a conventional cheonggukjang in cheonggukjang containing a high purity thrombolytic enzyme in the cheonggukjang. The final goal of the present invention is to establish a thrombolytic enzyme manufacturing process of thrombolytic ability and establish a quality standard to prepare a concentrate, and formulate a high concentration thrombolytic enzyme suitable for the consumer's taste into beads, jelly or capsules, etc. It is about developing products.
본 발명의 혈전용해효소는 활성이 매우 높고 효소가 위에서 실활 되지 않고 장까지 도달하여 혈행개선 효과가 뛰어나고, 청국장의 이취나 거부감이 없는 청국장 유래 혈전용해효소를 제공할 수 있다. 또한 비타민C, 유산균, 과일쥬스, 기타 영양성분 등과 융합으로 식감, 맛, 풍미, 색상 등 혈전용해효소제품의 풍미와 기호성이 증진된다.The thrombolytic enzyme of the present invention can provide a thrombolytic enzyme derived from cheonggukjang, which has a very high activity and the enzyme does not become inactivated in the stomach and reaches the intestine, has excellent blood circulation improvement effect, and has no odor or rejection. In addition, flavor and palatability of thrombolytic enzyme products such as texture, taste, flavor, and color are enhanced by fusion with vitamin C, lactic acid bacteria, fruit juice, and other nutrients.
도 1은 일본의 일반적인 낫토 제품을 나타낸다.
도 2는 본 발명의 혈전용해효소를 비드화제품, 젤리화제품 및 캡슐화제품이다.
도 3은 본 발명의 혈전용해효소의 효소활성을 나타낸 것이다.1 shows a typical natto product in Japan.
Figure 2 is a thrombolytic enzyme of the present invention is a beaded product, a jelly product and an encapsulated product.
Figure 3 shows the enzyme activity of the thrombolytic enzyme of the present invention.
본 발명의 목표는 최종 제조공정 확립 및 품질기준 설정을 통하여 청국장 발효액으로부터 혈전용해능이 우수한 혈전용해효소 농축액의 제조, 혈전용해능 및 관능성이 우수한 혈전용해효소를 제공할 수 있다. 본 발명은 청국장을 발효하여 혈전용해능이 우수한 혈전용해효소를 제조하는 단계와, 청국장 발효액으로부터 혈전용해효소를 추출 및 농축하는 단계, 상기 농축된 혈전용해효소를 비드나 젤리 또는 캡슐 등으로 제형화하는 단계로 구성된다.The objective of the present invention is to provide a thrombolytic enzyme having excellent thrombolytic enzyme concentration, thrombolytic ability and functionality from Chungcheongjang fermentation broth through establishment of a final manufacturing process and setting of quality standards. The present invention is a step of preparing a thrombolytic enzyme having excellent thrombolytic ability by fermenting cheonggukjang, and extracting and concentrating thrombolytic enzyme from the chunggukjang fermentation broth, and formulating the concentrated thrombolytic enzyme into beads, jelly or capsules, etc. It consists of stages.
