KR101856693B1 - Method for preparing fermented mixture comprising Laminaria japonica, Undaria pinnatifida Suringer and hizikia fusiforme and composition for enhancing antioxidant activity and immune activity comprising fermented mixture prepared thereby - Google Patents
Method for preparing fermented mixture comprising Laminaria japonica, Undaria pinnatifida Suringer and hizikia fusiforme and composition for enhancing antioxidant activity and immune activity comprising fermented mixture prepared thereby Download PDFInfo
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- KR101856693B1 KR101856693B1 KR1020170117188A KR20170117188A KR101856693B1 KR 101856693 B1 KR101856693 B1 KR 101856693B1 KR 1020170117188 A KR1020170117188 A KR 1020170117188A KR 20170117188 A KR20170117188 A KR 20170117188A KR 101856693 B1 KR101856693 B1 KR 101856693B1
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- seaweed
- activity
- bacillus subtilis
- fermented product
- fermented mixture
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Abstract
Description
본 발명은 다시마, 미역, 톳을 포함하는 해조류 혼합물을 바실러스 서브틸리스 균주로 발효시켜 항산화 활성이 뛰어날 뿐만 아니라, L-아르기닌, L-라이신 등의 아미노산을 다량 포함하여 우수한 면역 증강 기능을 갖는 복합 발효물을 제조하는 방법과, 이에 의해 제조된 복합 발효물을 유효성분으로 포함하는 항산화 및 면역 증진용 조성물에 관한 것이다. The present invention is based on the finding that a fermented seaweed mixture containing seaweed, marine algae, and seaweed is fermented with a strain of Bacillus subtilis, which not only excels in antioxidant activity but also contains a large amount of amino acids such as L-arginine and L- To a method for producing a fermented product, and to a composition for antioxidant and immunity enhancement comprising the complex fermented product thus produced as an effective ingredient.
해양 환경에서 서식하는 해조류는 고부가가치의 물질을 생산하고 인간생활의 질적 향상을 도모할 수 있는 자원의 하나로서, 예로부터, 아시아 지역에서 널리 섭취해 왔으며, 영양학적으로 열량은 매우 낮으면서 비타민과 무기질, 식이섬유소가 풍부하고, 육지 식물에는 없는 비소화성의 점질성 다당류를 다량 함유하고 있는 것으로 알려진 바 있다.Algae living in the marine environment is one of the resources that can produce high value-added materials and improve the quality of human life. It has been widely consumed in Asia since ancient times. It has nutritionally low calorific value, It is known that it contains a large amount of non-digestible, viscous polysaccharides rich in minerals, dietary fiber, and not found in land plants.
특히, 상기 해조류는 채소류에 비해 필수 아미노산과 불포화 지방산의 함량이 높고, 고지혈증 및 고혈압 예방, 항당뇨, 항돌연변이, 항암, 멜라닌 생성억제, 항염증 및 항응고, 면역조절작용 등 다양한 생리활성 효과 등을 나타내는 것으로 보고되면서, 최근에는 해조류를 건강기능성 식품원료로 인식하고 있으며, 해조류 유래의 고부가가치를 갖는 기능성 신소재에 대한 관심이 높아짐에 따라 해조류의 활용성을 증진시키기 위한 다양한 연구가 진행되고 있다.In particular, the above-mentioned seaweeds have a higher content of essential amino acids and unsaturated fatty acids than those of vegetables, and have various physiological activity effects such as prevention of hyperlipidemia and hypertension, anti-diabetes, antimutagenic, anti-cancer, melanin production inhibition, anti-inflammatory and anti- Recently, seaweeds have been recognized as a source of health functional food. Various interest in functional new materials having high added value derived from seaweeds has been increased, and various studies for improving the utilization of seaweed have been carried out.
종래에는 해조류를 열수 추출이나 알칼리, 산 또는 효소처리 등에 의하여 추출 후 가공하는 방법들을 사용하여 해조류의 생리활성 성분을 추출하여 이용하고자 하였으나, 이와 같은 추출공정은 해조류에 포함되어 있는 탄수화물의 대부분이 비소화성 복합 다당류로서 산이나 알칼리에 비교적 안정하고 특수한 미생물 효소에 의하지 않고서는 분해되기가 어려워 여러 가지 생체 활성물질의 변질 및 파괴 등을 수반하는 결과를 초래하거나 또는 효과적으로 추출해내지 못하는 단점이 있다. Conventionally, seaweed extracts were extracted by hot water extraction, alkali, acid or enzyme treatment, and then extracted and used for extracting physiologically active components of seaweed. However, in this extraction process, most of the carbohydrates contained in seaweed are arsenic It has a disadvantage in that it is relatively stable to acids and alkalis and is difficult to decompose without depending on a specific microbial enzyme, resulting in the deterioration or destruction of various bioactive substances, or it can not be extracted effectively.
한편, 인체의 대사과정 중 생성되는 활성 산소종(reactive oxygen species, ROS)은 체내의 세포막, 단백질 및 DNA 등의 손상을 일으켜 노화와 질병의 원인이 되는 물질 중 하나로서, 살아있는 유기체의 산화 진행 중에 부산물로써 생성될 수 있다. 상기 활성 산소종(ROS)은 정상적인 신진대사에서 많은 중요한 기능을 하지만, 산화 스트레스에 의해 유도되는 과도한 ROS는 단백질의 기능을 손상, 세포 거대 분자에 산화 손상을 가져오고, trigger cell의 사멸을 유도할 수 있다. 특히, 장기간의 산화 스트레스 상태는 심혈과 질환과 암 등 여러 질병의 발병을 유도할 수 있고, DNA, 단백질 및 기타 거대 분자 들은 산화적 스트레스로 인해 손상이 유발될 수 있으며, 나아가 노화로 이어지는 내적 손상의 주요 유형을 구성한다고 가정되어지고 있다. On the other hand, reactive oxygen species (ROS) generated during the metabolic process of the human body cause damage to the cell membrane, protein and DNA in the body and cause aging and disease. As a result, Can be generated as a byproduct. Although the active oxygen species (ROS) play many important functions in normal metabolism, excessive ROS induced by oxidative stress may impair protein function, cause oxidative damage to cellular macromolecules, induce trigger cell death . In particular, long-term oxidative stress may lead to the development of various diseases such as cardiovascular diseases and cancer, and DNA, proteins and other macromolecules may be damaged due to oxidative stress, It is assumed that it constitutes the main type of.
최근, 산화적 스트레스로 인해 발생되는 악영향을 최소화하고 건강한 삶을 영위하기 위한 목적으로 항산화 물질의 섭취량이 증가하고 있으며, 특히, 인체에 무해한 천연 항산화제에 대한 관심과 수요가 증가하고 있어 천연 물 유래의 다양한 항산화제의 탐색이 진행되고 있다.In recent years, the intake of antioxidants has been increasing for the purpose of minimizing adverse effects caused by oxidative stress and maintaining a healthy life. In particular, interest and demand for natural antioxidants harmless to humans are increasing, And a variety of antioxidants.