<청국장의 배양> <Cultivation of Cheonggukjang>
청국장발효에 사용되는 균주는 바실러스 서브틸리스(Bacilus subtilis), 또는 시판중인 청국장균 중에서 선택된 어느 하나 또는 둘 이상의 균주를 사용할 수 있다. 청국장의 배양은 일반적으로 알려진 방법에 따라 대두를 증자하여 배지를 만들고 상기 균주를 배양한 종균(Seed)을 접종하여 청국장 발효물을 만든 다음 정제수로 추출 여과하여 효소액을 얻는다. 이와 달리 액침배양 할 경우에 사용되는 배지는 탄소원으로 전분, 포도당, 과당, 설탕 중에서 선택된 어느 하나 또는 둘 이상을 사용할 수 있다. 질소원으로는 노랑콩, 흰콩, 검정콩, 탈지 대두, 고기 추출물 중에서 선택된 어느 하나 또는 둘 이상을 사용하는 것이 좋다. 무기염으로는 탄산칼슘, 염화마그네슘, 염화칼륨 중에서 선택된 어느 하나 또는 둘 이상을 사용하는 것이 좋다. 그밖에 식염수, 지방산 중에서 그 일부를 배지 성분으로 사용할 수도 있다. 청국장 발효는 35℃~45℃가 바람직하며, 37℃~43℃가 보다 바람직하며, 40℃ 전후가 가장 바람직하다. 배양시간은 24~72시간이 바람직하다.The strain used for fermentation of Cheonggukjang may be Bacilus subtilis , or any one or more strains selected from commercially available Cheonggukjang. Cultivation of Cheonggukjang is performed by increasing the soybean according to a generally known method to make a medium, inoculating the seed cultured with the strain, making a fermentation product of Cheonggukjang, and then extracting it with purified water to obtain an enzyme solution. On the other hand, the medium used for immersion culture can be any one or two or more selected from starch, glucose, fructose, and sugar as a carbon source. As a nitrogen source, it is preferable to use one or more selected from yellow beans, white beans, black beans, skim soybeans, and meat extracts. As the inorganic salt, it is preferable to use any one or two or more selected from calcium carbonate, magnesium chloride, and potassium chloride. In addition, some of the saline and fatty acids may be used as a medium component. The fermentation of Cheonggukjang is preferably 35°C to 45°C, more preferably 37°C to 43°C, and most preferably around 40°C. Incubation time is preferably 24 to 72 hours.
<혈전용해효소의 추출 및 농축><Extraction and concentration of thrombolytic enzymes>
청국장 균주의 최적 배양조건에서 최고의 용해활성을 보일 때까지 균주를 배양하여 혈전용해효소가 함유된 청국장 발효액을 얻는다. 상기의 발효액을 원심분리하여 균체를 제거한 다음 혈전용해효소액을 얻는다. 여과기의 벽면에 여과보조제로 규조토 또는 활성탄을 코팅시킨 다음 상기 혈전용해효소액을 여과시켜, 여과된 혈전용해효소액을 얻을 수 있다. 상기와 같이 얻어진 여과된 혈전용해효소액을 농축시키기 위하여 0.5μm 내지 0.2μm의 필터로 여과한 액을 역삼투압 농축기를 사용하여 분자량 100 이하의 대부분의 물질을 제거시킨다. 또 다른 방법으로 혈전용해효소액의 농축을 위해 막분리기(ultrafiltration unit, Sartorius, Germany)을 사용하여 분자량 100 이하의 물질을 제거함으로써 혈전용해효소액의 농축액을 얻는다. 농축과정에 미리 각 membrane을 사용하기 전에 1N-NaOH 용액과 1차 증류수를 이용하여 막분리기를 세척한다. 분리과정 중 효소활성이 실활 되지 않도록 냉각기를 이용하여 4℃로 유지하면서 초기 혈전용해효소액의 부피를 1/10까지 농축하여 혈전용해효소용액을 제조한다.The strain is cultivated until the best solubility is exhibited under the optimum culture conditions for the strain of Chungkukjang to obtain the Chungkukjang fermentation broth containing thrombolytic enzyme. The fermentation broth is centrifuged to remove the cells, and then a thrombolytic enzyme solution is obtained. The diatomaceous earth or activated carbon is coated with a filter aid on the wall surface of the filter, and then the thrombolytic enzyme solution is filtered to obtain a filtered thrombolytic enzyme solution. In order to concentrate the filtered thrombolytic enzyme solution obtained as described above, a liquid filtered with a filter of 0.5 μm to 0.2 μm is removed using a reverse osmosis concentrator to remove most substances having a molecular weight of 100 or less. Another method for concentration of the thrombolytic enzyme solution is to remove a substance having a molecular weight of 100 or less by using a membrane separator (ultrafiltration unit, Sartorius, Germany) to obtain a concentrate of the thrombolytic enzyme solution. Prior to using each membrane in the concentration process, the membrane separator is washed with 1N-NaOH solution and primary distilled water. During the separation process, the initial thrombolytic enzyme solution is concentrated to 1/10 of the thrombolytic enzyme solution while maintaining the temperature at 4°C using a cooler so as not to deactivate the enzyme activity.