기존에 해조류는 항산화 효과를 나타낼 뿐만 아니라 다양한 생리활성 효능을 나타내는 천연물질로 보고되고 있어 해조류에 대한 다양한 연구가 진행되고 있으나, 종래에는 3종 이상의 해조류를 동시에 복합적으로 발효하여 항산화 활성이 우수한 복합 발효물에 관한 기술 내용은 개시된 바 없어 이에 대한 연구가 필요하다.Conventionally, seaweeds have been reported to be natural substances exhibiting various physiologically active effects as well as antioxidative effects. Various studies on seaweeds have been carried out, but conventionally, three or more kinds of seaweeds have been mixed and fermented at the same time, The technical content of water has not been disclosed yet and needs to be studied.
본 발명의 발명자들은 다시마, 미역 및 톳을 포함하는 해조류를 특정 환경에서 선별한 바실러스 서브틸리스(Bacillus subtilis) 균주로 발효시킨 복합 발효물이 항산화 활성 효능이 우수하고, L-라이신과 L-아르기닌 등과 같은 아미노산의 함량이 현저히 증가하여 면역 증강 작용 등과 같은 건강 기능성을 갖는다는 사실을 확인하였다.The inventors of the present invention have found that a complex fermented product obtained by fermenting Bacillus subtilis strain selected from marine algae containing seaweed, marine algae and seaweed in a specific environment has excellent antioxidative activity, and L-lysine and L-arginine And the like, and thus have health functionalities such as immunity enhancement and the like.
따라서, 본 발명은 다시마, 미역 및 톳을 포함하는 해조류를 바실러스 서브틸리스 균주로 발효시켜 복합 발효물을 제조하는 방법과 이에 의해 제조된 복합 발효물을 유효성분으로 포함하여 항산화 활성 및 면역 증강 기능을 갖는 조성물에 대한 기술 내용을 제공하고자 하는 것이다.Accordingly, the present invention relates to a method for producing a complex fermented product by fermenting seaweeds including sea tangle, seaweed, and garlic with a strain of Bacillus subtilis, and a method for producing a complex fermented product comprising the complex fermented product thus prepared as an active ingredient, The present invention is directed to a composition having the above-mentioned composition.
상기한 바와 같은 기술적 과제를 달성하기 위해서 본 발명은, 다시마, 미역, 톳 및 물을 포함하는 해조류 혼합물에 바실러스 서브틸리스 균주를 접종하여 발효시키는 단계를 포함하는 복합 발효물의 제조방법을 제공한다.According to an aspect of the present invention, there is provided a method for preparing a complex fermented product, comprising the step of inoculating a Bacillus subtilis strain into a seaweed mixture containing kelp, sea must, tortoise and water and fermenting the same.
또한, 상기 해조류 혼합물은 다시마 150 내지 200 중량부, 미역 50 내지 150 중량부, 톳 100 내지 150 중량부 및 물 100 내지 600 중량부를 포함하는 것을 특징으로 한다.Also, the seaweed mixture comprises 150-200 parts by weight of sea tangle, 50-150 parts by weight of seaweed, 100-150 parts by weight of seaweed, and 100-600 parts by weight of water.
또한, 상기 발효시키는 단계는 상기 해조류 혼합물 100 중량부에 대하여 1 내지 10 중량부에 해당하는 바실러스 서브틸리스 균주를 접종하여 발효시킨 것을 특징으로 한다.The fermentation step may be performed by inoculating a Bacillus subtilis strain corresponding to 1 to 10 parts by weight with respect to 100 parts by weight of the seaweed mixture.
또한, 상기 발효시키는 단계는 20 내지 40 ℃에서 48 내지 120시간 동안 수행하는 것을 특징으로 한다.Also, the fermentation step is performed at 20 to 40 DEG C for 48 to 120 hours.
또한, 본 발명은 상기에 기재된 방법으로 제조한 복합 발효물을 유효성분으로 포함하는 항산화 및 면역 증진용 조성물을 제공한다.The present invention also provides a composition for antioxidant and immunity enhancement comprising the complex fermented product produced by the above-described method as an effective ingredient.
본 발명의 복합 발효물의 제조방법에 따르면, 다시마, 미역 및 톳을 포함하는 해조류 혼합물을 특정 바실러스 서브틸리스 균주로 발효시켜 L-아르기닌, L-라이신 등과 같은 면역 증강 기능을 갖는 아미노산을 다량 포함할 뿐만 아니라 항산화 활성 효능이 우수한 복합 발효물을 제조할 수 있다.According to the method for producing a complex fermented product of the present invention, a seaweed mixture including kelp, sea must, and red pepper is fermented with a specific strain of Bacillus subtilis to contain a large amount of amino acids having immunity enhancing function such as L-arginine and L-lysine In addition, a complex fermented product having excellent antioxidant activity can be produced.
상기한 복합 발효물은 항산화 활성 기능 및 면역 증진 기능을 나타내어 식품, 화장품 등의 제조를 위해 손쉽게 활용될 수 있다.The complex fermented product exhibits an antioxidative activity function and an immunity enhancing function and can be easily used for the production of foods, cosmetics and the like.
도 1은 실시예 및 비교예에 따른 발효물 시료와 대조군(BHT)의 DPPH 라디칼 소거능의 측정한 결과이다.
도 2는 실시예 및 비교예에 따른 복합 발효 추출물과 대조군(아스코르브산)의 ABTS 라디칼 소거능의 측정한 결과이다.
도 3은 실시예 및 비교예에 따른 복합 발효 추출물과 대조군(아스코르브산)의 아질질산(nitrite) 라디칼 소거능의 측정한 결과이다.FIG. 1 shows the results of measurement of the DPPH radical scavenging activity of the fermented sample and the control (BHT) according to the examples and the comparative examples.
FIG. 2 shows the results of measurement of the ABTS radical scavenging activity of the complex fermentation extract and the control group (ascorbic acid) according to the examples and the comparative examples.
FIG. 3 shows the results of measurement of the nitrite radical scavenging ability of the complex fermentation extract and the control group (ascorbic acid) according to the examples and the comparative examples.
본 발명에서는 생리활성이 뛰어난 것으로 알려진 다시마, 미역 및 톳을 포함하는 해조류를 바실러스 서브틸리스(Bacillus subtilis)로 발효시켜 복합 발효물을 제조하는 방법과, 이에 의해 제조된 복합 발효물을 유효성분으로 포함하여 우수한 항산화 활성 및 면역 증강 기능을 갖는 조성물에 관한 기술 내용을 제공하고자 한다. In the present invention, a method of producing a complex fermented product by fermenting seaweeds containing tides, seaweeds and tapes, which are known to have excellent physiological activity, with Bacillus subtilis , and a method of producing a complex fermented product And a composition having an excellent antioxidative activity and an immunostimulating function.
본 발명에서 "발효"란, 미생물이 자신이 가지고 있는 효소를 이용해 다시마, 미역 및 톳을 포함하는 해조류에 포함된 각종 유기물을 분해시키는 과정을 의미하는 것이다.The term "fermentation" in the present invention means a process in which a microorganism decomposes various organic substances contained in seaweeds including kelp, wakame, and tortoise using an enzyme of its own.
이하, 본 발명을 상세히 설명하도록 한다.Hereinafter, the present invention will be described in detail.
본 발명은, 다시마, 미역, 톳 및 물을 포함하는 해조류 혼합물에 바실러스 서브틸리스 균주를 접종하여 발효시키는 단계를 포함하는 복합 발효물의 제조방법을 제공한다.The present invention provides a method for producing a complex fermented product comprising a step of inoculating a strain of Bacillus subtilis into a seaweed mixture containing kelp, sea mustard, water and water to ferment.
상기 다시마(Laminaria japonica)는, 갈조식물문 다시마과 갈조류에 속하는 한류성 해조류로서 인체에 필요한 수십 종의 미량원소와 칼륨, 칼슘, 마그네슘 및 인산 등과 같은 미네랄이 풍부하다. 특히, 상기 다시마는 항혈액 응고 작용, 항암작용, 혈청 콜레스테롤 저하작용, 유해 중금속의 체내 흡수 방지 및 배출 기능 뿐 아니라 면역력 증가, 항산화 활성 등의 효능을 나타낼 수 있을 뿐만 아니라, 다른 해조류에 비해 요오드의 함량이 높아 갑상선암 및 각기병 예방 등에 효능이 있는 건강식품으로 각광받고 있는 주요 수산식물로서, 최근 일본의 원자력 발전소 방사능 유출 사고로 인해 다시마의 소비가 촉진되고 있다.The seaweed ( Laminaria japonica ) is a seaweed belonging to the seagrass, kelp and brown algae, and is rich in various kinds of trace elements necessary for the human body and minerals such as potassium, calcium, magnesium and phosphoric acid. Particularly, the kelp can exhibit not only an anti-blood coagulation action, an anti-cancer action, a serum cholesterol lowering effect, an anti-absorption and excretion function of harmful heavy metals, an immunity increase and an antioxidant activity, It is a major fishery plant that has high content and is attracting attention as a health food that is effective for prevention of thyroid cancer and angiopathy. Recently, consumption of kelp has been promoted due to radioactive leak accident in nuclear power plant in Japan.
상기 미역(Undaria pinnatifida Suringer)은, 다시마목 미역과에 속하는 갈조류로서, 나트륨(Na), 칼륨(K), 칼슘(Ca), 마그네슘(Mg), 인(P), 황(S) 등이 풍부하고, 식이섬유소, 리놀산, 비타민 등이 풍부한 알칼리성 건강식품으로 생리활성 물질이 다량 함유되어 있다. 특히, 미역에 들어 있는 점질 다당류인 알긴산(alginic acid)은 중금속 및 방사능 물질의 체외배출, 콜레스테롤 저하, 비만 및 변비방지 효능과 더불어 혈압이나 당뇨 예방효과가 우수한 것으로 알려져 있으며, 미역에 포함된 후코이단(fucoidan)은 항암, 항콜레스테롤, 혈액응고저해, 혈압조절 등의 혈류개선작용이 우수하며, 지질대사 개선에도 효과가 있다. The undaria pinnatifida suringer is a brown alga belonging to the sea tangle mackerel and is rich in sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), phosphorus (P) , And is an alkaline health food rich in dietary fiber, linoleic acid, and vitamins, and contains a large amount of physiologically active substances. In particular, alginic acid, a viscous polysaccharide contained in seaweed, is known to exert excellent effects on blood pressure and diabetes prevention as well as efficacy of preventing heavy metals and radioactive substances from excretion, cholesterol lowering, obesity and constipation prevention, and fucoidan fucoidan) is excellent in improving blood flow such as anticancer, anticholesterol, inhibition of blood clotting and blood pressure control, and is also effective in improving lipid metabolism.
상기 톳(Hijikia fusiforme, Tott)은 갈조류 모자반과에 속하는 다년생 해조류로서, 독특한 맛과 함께 칼슘, 비타민 A 및 식이섬유소 함량이 풍부하여 당뇨병, 고혈압 예방, 대장암 및 변비 등에 효과가 좋으며 요오드 성분을 다량 포함하여 갑상선암 및 각기병 예방에 효과가 있다고 알려져 있다.The Hijikia fusiforme (Tott) is a perennial seaweed belonging to the brown algae and is rich in calcium, vitamin A, and dietary fiber content along with its unique taste. It is effective for prevention of diabetes, hypertension, colon cancer and constipation. And is known to be effective in preventing thyroid carcinoma and otolaryngology.
상기 다시마, 미역 및 톳은 시중에 시판되고 있는 통상적인 소재라면 제한받지 않고 사용할 수 있으며, 상기와 같은 다시마, 미역 및 톳은 세척 과정을 거쳐 염분을 충분히 제거한 것을 사용할 수 있으며, 상기와 같은 해조류는 바실러스 서브틸리스에 의한 균일한 발효가 유도될 수 있도록 바람직하게는 1 내지 20 mm 크기를 갖도록 분쇄한 것을 사용할 수 있다.The seaweed, the seaweed, and the seaweed can be used without restriction as long as it is a common material commercially available in the market. The seaweed, the seaweed and the seaweed may be washed with a sufficient amount of salinity, And pulverized to have a size of preferably 1 to 20 mm so that uniform fermentation by Bacillus subtilis can be induced can be used.
또한 상기와 같이 분쇄한 다시마, 미역 및 톳의 분쇄 혼합물을 정제수에 혼합하여 함수량을 증가시키고, 여기에 바실러스 서브틸리스를 접종하여 발효를 수행할 수 있다.Also, the pulverized mixture of crushed kelp, wakame, and garlic may be mixed with purified water to increase the water content, and the fermentation can be performed by inoculating Bacillus subtilis.
기존에 발효 방법에서는 별도의 탄소원을 첨가하거나, 다시마, 미역 및 톳을 각각 발효시켜 발효물을 제조하였으나, 본 발명에서는 상기 다시마, 미역 및 톳을 모두 포함하는 혼합물을 한번에 발효시켜 항산화 활성 효능을 나타낼 뿐만 아니라, 면역 증강 기능을 갖는 복합 발효물을 제조할 수 있다.In the conventional fermentation method, a separate carbon source is added, or a fermented product is produced by fermenting kelp, wakame, and red pepper, respectively. In the present invention, however, the mixture containing the kelp, wakame, and red pepper is fermented at once to exhibit antioxidant activity In addition, a complex fermentation product having an immune enhancing function can be produced.