<혈전용해효소의 제형> ; 페이스트, 분말, 과립, 정제, 캡슐, 비드, 젤리<Formulation of thrombolytic enzyme>; Paste, powder, granule, tablet, capsule, bead, jelly
수득된 혈전용해효소의 농축액은 재차 농축시켜 페이스트상으로 만들 수 있다. 또한, 수득된 농축액에 대하여 적절한 양의 수용성 식이섬유, 유당, 셀룰로스 등을 첨가하여 동결건조시켜 분말상 또는 과립상의 청국장 유래 혈전용해효소액을 제조할 수 있다. 또한, 상기의 농축액, 페이스트상, 분말상 및 과립상의 추출물을 캡슐에 밀봉시켜 혈전용해효소액을 함유하는 캡슐을 만들 수 있다. 또한, 이들 표면을 당의정으로 코팅하여 정제로 만들 수 있다.The concentrate of the obtained thrombolytic enzyme can be concentrated again to make a paste. In addition, an appropriate amount of water-soluble dietary fiber, lactose, cellulose, and the like may be added to the obtained concentrate to freeze-dry to prepare a thrombolytic enzyme solution derived from a powdery or granular cheonggukjang. In addition, the above-described concentrate, paste, powder and granular extracts can be sealed in capsules to make capsules containing thrombolytic enzyme solution. In addition, these surfaces can be coated with dragees to form tablets.
또한 혈전용해효소 농축액에 에틸알콜을 85~90%(w/v)가 되도록 첨가하여 결정화한다. 이때 혈전용해효소에 에틸알콜을 사용하여 결정화를 하게 되면 청국장 냄새를 제거할 수 있는 장점이 있다. 또한 혈전용해효소에 에틸알콜을 사용하여 결정화를 하게 되면 용매 효과가 좋아지고, 맛이 좋으며, 과일 본연의 색소로 착색효과를 얻을 수 있다, 농축된 혈전용해효소액을 알긴산나트륨(sodium alginate) 1%(w/w) 내지 3%(w/w) 용액에 넣고 완전히 녹이고 air-atomizing device (Spray Systems Co., Incheon,Korea)를 사용하여 0.5M-CaCl2 용액에 분사시켜 미세 비드(bead)를 형성하거나 주사기를 사용하여 비드(bead)를 만든다. 형성된 beads는 30분 동안 CaCl2 용액에 방치하고 증류수로 세척하여 동결건조한다. 미세캡슐 bead화 제품의 물리적 특성으로 건조된 미세캡슐의 morphology 형성유무는 주사전자현미경(SEM) 측정을 통해 관찰한다.In addition, ethyl alcohol is added to the thrombolytic enzyme concentrate to be 85 to 90% (w/v) to crystallize. At this time, crystallization using ethyl alcohol in the thrombolytic enzyme has the advantage of removing the odor of cheonggukjang. In addition, when ethyl alcohol is used for thrombolytic enzyme, crystallization improves the solvent effect, tastes good, and provides a coloring effect with the fruit's natural pigment. Sodium alginate in concentrated thrombolytic enzyme solution 1% (w/w) to 3% (w/w) solution, completely dissolved, and sprayed with 0.5M-CaCl 2 solution using an air-atomizing device (Spray Systems Co., Incheon,Korea) to fine beads. Form or make a bead using a syringe. The formed beads are left in CaCl 2 solution for 30 minutes, washed with distilled water and lyophilized. The presence or absence of morphology formation of the dried microcapsules due to the physical properties of the microcapsule beaded product is observed through a scanning electron microscope (SEM) measurement.
<실시예 1> <Example 1>
배지 전체 중량에 대하여, 폴리펩톤 1 중량%, 포도당 1 중량%, 고기추출물 O.5 중량% 및 NaCl O.2 중량%를 넣고 pH 7.O의 배지를 만들어 고초균인 바실러스 서브틸리스(KACC 91957P)를 접종한 후, 36℃에서 20시간 동안 배양하였다. 낫토바실러스 배양액을 상기와 동일한 배지를 종배양조에 접종하고, 24시간 동안 배양하여 종배양액을 얻었다.With respect to the total weight of the medium, 1% by weight of polypeptone, 1% by weight of glucose, O.5% by weight of meat extract, and 2% by weight of NaCl were added to make a medium having a pH of 7.O. Bacillus subtilis (KACC 91957P) ), and incubated at 36°C for 20 hours. The nattobacillus culture was inoculated with the same medium as the above in a seed culture tank, and cultured for 24 hours to obtain a seed culture solution.
본 배양을 위하여 정제수 100 중량부에 대하여 삶은콩 10중량부, 발아 겉보리 분말 5중량부, 탄산칼슘 0.5중량부, 대두유 3.5중량부 및 실리콘 0.01중량부를 넣고 pH 7.0으로 맞추어 본 배양조에 넣고, 종배양액을 첨가하여 통기량 O.5VVM 및 온도 39℃에서 70시간 동안 본배양을 실시하여 혈전용해효소를 함유하는 청국장 발효액을 제조하였다.For this culture, 10 parts by weight of boiled beans, 5 parts by weight of germinated barley powder, 0.5 parts by weight of calcium carbonate, 3.5 parts by weight of soybean oil, 0.01 parts by weight of silicon and 0.01 parts by weight of silicon are added to 100 parts by weight of purified water. Was added, and the main culture was carried out for 70 hours at an aeration amount of O.5VVM and a temperature of 39°C to prepare a cheonggukjang fermentation broth containing a thrombolytic enzyme.
<실시예 2><Example 2>
혈전용해효소를 생산하는 청국장 균주로 바실러스 서브틸리스(KCCM 11053P)를 사용한 것을 제외하고는 실시예 1과 동일하게 종배양하여 얻은 종배양액과 본배양액을 실시하여 혈전용해효소를 함유하는 청국장 발효액을 제조하였다.Cheonggukjang fermentation broth containing thrombolytic enzymes by performing a seed culture and a main culture solution obtained in the same manner as in Example 1, except that Bacillus subtilis (KCCM 11053P) was used as a strain for producing thrombolytic enzymes. It was prepared.
<실시예 3> <Example 3>
혈전용해효소를 생산하는 청국장 균주로 바실러스 서브틸리스(KCTC 3014)를 사용한 것을 제외하고는 실시예 1과 동일하게 종배양하여 얻은 종배양액과 본배양액을 실시하여 혈전용해효소를 함유하는 청국장 발효액을 제조하였다.Cheonggukjang fermentation broth containing the thrombolytic enzyme was performed by performing the seed culture and the main culture as in Example 1, except that Bacillus subtilis (KCTC 3014) was used as the strain for producing the thrombolytic enzyme. It was prepared.
<실시예 4><Example 4>
종래 청국장 발효방법을 이용하여 정제수 100 중량부에 대하여 삶은콩 10중량부에 발아 겉보리 5중량부를 혼합한 후 <실시예 1>의 종배양액을 잡종하여 41℃에서 40시간 배양하여 청국장 발효물을 얻었다. 이 발효물에 정수를 가하여 혈전용해 효소를 추출하였다.Using conventional Cheonggukjang fermentation method, germination of 10 parts by weight of boiled beans to 5 parts by weight of boiled soybeans was mixed with 100 parts by weight of purified water. . To this fermentation product, purified water was added to extract thrombolytic enzymes.
<대조군><control group>
혈전용해효소를 생산하는 청국장 균주로 시중에서 유통되고 있는 바실러스 서브틸리스(G마켓)를 사용한 것을 제외하고는 실시예 1과 동일하게 종배양하여 얻은 종배양액과 본배양액을 실시하여 혈전용해효소를 함유하는 청국장 발효액을 제조하였다.The thrombolytic enzyme was prepared by performing the seed culture solution and the main culture solution obtained in the same manner as in Example 1, except that Bacillus subtilis (G market), which is commercially available as a strain for producing thrombolytic enzyme, was used. A containing fermentation broth of Cheonggukjang was prepared.