또한, 기존에 통상적인 발효 방법을 통해 다시마, 미역 및 톳의 혼합물을 발효시킬 경우 발효 과정 중에 산성화가 유도되어 발효물의 산도가 낮아지는 특성이 나타나며, 이에 의해 약알칼리 환경에서 생육활성을 나타내는 바실러스 서브틸리스 균주를 이용한 발효가 저해되어 생리활성 물질의 생성이 급증하지 못하는 문제가 있다.In addition, when fermentation of a mixture of kelp, sea must, and red pepper with conventional fermentation methods, acidification is induced during the fermentation process and the acidity of the fermented product is lowered. As a result, Bacillus subtilis There is a problem that the fermentation using the Tillers strain is inhibited and the production of the physiologically active substance is not increased rapidly.
이에 본 발명에서는, 특정 조건에서 선별한 바실러스 서브틸리스 균주를 이용해 해조류 혼합물을 발효시킴에 따라, 항산화 물질이 다량 생성되어 항산화 활성 효능이 우수하고, L-아르기닌, L-라이신 등과 같이 면역 증강 기능을 갖는 아미노산을 다량 포함하는 복합 발효물을 제조하도록 구성할 수 있다.Therefore, in the present invention, the fermentation of the seaweed mixture using the Bacillus subtilis strains selected under specific conditions produces a large amount of antioxidant substances, which is excellent in antioxidant activity, and has an immunostimulatory function such as L-arginine and L- ≪ RTI ID = 0.0 > a < / RTI >
특히, 상기 L-아르기닌(L-arginine)은 구아니딘(guanidine) 작용기로 인해 강한 염기성을 나타내는 물질로서, 생체 내 오르니틴(ornithine) 순환과정의 대사경로에서 시트룰린(citrulline)과 아스파르트산(aspartic acid)으로부터 생성되고, 아르긴 분해효소(arginase)의 작용에 의해 요소와 오르니틴으로 분해되어 생성되며, 혈중 암모니아를 요소로 배설하는데 도움을 주기 때문에 암모니아에 의해 유발되는 혈중 간기능 장애에 유익하며, 혈압, 장운동의 조절, 혈소판 응고, 식균세포의 기능에 관여하는 질산(NO)의 전구체 역할을 한다. 상기 L-아르기닌은 체내에서 정상치 이하일 경우 생리적 욕구를 충족시키기 위해 체내에서 합성되지만 필요량을 충족시키기 위해 식사의 형태로 적당량 섭취해야 하는 물질로서, 면역 세포 및 성장호르몬의 분비를 증가시켜 면역기능 개선에 도움을 주고, B-세포 또는 T-세포 등의 면역세포 성장과 발현을 증가시켜 신체 저항력을 증진시키며, 항노화 호르몬으로 알려진 성장 호르몬의 분비를 증가시켜 지방연소. 근육량 및 골밀도 증가, 면역기능 개선에 유의적인 효능을 나타낼 수 있다.In particular, the L-arginine is a substance having a strong basicity due to a guanidine functional group, and it is a substance which is produced by citrulline and aspartic acid in the metabolic pathway of the ornithine circulation in vivo. Which is produced by decomposition of urea and ornithine by the action of arginase and which helps to excrete blood ammonia into the urea and thus is beneficial to the blood liver dysfunction caused by ammonia, , The regulation of intestinal motility, platelet clotting, and nitric acid (NO), which is involved in the function of phagocyte cells. The L-arginine is synthesized in the body to meet the physiological needs when it is below the normal level in the body, but it needs to be taken in an appropriate amount in the form of a meal in order to meet the required amount. It enhances secretion of immune cells and growth hormone, It helps to increase the body's resistance by increasing the immune cell growth and expression of B-cells or T-cells, and by increasing the secretion of growth hormone known as anti-aging hormone. Muscle mass, bone density, and immune function.
또한, L-라이신(L-Lysine)은 인체 내 필수 아미노산으로 특히 식물성 곡류를 주식으로 하는 동양계에서는 부족해지기 쉬운 영양소로서, 체내 골격형성을 위한 단백질 전구체로써 작용하며, 생리적으로는 지방산 대사 촉진에 관여하는 카르니틴 생합성, 성장 무기 요소인 칼슘 흡수촉진 및 다른 글루타민산, 아르기닌과 병행 섭취시 장내 발달 촉진과 더불어 면역력 증강 효과를 나타낼 수 있다.In addition, L-Lysine is an essential amino acid in the human body. It is a nutrient that tends to become scarce in the Asian diet, especially in vegetable grains. It acts as a protein precursor for the formation of the body skeleton and is physiologically involved in the promotion of fatty acid metabolism Carnitine biosynthesis, accelerating the absorption of calcium, a growth inorganic element, and promoting intestinal development in combination with other glutamate and arginine, may also have the effect of enhancing the immune system.
바람직하게는, 상기와 같은 생리활성 기능을 나타내는 복합 발효물은 다시마 150 내지 200 중량부, 미역 50 내지 150 중량부, 톳 100 내지 150 중량부 및 물 100 내지 600 중량부를 포함하는 해조류 혼합물에 통상적인 다양한 바실러스 서브틸리스균주를 접종하여 발효시켜 제조할 수 있으며, 상기 해조류 혼합물 100 중량부에 대하여 1 내지 10 중량부에 해당하는 바실러스 서브틸리스 균주를 접종하여 발효시킬 수 있다. 보다 바람직하게는, 다시마, 180 중량부, 미역 100 중량부, 톳 120 중량부 및 물 200 중량부를 포함하는 혼합물에 30 중량부의 바실러스 서비틸리스 균주를 접종하여 발효를 수행할 수 있다.Preferably, the complex fermented product exhibiting the above-mentioned physiological activity function is added to a seaweed mixture containing 150 to 200 parts by weight of seaweed, 50 to 150 parts by weight of marrow, 100 to 150 parts by weight of potato and 100 to 600 parts by weight of water, The fermentation can be carried out by inoculating various Bacillus subtilis strains and fermenting them. The fermentation can be carried out by inoculating 1 to 10 parts by weight of Bacillus subtilis strains to 100 parts by weight of the seaweed mixture. More preferably, 30 parts by weight of Bacillus subtilis strain may be inoculated into a mixture comprising kelp, 180 parts by weight, 100 parts by weight of seaweed, 120 parts by weight of potato, and 200 parts by weight of water.
특히, 상기 바실러스 서브틸리스 균주는 pH 3 내지 6의 산성 환경에서도 다시마, 미역 및 톳을 효과적으로 발효시킬 수 있는 선별된 균주를 사용할 수 있고 이와 같은 균주는 다시마, 미역 및 톳에 포함된 각종 다당류 및 단백질을 효과적으로 분해하여 항산화 활성 및 면역 증강 기능을 갖는 물질을 과량 생성할 수 있으며, 바람직하게는, pH가 4 내지 5인 조건에서도 안정적으로 발효를 수행할 수 있는 바실러스 서브틸리스 균주를 선별하여 다시마, 미역 및 톳을 포함하는 해조류 혼합물을 발효하도록 구성할 수 있다.In particular, the Bacillus subtilis strain can use a selected strain capable of effectively fermenting kelp, waxy sea bass and roots in an acidic environment having a pH of 3 to 6, and such a strain can be used for various polysaccharides, Bacillus subtilis strains capable of stably fermenting under conditions of pH 4 to 5 can be selected and used for the production of kelp , Seaweed and peat can be fermented.
상기와 같은 조성을 갖는 혼합물에 바실러스 서브틸리스 균주를 접종하여 발효시키면, 다시마, 미역, 톳 각각에 포함된 부족한 영양성분을 서로 보충하는 효과를 나타내어 바실러스 서브틸리스 균주에 의한 발효 효율을 촉진시킬 수 있으며, 별도의 탄수화물 공급원을 첨가하지 않아도 바실러스 서브틸리스 균주가 다시마, 미역 및 톳에 포함된 각종 다당류와 단백질을 영양분으로 활용하여 고분자를 저분자화시켜 분해시킴에 따라, 항산화 활성 및 면역 증강 기능을 갖는 각종 생리활성 물질을 과량 생성할 수 있다.When the Bacillus subtilis strain is inoculated and fermented in the mixture having the above composition, the deficient nutrients contained in each of kelp, seaweed, and tortoise are supplemented to promote the fermentation efficiency by the Bacillus subtilis strain Bacillus subtilis strains can be used as nutrients for various polysaccharides and proteins contained in seaweeds, seaweeds, and seaweeds to decompose them into small molecules, thus providing antioxidant and immunostimulating functions without additional carbohydrate sources. It is possible to generate an excessive amount of various physiologically active substances.
상기 발효는 바람직하게는 20 내지 40 ℃에서 48 내지 120시간 동안 수행하도록 구성할 수 있으며, 상기 발효 온도가 너무 높거나 낮을 경우 원활한 발효 공정이 일어나지 않고 시간적, 비용적인 면에서 효율성이 떨어지며 바실러스 서브틸리스 균주를 이용하여 상기 해조류를 발효시켜 항산화 효과를 극대화시키고, 각종 아미노산의 생성을 증가시키기 위해서는 상기 범위 내에서 발효시키는 것이 중요하며, 보다 바람직하게는, 상기 발효는 34 ℃의 온도에서 48 내지 72시간 동안 발효 공정을 수행할 수 있다.If the fermentation temperature is too high or low, a smooth fermentation process will not take place, and efficiency in terms of time and cost will be inadequate. If the fermentation temperature is too high or low, the Bacillus subtilis In order to maximize the antioxidative effect and increase the production of various amino acids, it is important to ferment the seaweeds in the above-mentioned range. More preferably, the fermentation is carried out at a temperature of 34 ° C in the range of 48 to 72 The fermentation process can be carried out for a period of time.
또한, 본 발명에서는 상기와 같이 해조류에 특정 바실러스 서브틸리스 균주를 이용해 발효시킨 발효물을 60 내지 90 ℃의 온도에서 일정 시간동안 열처리시켜 바실러스 서브틸리스 균주의 활성을 불활성화 시킬 수 있으며, 이와 같이 균주가 불활성화된 발효물을 용매 추출법, 초임계 추출법 또는 초음파 추출법 등의 방법으로 추출하여 복합 발효 추출물을 수득하거나, 발효물을 여과지로 여과하여 복합 발효물을 수득하도록 구성할 수 있다.In addition, in the present invention, the fermentation product fermented by using a strain of Bacillus subtilis in a seaweed as described above can be heat-treated at a temperature of 60 to 90 ° C for a predetermined time to inactivate the activity of the Bacillus subtilis strain. A fermentation product in which the strain is inactivated as described above may be extracted by a method such as solvent extraction, supercritical extraction or ultrasonic extraction to obtain a complex fermentation extract, or the fermentation product may be filtered with a filter paper to obtain a complex fermentation product.
상기와 같은 방법으로 제조된 복합 발효물은 다시마, 미역 및 톳에 포함된 각종 항산화 물질과 각종 아미노산의 생성이 촉진되어 우수한 항산화 활성 효능과 면역 증강 기능을 나타내어 화장료, 피부 외용제 등의 제조에 활용할 수 있을 뿐만 아니라 수득한 복합 발효물을 농축하여 음료, 식품 제조에 활용할 수 있는 다양한 형태의 건강 기능성 조성물의 제조를 위해 활용할 수도 있다.The complex fermented product produced by the above method promotes the production of various antioxidants and various amino acids contained in sea tangle, seaweed and seaweed, exhibits excellent antioxidative activity and immunity enhancement, and can be used for the production of cosmetics, external preparation for skin and the like And may be utilized for the production of various types of health functional compositions which can be used for beverage and food production by concentrating the resulting complex fermented product.
구체적으로, 상기와 같은 방법으로 제조한 복합 발효물은 생리활성 기능을 갖는 각종 아미노산의 함량이 증가하여 우수한 건강 기능성을 나타낼 수 있을 뿐만 아니라, 특히, L-아르기닌의 함량은 90% 이상 증가하고, L-라이신의 함량은 230% 이상 증가하여 우수한 면역 증강 기능을 나타낼 수 있으며, 각종 라디칼을 효과적으로 제거할 수 있어 우수한 항산화 활성 효능을 나타낸다.Specifically, the complex fermented product produced by the above-mentioned method has an increased content of various amino acids having a physiologically active function and thus exhibits excellent health functionalities. In particular, the content of L-arginine is increased by 90% or more, The content of L-lysine is increased by 230% or more, so that it can exhibit excellent immune enhancing function and can effectively remove various radicals, thus exhibiting excellent antioxidative activity.
또한, 본 발명은 다시마, 미역 및 톳을 포함하는 해조류 혼합물을 바실러스 서브틸리스 균주로 발효시킨 복합 발효물을 유효성분으로 포함하여 항산화 및 면역 증진 기능을 갖는 조성물을 제공하며, 상기 조성물은 항산화 및 면역 증긴 기능을 유도하기 위한 건강 기능성 식품, 항산화 화장품 제조를 위해 활용될 수 있다.Also, the present invention provides a composition having antioxidant and immunity enhancing functions, which comprises a complex fermented product obtained by fermenting a seaweed mixture containing kelp, sea must, and garlic as a strain of Bacillus subtilis, as an active ingredient, Health functional food to induce immune-enhanced function, and antioxidant cosmetics.
상기한 바와 같은 본 발명에 따른 조성물은 다시마, 미역 및 톳의 천연 해조류에 특정 바실러스 서브틸리스 균주로 발효시켜 제조한 복합 발효물을 유효성분으로 포함함에 따라 항산화 활성 및 면역 증진 기능을 나타낼 뿐만 아니라 장기 복용시 또는 피부에 도입하여도 부작용이 없는 장점을 갖는다.As described above, the composition according to the present invention not only exhibits antioxidant activity and immunity enhancing function by including a complex fermented product produced by fermenting a natural seaweed of sea tangle, seaweed and garlic with a specific strain of Bacillus subtilis as an active ingredient It has the advantage that there is no side effect even when it is taken for a long time or when it is introduced into the skin.