<실시예 5><Example 5>
상기의 실시예 1 내지 4와 같이 발효하여 얻은 청국장 발효액을 감압농축하여 한외여과(ultrafiltration)후 역삼투압농축기(reverse osmosis)를 이용하여 혈전용해효소 농축액 200g를 얻었다. 이 농축액 200g에 알긴산나트륨 3g, 감귤쥬스 1L를 섞은 다음, 2%(w/w)의 염화칼슘(CaCl2)용액에 낙하시켜 비드를 제조하였다. 형성된 비드를 30분간 숙성한 다음 비드를 분리하고 증류수 세척에 의해 염화칼슘을 제거하고 UV 살균기로 살균하여 식용 가능한 비드를 제조하였다.The fermentation broth of Cheonggukjang obtained by fermentation as in Examples 1 to 4 was concentrated under reduced pressure to obtain 200 g of thrombolytic enzyme concentrate using ultrafiltration followed by reverse osmosis. Beads were prepared by mixing 3 g of sodium alginate and 1 L of citrus juice in 200 g of the concentrated solution, and then dropping it in a 2% (w/w) calcium chloride (CaCl 2 ) solution. The formed beads were aged for 30 minutes, then the beads were separated, calcium chloride was removed by washing with distilled water, and sterilized with a UV sterilizer to prepare edible beads.
<실시예 6><Example 6>
상기의 실시예 1 내지 4와 같이 발효하여 얻은 혈전용해효소액을 여과한 후 감압농축하여 얻은 혈전용해효소액 100중량부(농도 65%(w/w))에 대하여 95%(w/v) 에탄올 용액 500중량부를 첨가하여 혈전용해효소를 결정화하여 80중량부를 얻었다. 건조후 32 mesh 체로 체별하여 혈전용해효소 분말 60중량부를 얻었다. 혈전용해효소 분말 100 중량부에 대하여 3%(w/w) 알긴산나트륨 200중량부를 첨가하여 코팅시킨 혈전용해효소를 건조한 다음 캡슐화하였다.95% (w/v) ethanol solution with respect to 100 parts by weight of the thrombolytic enzyme solution (concentration 65% (w/w)) obtained by filtration and concentration under reduced pressure after filtering the thrombolytic enzyme solution obtained by fermentation as in Examples 1 to 4 above Thrombolytic enzyme was crystallized by adding 500 parts by weight to obtain 80 parts by weight. After drying, it was sieved through a 32 mesh sieve to obtain 60 parts by weight of thrombolytic enzyme powder. The coated thrombolytic enzyme was dried and encapsulated by adding 200 parts by weight of 3% (w/w) sodium alginate to 100 parts by weight of the thrombolytic enzyme powder.
<실시예 7><Example 7>
젤리베이스의 전체 중량에 대하여 젤라틴 15 중량%, 펙틴 3 중량%, 설탕 2 중량%, 조청 10 중량%를 섞어 젤리베이스를 만든 다음, 121℃에서 20분간 살균하여 혈전용해효소 젤리의 전체 중량에 대하여 비드 30 중량%를 첨가한 후, 금형틀에 넣어 원형, 다각형 등 여러 가지 형태의 혈전용해효소 젤리로 만들었다.With respect to the total weight of the jelly base, 15% by weight of gelatin, 3% by weight of pectin, 2% by weight of sugar, and 10% by weight of crude water are mixed to make a jelly base, and sterilized at 121°C for 20 minutes, based on the total weight of the thrombolytic enzyme jelly. After adding 30% by weight of beads, it was put into a mold and made into various types of thrombolytic enzyme jelly such as round and polygon.
<적용예><Application example>
상기와 같이 만든 혈전용해효소를 적당량의 비타민C, 유산균, 과일즙 등을 혼합하여 공지의 방법으로 페이스트형, 분말, 과립으로 만들어 풍미와 기호성을 증진시켰다. 과일즙은 포도즙, 감귤즙, 석류즙, 복분자즙, 복숭아즙, 매실즙, 레몬즙 등에서 선택된 어느 하나를 사용할 수 있다.The thrombolytic enzyme made as described above was mixed with an appropriate amount of vitamin C, lactic acid bacteria, fruit juice, and the like to form a paste, powder, or granule in a known manner to enhance flavor and palatability. As the fruit juice, any one selected from grape juice, citrus juice, pomegranate juice, bokbunja juice, peach juice, plum juice, and lemon juice can be used.