구체적으로, 상기 조성물은 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공하여 건강 기능성 식품 제조를 위해 활용할 수 있으며, 음료, 스낵류, 빵류, 비타민 복합제, 인스턴트 식품 등의 제조에 활용될 수 있다.Specifically, the composition can be used for producing a health functional food by preparing and processing in the form of tablets, capsules, powders, granules, liquids, and rings using raw materials and components having useful functions in the human body, , Breads, vitamin complexes, instant foods, and the like.
또한, 상기 조성물은 상기 복합 발효물을 지방 유기 용매, 용해제, 농축제, 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온 봉쇄제 및 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 계면 활성제, 지질 소낭 또는 화장품에 통상적으로 사용되는 보조제 등과 혼합하여 화장품 제조를 위해 활용할 수 있으며, 상기 화장품은 용액, 겔, 고체 또는 반죽 무수 생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는 이온형(리포좀), 비이온형의 소낭 분산제의 형태, 크림, 스킨, 로션, 파우더, 연고, 스프레이 또는 콘실 스틱의 형태를 가질 수 있다.In addition, the composition may be prepared by mixing the complex fermented product with at least one selected from the group consisting of a fatty organic solvent, a solubilizing agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, It may be mixed with an ionic emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a blocking agent, a wetting agent, an essential oil, a dye, a pigment, a hydrophilic or lipophilic surfactant, The cosmetic may be in the form of a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic (liposome) A cream, a skin, a lotion, a powder, an ointment, a spray, or a cone stick.
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 embodiments presented are only a concrete example of the present invention and are not intended to limit the scope of the present invention.
<실시예 1> 복합 발효물의 제조Example 1: Preparation of complex fermented product
(1) 바실러스 서브틸리스 균주의 선별(1) Selection of Bacillus subtilis strains
본 발명에서는 다시마, 미역 및 톳을 포함하는 해조류를 효과적으로 분해하는 균주를 선별하기 위한 목적으로 해조류의 분해 활성을 확인하기 위해 3.0% 미역, 다시마 및 톳의 혼합 추출물을 첨가한 LB 고체배지를 균주 배양을 위한 배지로 활용하였으며, LB 배지에 멸균된 디옥시숙신산(C4H6O6, 20%, w/v)을 첨가하여 배지의 pH를 4.5로 조정하였다.In the present invention, in order to isolate strains that effectively decompose seaweeds including sea tangle, seaweed and seaweed, LB solid medium supplemented with 3.0% seaweed, sea tangle, And the pH of the medium was adjusted to 4.5 by adding sterile dioxysuccinic acid (C 4 H 6 O 6 , 20%, w / v) to the LB medium.
볏짚에서 채취한 시료를 멸균된 생리식염수(0.85% NaCl)에 현탁시켰으며, 희석액 0.1 ml를 LB 고체 배지에 도말하고, 37 ℃에서 배양하여 큰 투명환(clear zone)을 형성하는 균주들을 일차로 선별하였다. Samples collected from rice straw were suspended in sterilized physiological saline (0.85% NaCl), 0.1 ml of the diluted solution was plated on LB solid medium, and cultured at 37 ° C to form a large clear zone Respectively.
한천을 제외한 산성 LB 액체배지에 선별된 균주를 재접종하여 활성이 강하고 생육정도가 가장 우수한 균주를 2차로 선별하였으며, 선별된 균주의 동정을 위해 그람 염색, 포자염색 및 주사 전자현미경(JEOL, JSM-5600) 분석을 통해 형태적 특성을 확인하였다. 그 결과 선별된 균주는 그람양성의 미생물이며, 내생포자 형성이 가능하고, 장간균(rod shape)의 형태를 가지고 있는 바실러스 서브틸리스 균주인 것을 확인하였고, 산성 배지 조건에서도 강한 활성을 갖는 선별된 바실러스 서브틸리스 균주를 해조류 발효를 위한 종균으로 활용하였다.In order to identify the selected strains, Gram stain, spore staining, and scanning electron microscope (JEOL, JSM, and JSM) were used for the identification of the strains which were strongest in activity and the best in growth, -5600), and the morphological characteristics were confirmed. As a result, it was confirmed that the selected strains were Gram positive microorganisms and that Bacillus subtilis strains capable of forming endospores and having a rod shape were selected, Bacillus subtilis strains were used as seeds for seaweed fermentation.
(2) 복합 발효물의 제조(2) Production of complex fermented product
복합 발효물을 제조하기 위해서, 하기 표 1에 나타낸 바와 같은 조성으로 다시마, 미역, 톳을 포함하는 해조류 원재료를 바실러스 서브틸리스(Bacillus subtilis)로 발효시켰다.In order to prepare a complex fermented product, raw materials of seaweeds including kelp, wakame, and tortoise were fermented with Bacillus subtilis in the composition shown in Table 1 below.
이를 위해, 다시마, 미역, 톳을 각각 분쇄하여 평균 입자 크기가 5 내지 10 mm인 분쇄물을 제조하고, 제조한 분쇄물을 세척하였다. Bacillus subtilis에 정제수를 넣고 분쇄한 다시마, 미역, 톳의 분쇄물을 혼합한 후 34 ℃의 온도조건에서 72시간 동안 incubator에서 배양하여 발효물을 제조하였다. 제조한 발효물을 60 ℃에서 20분간 열처리하여 발효를 불활성화시키고 여과지로 여과한 후 회전감압농축기(rotary vacuum evaporator)로 농축하여 복합 발효물을 제조하였다.For this purpose, kelp, wakame, and starch were respectively pulverized to prepare pulverized products having an average particle size of 5 to 10 mm, and the pulverized products thus prepared were washed. Purified water was added to Bacillus subtilis , and the pulverized kelp, wakame, and red pepper powder were mixed and cultured in an incubator at 34 ° C for 72 hours. The fermented product was heat-treated at 60 ° C for 20 minutes to inactivate fermentation, which was then filtered through a filter paper and concentrated by a rotary vacuum evaporator to prepare a composite fermented product.
<비교예 1 내지 3> ≪ Comparative Examples 1 to 3 >
하기의 표 2에 나타낸 바와 같은 조성을 갖는 원재료를 실시예와 동일한 방법으로 발효시켜 발효물을 제조하였다.The raw materials having compositions as shown in the following Table 2 were fermented in the same manner as in Example to prepare fermented products.
<실험예 1> 복합 발효물의 산도 측정<Experimental Example 1> Measurement of acidity of complex fermented product
실시예에 따른 방법으로 제조하여 다시마, 미역 및 톳을 포함하는 분쇄물을 정제수에 혼합한 혼합물과, 선별된 균주로 24시간, 48시간 및 72시간 동안 발효시킨 복합 발효물의 산도를 측정하였다.The acidity of the composite fermented product obtained by the method according to the Example and the fermented product obtained by mixing the crushed product including the sea tangle, the seaweed, and the ground product into the purified water and the selected strain for 24 hours, 48 hours, and 72 hours was measured.
그 결과, 혼합물의 산도는 pH 5.8 정도인 것을 확인하였으며, 24시간 동안 발효시킨 복합 발효물의 경우, pH 5.2, 48시간 동안 발효시킨 복합 발효물의 경우 pH 4.9, 72시간 동안 발효시킨 복합 발효물의 경우 pH 4.5인 것을 확인할 수 있었으며, 제조한 복합 발효물의 산도가 크게 낮아진다는 사실을 확인할 수 있었다.As a result, the acidity of the mixture was confirmed to be about 5.8, and for the complex fermented product fermented for 24 hours, the pH of the mixed fermented product fermented for 48 hours was pH 4.9, 4.5, and it was confirmed that the acidity of the composite fermented product was significantly lowered.
이와 같은 사실을 통해서, 선별한 바실러스 서브틸리스 균주에 의해 다시마, 미역 및 톳을 포함하는 혼합물의 발효가 활발히 진행되며, 알칼리성에서 생육 활성을 갖는 것으로 알려진 통상적인 바실러스 서브틸리스 균주를 활용하는 것에 비해 발효가 더욱 촉진될 것으로 판단되었다.As a result, the fermentation of a mixture containing kelp, sea must, and tortoise is actively carried out by the selected Bacillus subtilis strain, and the use of a conventional Bacillus subtilis strain known to have a viability in alkalinity The fermentation was further promoted.
<실험예 2> 제조한 발효물의 항산화 활성 효능 분석<Experimental Example 2> Antioxidative activity of the fermented product
(1) DPPH radical-scavenging activity 측정(1) Measurement of DPPH radical-scavenging activity
실시예 및 비교예 1 내지 3에 따른 방법으로 제조한 발효물이 갖는 항산화 활성 효능을 분석하기 위해서, DPPH 라디컬 소거 활성(DPPH radical scavenging activity)을 분석하였다. DPPH 라디컬 소거 활성 분석은 Jeong의 (Jeong et al. 2009) 실험방법을 이용하여 수행하였으며, 산화방지제로 알려진 Butylated hydroxytoluene(BHT)를 양성 대조군으로 활용하고, 0.12 내지 2 mg/mL의 농도로 각각 처리하여 DPPH 라디컬 소거 활성을 분석하였다. DPPH radical scavenging activity was analyzed in order to analyze the antioxidant activity of the fermented products prepared by the method according to Examples and Comparative Examples 1 to 3. DPPH radical scavenging activity assay was performed using Jeong's (Jeong et al. 2009) experimental method. Butylated hydroxytoluene (BHT), which is known as an antioxidant, was used as a positive control. And analyzed for DPPH radical scavenging activity.
먼저, 농도별로 만든 발효물 시료와 BHT를 96 well plate에 각각 80 ㎕씩 각각 분주한 후 증류수(DW), 메탄올(methanol), DPPH의 순서로 80 ㎕씩 분주한 후 빛과 반응하지 못하게 호일(foil)로 감싸준 뒤 실온에서 30분간 방치한 후 ELISA reader를 이용해 517 nm 흡광도를 측정한다. DPPH 라디칼 소거능은 하기 식 1을 이용하여 분석하였으며, 그 결과를 도 1에 나타내었다.First, 80 μl of fermented samples and BHT were placed in 96-well plates, and 80 μl of distilled water (DW), methanol, and DPPH were dispensed in this order. Then, foil foil), incubate at room temperature for 30 minutes, and measure the absorbance at 517 nm using an ELISA reader. The DPPH radical scavenging activity was analyzed using the following
[식 1][Equation 1]
도 1에 나타난 바와 같이, 실시예에 따른 방법으로 제조한 복합 발효물은 0.12 내지 2 mg/mL의 농도에서 농도 의존적으로 DPPH 라디칼 소거능을 나타낸다는 사실을 확인할 수 있었으며, 특히, 농도 2 mg/mL에서 가장 높은 DPPH 라디칼 소거능이 나타난 것을 확인할 수 있었고, 해조류의 단일 발효물인 비교예 1 내지 3의 발효물과 비교했을 때 현저히 향상된 항산화 활성 효능을 나타낸다는 사실을 확인할 수 있었다.As shown in FIG. 1, it was confirmed that the complex fermented product prepared by the method according to the Example exhibited a DPPH radical scavenging ability in a concentration-dependent manner at a concentration of 0.12 to 2 mg / mL, Showed the highest DPPH radical scavenging activity and showed remarkably improved antioxidant activity as compared with the fermented products of Comparative Examples 1 to 3, which is a single fermented product of seaweeds.
(2) ABTS radical-scavenging activity 측정(2) Measurement of ABTS radical-scavenging activity
실시예 및 비교예 1 내지 3에 따른 방법으로 제조한 발효물이 갖는 항산화 활성 효능을 분석하기 위해서, ABTS 라디칼 소거능을 분석하였다.To analyze the antioxidant activity of the fermented products prepared by the method according to the Examples and Comparative Examples 1 to 3, the ABTS radical scavenging ability was analyzed.
ABTS 라디칼 소거능은 항산화제에 의한 라다칼의 감소를 기반으로써 Re et al.(1999)에 개시된 방법에 따라서 수행되었다. The ABTS radical scavenging activity was performed according to the method described in Re et al. (1999) based on the reduction of ladalacid by antioxidants.
이를 위해, 먼저, 증류수에 용해한 7 mM의 ABTS와 2.45 mM의 과황화 칼륨(Potassium persulfate)을 혼합하여 혼합물을 제조한 후, 안정화를 위해 암실상태에서 12 내지 16시간 방치하였다. 734 nm에서 혼합물이 0.70 ㅁ 0.02이 되도록 PBS(0.01M, pH 7.4)로 희석하였고, 발효물 시료와 아스코르빈산 표준물질(ascorbic acid standard, 0.12 내지 2 mg/mL) 0.2 ㎖을 0.8㎖의 혼합물(ABTS+ solution)과 혼합 후 상온에서 5분 반응 시킨 뒤 blank에 대하여 734 nm 파장의 빛으로 흡광도를 측정하였다. 대조군은 시료의 추출물 대신 용매가 사용되었으며, ABTS 라디칼의 소거 활성은 상기 식 1과 동일한 방법으로 계산하였으며, 그 결과를 도 2에 나타내었다.To this end, 7 mM ABTS dissolved in distilled water and 2.45 mM potassium persulfate were mixed to prepare a mixture. The mixture was allowed to stand for 12-16 hours in a dark room for stabilization. At 734 nm, the mixture was diluted with PBS (0.01 M, pH 7.4) to 0.70 and 0.02, and 0.2 ml of the ascorbic acid standard (ascorbic acid standard, 0.12 to 2 mg / mL) (ABTS + solution), and reacted at room temperature for 5 minutes. Absorbance was measured by light of 734 nm wavelength against blank. As a control, a solvent was used instead of the extract of the sample, and the scavenging activity of the ABTS radical was calculated in the same manner as in the
도 2에 나타난 바와 같이, 실시예에 따른 방법으로 제조한 복합 발효물은 농도 의존적으로 ABTS 라디칼 소거능을 나타내었으며, 강력한 항산화능이 있는 아스코르빈산(ascorbic acid)보다 실시예에 따른 복합 발효물의 소거능이 1 mg/ml 농도에서 부터 더욱 뛰어난 항산화 효능을 나타낸다는 사실을 확인할 수 있었으며, 특히, 2 mg/ml에서 가장 높은 것으로 확인되었다. As shown in FIG. 2, the complex fermented product prepared by the method according to the Example exhibited ABTS radical scavenging ability in a concentration-dependent manner, and the scavenging ability of the complex fermentation product according to the Example was higher than that of ascorbic acid having
상기와 같은 결과를 통해, 실시예에 따른 복합 발효물은 전자 기증자 역할을 하는 물질을 포함하고, 더 안정적인 물질로 자유 라디칼을 바꾸고, 라디칼 연쇄 반응을 종결시킬 수 있다는 사실을 확인할 수 있었다.As a result, it can be confirmed that the complex fermented product according to the present invention contains a substance serving as an electron donor and can change free radicals as a more stable substance and terminate the radical chain reaction.