<시험예 1>; 혈전용해효소액의 혈전용해능 방출시험<Test Example 1>; Thrombolytic ability release test of thrombolytic enzyme solution
미세캡슐은 동결건조 후에 냉동 보관하면서 실험에 사용한다. 혈전용해효소액의 release를 위해 10mM 인산완충용액(pH 7.4) 에 분산시켜 3시간 동안 교반한 후, 원심분리하여 wall material을 제거하고 상등액의 혈전용해활성을 fibrin plate법에 의해 측정한다.The microcapsules are used for experiments while being stored frozen after lyophilization. To release the thrombolytic enzyme solution, it is dispersed in 10 mM phosphate buffer solution (pH 7.4) and stirred for 3 hours, then centrifuged to remove the wall material and the thrombolytic activity of the supernatant is measured by the fibrin plate method.
<시험예 2>; 혈전용해효소의 활성측정<Test Example 2>; Measurement of thrombolytic enzyme activity
혈전용해효소를 피브린에 작용시키고 피브린의 분해에 따라 증가하는 tyrosine 양을, 자외선(275nm) 흡광도를 측정하여 구하는 방법으로 측정하였다. 1 Unit는 혈전용해효소 1ml가 1분 동안에 1㎍의 tyrosine을 생성하는 능력이다. 피브리노겐 수용액은 50mM 붕사 완충액(pH 8.5, 150mM NaCl 함유) 10ml에 피브리노겐(시그마 코포레이션(Sigma Corp.)제, 혈장 유래 피브리노겐 분획 I, TYPE I-S) 72mg 상당분을 용해시켜 0.72%(w/w) 피브리노겐 수용액을 조제하였다. 트롬빈 용액은 트롬빈(시그마 코포레이션제, 소 혈장 유래)을 50mM 붕사 완충액에 용해시켜 1000U/ml의 농도로 조제하고, 사용할 때에 동일한 붕사 완충액을 사용하여 50배로 희석( 20U/ml)하여 트롬빈 용액을 제조하였다. 나토키나제의 활성 측정은 시험관에 50mM 붕사 완충액 1.4ml 및 피브리노겐 수용액 0.4ml를 취하고, 37℃±O.3℃의 항온 수조에서 5분 동안 가온한 다음, 트롬빈 용액 0.1ml를 가하여 교반한다. 혼합액을 10분 동안 이러한 항온 수조에 방치한 다음, 시료 용액 O.1ml를 혼합액에 가하고 5초 동안 교반하여 항온 수조에서 방치한다. 시료 용액을 첨가하고 나서 20분 후 및 40분 후에 각각 5초 동안 교반하며, 60분 후에 O.2M 트리클로로아세테이트 용액 2ml를 가하여 교반한 다음, 추가로 20분 동안 방치한다. 이러한 반응액을 15,000×g에서 5분 동안 원심분리하여 상등액의 275nm에서의 흡광도(Ar)를 측정한다. 한편, 50mM 붕사 완충액 1.4ml 및 피브리노겐 수용액 0.4ml를 취하고, 37℃±0.3℃의 항온 수조에서 5분 동안 가온한 다음, 트롬빈 용액 0.1ml를 가하여 교반한다. 혼합액을 10분 동안 이러한 항온 수조에 방치한 다음, O.2M 트리클로로아세테이트 용액 2ml를 혼합액에 가하여 교반한다. 다음에, 시료 용액을 0.1ml 가하여 교반하고 20분 동안 방치한다. 이러한 반응액을 15,000×g에서 5분 동안 원심분리하여 상등액의 275nm에서의 흡광도(Ab)를 측정하고 대조군으로 한다.The thrombolytic enzyme acted on the fibrin and the amount of tyrosine increased with the decomposition of fibrin was measured by measuring the absorbance of ultraviolet (275 nm). 1 Unit is the ability of 1 ml of thrombolytic enzyme to generate 1 μg of tyrosine in 1 minute. The fibrinogen aqueous solution was dissolved in 50 ml of borax buffer (pH 8.5, 150 mM NaCl) in 10 ml of fibrinogen (Sigma Corp., plasma-derived fibrinogen fraction I, TYPE IS) 72 mg equivalent dissolved 0.72% (w/w) fibrinogen An aqueous solution was prepared. The thrombin solution is prepared by dissolving thrombin (Sigma Corporation, bovine plasma) in 50 mM borax buffer to a concentration of 1000 U/ml, and diluting it 50 times using the same borax buffer (20 U/ml) to prepare the thrombin solution. Did. To measure the activity of nattokinase, take 1.4ml of 50mM borax buffer and 0.4ml of fibrinogen aqueous solution in a test tube, warm in a constant temperature water bath at 37°C±0.3°C for 5 minutes, then add 0.1ml of thrombin solution and stir. The mixed solution was left in this constant temperature water bath for 10 minutes, and then 0.1 ml of the sample solution was added to the mixed solution and stirred for 5 seconds to stand in the constant temperature water bath. After the sample solution is added, the mixture is stirred for 5 seconds after 20 minutes and 40 minutes, and after 60 minutes, 2 ml of an O.2M trichloroacetate solution is added and stirred, and then left for an additional 20 minutes. The reaction solution was centrifuged at 15,000 x g for 5 minutes to measure the absorbance (Ar) of the supernatant at 275 nm. Meanwhile, 1.4 ml of 50 mM borax buffer and 0.4 ml of fibrinogen aqueous solution were taken, heated in a constant temperature water bath at 37° C.±0.3° C. for 5 minutes, and then 0.1 ml of thrombin solution was added and stirred. The mixture was left in this constant temperature water bath for 10 minutes, and then 2 ml of an O.2M trichloroacetate solution was added to the mixture and stirred. Next, 0.1 ml of the sample solution is added, stirred, and left for 20 minutes. This reaction solution was centrifuged at 15,000 x g for 5 minutes to measure the absorbance (Ab) of the supernatant at 275 nm and serve as a control.
혈전용해효소의 효소 활성(A)은 하기 식으로 구한다.The enzyme activity (A) of the thrombolytic enzyme is obtained by the following formula.
A(FU/ml)= [(Ab-Ao)/(0.01×60×O.l)]×D, 상기식에서 D는 희석 배율이다.A(FU/ml)= [(Ab-Ao)/(0.01×60×O.l)]×D, where D is the dilution factor.
본 발명의 실시예 1 내지 4은 청국장 발효액은 시판균주(대조군)에 비하여 혈전용해효소 활성이 7 내지 10배, 배양액추출물은 대조군에 비하여 15 내지 17배, 추출액분말은 12 내지 13배로 높은 것으로 나타났다.In Examples 1 to 4 of the present invention, the fermentation broth of Cheonggukjang showed 7 to 10 times higher thrombolytic enzyme activity, 15 to 17 times higher than the control group, and 12 to 13 times higher extract powder than the commercially available strain (control). .
<시험예>; 관능평가<Test Example>; Sensory evaluation
관능검사에 대해 잘 훈련된 panel 요원(20대, 30대, 40대 남여 각 5명)으로 하여금 본 발명의 혈전용해효소가 함유된 페이스트형, 분말형, 과립형, 캡슐형, 젤리형 등의 제형별로 색, 맛, 냄새, 종합적 기호도를 9점 기호척도법으로 하여 평가하였다. 그 결과는 다음의 표 2에 나타냈다.For well-trained panel agents (5 people in their 20s, 30s and 40s) for sensory testing, paste, powder, granule, capsule, jelly, etc. containing the thrombolytic enzyme of the present invention The color, taste, smell, and overall preference for each formulation were evaluated using a nine-point preference scale. The results are shown in Table 2 below.
상기의 결과로부터 페이스트형 5.5이고, 분말, 과립, 캡슐, 젤리는 모두 6.1 내지 6.5 이상을 나타내고 있어서 관능 목표 6.0 이상을 유지하는 것으로 나타났다.From the above results, the paste was 5.5, and powders, granules, capsules, and jellies all exhibited 6.1 to 6.5 or more, thus maintaining a sensory target of 6.0 or more.