(3) 아질산염 소거 활성 측정(3) Measurement of nitrite scavenging activity
실시예 및 비교예 1 내지 3에 따른 방법으로 제조한 발효물이 갖는 항산화 활성 효능을 분석하기 위해서, 아질산염 소거능을 분석하였으며, 아질산염 소거능은 Griess 시약을 사용하여 측정하였다(Choi et al. 2008). In order to analyze the antioxidant activity of the fermented products prepared according to the methods of Examples and Comparative Examples 1 to 3, nitrite scavenging activity was assayed and nitrite scavenging activity was measured using a Griess reagent (Choi et al. 2008).
이를 위해, 0.25 mg/mL에 다양한 농도의 시료 또는 표준 시약 1㎖와 1mM의 NaNO2 1㎖로 혼합 후, 8 ㎖의 0.2M 시트르산염 완충용액(citrate buffer, pH 3.0)를 넣고 혼합한 후, 37 ℃에서 1시간 동안 incubation한 후에 혼합물 중 1㎖를 다른 시료에 옮긴 뒤 2% 아세트산(acetic acid) 2 ㎖과 Griess reagent를 0.4 ㎖ 넣고 혼합하였다. 그 후, 15분 동안 실온에서 반응 시킨 후, ELISA reader로 520 nm 파장의 빛으로 흡광도를 측정하고, 상기 식 1에 근거하여 아질산염 소거 활성을 측정하였으며, 측정 결과를 도 3에 나타내었다.For this, 8 ml of 0.2 M citrate buffer (pH 3.0) was added to 0.25 mg / ml of various concentrations of samples or 1 ml of standard reagent and 1 ml of NaNO2, After incubation for 1 hour at room temperature, 1 ml of the mixture was transferred to another sample, and then 2 ml of acetic acid and 0.4 ml of Griess reagent were added to the mixture. Thereafter, the reaction was carried out at room temperature for 15 minutes, and the absorbance was measured with light of 520 nm wavelength by an ELISA reader. The nitrite scavenging activity was measured based on the
도 3에 나타난 바와 같이, 실시예에 따른 방법으로 제조한 복합 발효물은 농도 의존적으로 nitrite 소거능을 나타낸다는 사실을 확인할 수 있었으며, 비교예 1 내지 3과 같은 해조류의 단일 발효물과 비교했을 때 상당히 뛰어난 nitrite 소거능을 나타낸다는 사실을 확인할 수 있었다.As shown in FIG. 3, it was confirmed that the composite fermented product prepared by the method according to the Example exhibited nitrite scavenging ability in a concentration-dependent manner. Compared with the single fermented product of seaweeds as in Comparative Examples 1 to 3, And it shows excellent nitrite scavenging ability.
<실험예 2> 제조한 발효물에 포함된 아미노산 함량 분석<Experimental Example 2> Analysis of Amino Acid Contents in the Fermented Product
실시예 및 비교예 1 내지 3에 따른 발효물에 포함된 L-아르기닌과 L-라이신 함량을 공인기관에 요청하여 분석하였으며, 분석 결과를 하기의 표 3에 나타내었다.The content of L-arginine and L-lysine contained in the fermented product according to Examples and Comparative Examples 1 to 3 was requested and analyzed by a public agency, and the results of the analysis are shown in Table 3 below.
표 3에 나타난 바와 같이, 실시예에 따른 방법으로 제조한 복합 발효물의 경우, 비교예 1 내지 3에 따른 발효물에 비해 현저히 높은 함량으로 L-아르기닌과 L-라이신을 포함한다는 사실을 확인할 수 있었다.As shown in Table 3, it was confirmed that the complex fermented product prepared by the method according to the Example contained L-arginine and L-lysine at a significantly higher content than the fermented product according to Comparative Examples 1 to 3 .
상기와 같은 결과를 통해서, 다시마, 미역, 톳을 포함하는 혼합물을 발효시킬 경우, 단일 해조류를 발효시키는 것 보다 혼합물을 발효시킬 경우, 항산화 활성 및 면역 증강 기능을 갖는 물질의 생성에 시너지 효과가 발생되어 항산화 활성 및 면역 증강 기능이 우수한 복합 발효물을 제조할 수 있다는 사실을 확인할 수 있었다.From the above results, when fermenting a mixture containing kelp, seaweed, and tortoise, fermenting the mixture rather than fermenting a single algae produces a synergistic effect on the production of a substance having antioxidative and immune enhancing functions Thus, it was confirmed that a complex fermented product having excellent antioxidant activity and immunity enhancing function can be produced.
Claims (5)
상기 바실러스 서브틸리스 균주는 디옥시숙신산을 포함하는 pH 4.5의 LB 배지에 볏짚에서 채취한 시료를 배양하여 투명환을 형성하는 균주를 1차 선별하고, 1차 선별한 균주를 LB 배지에 재접종하고 2차 선별하여 수득한 것을 특징으로 하는 복합 발효물의 제조방법.150 to 200 parts by weight of marine algae, 50 to 150 parts by weight of seaweed, 100 to 150 parts by weight of seaweed, and 100 to 600 parts by weight of water is added to 1 to 10 parts by weight of Bacillus subtilis Fermenting the strain at 20 to 40 DEG C for 48 to 120 hours,
The strain of Bacillus subtilis was cultivated in rice straw at pH 4.5 with deoxysuccinic acid and cultivated in rice straw. The strains forming the transparent ring were firstly screened, and the first selected strains were inoculated into the LB medium And a second step of screening the resulting complex fermentation product.
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