본 발명의 혈전용해효소는 혈행개선 효과가 뛰어나고, 청국장의 이취나 거부감이 없는 청국장 유래 효소를 제공할 수 있다.The thrombolytic enzyme of the present invention has excellent blood circulation improving effect, and can provide an enzyme derived from Cheonggukjang without odor or rejection of Cheonggukjang.
Claims (8)
상기 청국장 발효액으로부터 혈전용해효소를 추출 및 농축하는 단계는, 상기 청국장 발효액을 원심분리하여 균체를 제거하는 단계와, 여과기의 벽면에 규조토 또는 활성탄의 여과보조제를 코팅시켜 상기의 원심분리액을 여과한 후, 상기 여과된 혈전용해효소액을 0.5μm 내지 0.2μm의 막 필터가 포함된 역삼투압 농축기를 사용하여 분자량 100 이하의 물질을 제거하여, 상기 혈전용해효소액의 부피를 1/10까지 농축하는 단계로 구성되고,
상기 제형화는 젤리형이며,
상기 젤리형은 상기 농축된 혈전용해효소를 한외여과한 다음, 역삼투압농축기로 농축하는 단계와,
상기 농축한 혈전용해효소액 200g에 알긴산나트륨 3g, 감귤쥬스 1 L를 섞은 다음, 2%(w/w)의 염화칼슘용액에 낙하시켜 비드를 제조하는 단계와,
상기 제조된 비드를 30분간 숙성한 다음 비드를 분리하고 증류수로 세척하여 염화칼슘을 제거하고 UV 살균하여 비드를 제조하는 단계와,
젤리베이스의 전체 중량에 대하여 젤라틴 15 중량%, 펙틴 3 중량%, 설탕 2 중량%, 조청 10 중량%를 섞어 젤리베이스를 만든 다음 살균하여, 혈전용해효소의 전체 중량에 대하여 상기 비드 30 중량%를 첨가한 후, 금형틀에 넣어 혈전용해효소 젤리를 만드는 단계로 구성되는 것을 특징으로 하는 혈행개선 증진용 혈전용해효소의 제조방법.Comprising a step of fermenting Cheonggukjang strain in a medium to prepare a Cheonggukjang fermentation broth containing a thrombolytic enzyme, extracting and concentrating thrombolytic enzymes from the Cheonggukjang fermentation broth, and formulating the concentrated thrombolytic enzyme As a method of manufacturing a thrombolytic enzyme for improving blood circulation,
The step of extracting and concentrating the thrombolytic enzyme from the cheonggukjang fermentation broth, removing the cells by centrifuging the cheonggukjang fermentation broth, and filtering the centrifugation solution by coating a filter aid of diatomaceous earth or activated carbon on the wall of the filter Thereafter, the filtered thrombolytic enzyme solution is removed by using a reverse osmosis concentrator containing a membrane filter of 0.5 μm to 0.2 μm to remove substances having a molecular weight of 100 or less, and concentrating the volume of the thrombolytic enzyme solution to 1/10. Composed,
The formulation is a jelly type,
The jelly type is the step of ultrafiltration of the concentrated thrombolytic enzyme, and then concentrated with a reverse osmosis concentrator,
Preparing beads by mixing 3 g of sodium alginate and 1 L of citrus juice in 200 g of the concentrated thrombolytic enzyme solution, and then dropping it in 2% (w/w) calcium chloride solution;
After aging the prepared beads for 30 minutes, separating the beads and washing with distilled water to remove calcium chloride and UV sterilizing to prepare beads,
15% by weight of gelatin, 3% by weight of pectin, 2% by weight of sugar, and 10% by weight of crude water relative to the total weight of the jelly base to make a jelly base and sterilized, and 30% by weight of the beads relative to the total weight of the thrombolytic enzyme After adding, the method of manufacturing a thrombolytic enzyme for improving blood circulation, characterized by comprising a step of making a thrombolytic enzyme jelly in a mold.
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