KR20230030940A - A cosmetic composition containing a sea urchin roe hydrolyzate or a sea urchin roe hydrolyzed fermented product, which has a function of improving wrinkles and regenerating skin, and a method for producing the same - Google Patents
A cosmetic composition containing a sea urchin roe hydrolyzate or a sea urchin roe hydrolyzed fermented product, which has a function of improving wrinkles and regenerating skin, and a method for producing the same Download PDFInfo
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- KR20230030940A KR20230030940A KR1020210113219A KR20210113219A KR20230030940A KR 20230030940 A KR20230030940 A KR 20230030940A KR 1020210113219 A KR1020210113219 A KR 1020210113219A KR 20210113219 A KR20210113219 A KR 20210113219A KR 20230030940 A KR20230030940 A KR 20230030940A
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- sea urchin
- hydrolyzate
- cosmetic composition
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- sea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dermatology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Cosmetics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
본 발명은 성게알 가수분해물 또는 성게알 가수분해 발효물을 유효성분으로 포함하여, 주름개선 및 피부재생 기능성을 갖는 화장료 조성물과 이의 제조방법에 관한 것이다. The present invention relates to a cosmetic composition containing a sea urchin hydrolyzate or fermented sea urchin hydrolyzed product as an active ingredient, having wrinkle-improving and skin regenerating functions, and a manufacturing method thereof.
그동안 화장품 원료의 개발은 주로 육상 식물성 자원에서 추출 및 분리되어 왔다. 육상 식물을 이용한 원료 개발은 오랜 기간 동안 많은 연구가 진행되어 새로운 소재 연구의 한계에 다다랐다. 이러한 문제를 해결하기 위해서 해양자원을 이용한 원료 개발이 매우 절실한 때이다. 남해안의 청정지역을 가진 우리나라에서 해양생물을 이용한 자원연구가 진행되고 있으나 해양 동물을 이용한 자원의 개발기술은 미약하여, 멍게, 군소, 불가사리, 동물성 플랑크톤, 캐비어, 연어 등에 대해서만 일부 연구가 이루어지고 있다. In the meantime, the development of cosmetic raw materials has been mainly extracted and separated from terrestrial plant resources. The development of raw materials using terrestrial plants has reached the limit of new material research as many studies have been conducted for a long period of time. In order to solve these problems, it is very urgent to develop raw materials using marine resources. Resource research using marine life is being conducted in Korea, which has a clean area on the south coast, but the technology for developing resources using marine animals is weak. Some research is being conducted only on sea squirts, sea urchins, starfish, zooplankton, caviar, and salmon. .
한편, 성게는 수분, 단백질, 지방, 비타민 B군과 비타민 C, 철분, 마그네슘 및 칼슘 등이 함유되어있고, 단백질은 해삼부터 많이 함유되어 있다. 특히, 성게알은 생식소 부위로서, 식용되는 생식소(성게알) 부위가 약 20%이고 나머지 80%는 성게껍질로 구성되어 있다. On the other hand, sea urchin contains moisture, protein, fat, vitamin B group, vitamin C, iron, magnesium and calcium, and contains a lot of protein from sea cucumber. In particular, sea urchin roe is a gonadal part, and the gonadal (sea urchin) part that is edible is about 20% and the remaining 80% is composed of sea urchin shell.
이와 관련하여, 성게에 관한 연구는 대부분 성게의 성분 및 생산, 가공에 관한 것으로 Nam(J. Korea Oil Chemists`Soc 3. 33-37 (1986)) 및 De la Cruz-Garcia 등의 문헌(J. Sci Food Agric 80, 1189-1192.(2000))에서는 성게와 통조림 제품의 단백질, 아미노산 및 지방산 조성에 대하여 보고하였고, Yoo 등의 문헌(Bull. Korean Fish Soc 15, 345-358 (1982))에서는 성게의 산란 및 성장에 대해 보고된바 있다. 이외에 성게를 이용한 서남해역 저질 오염 검정(Wui et al., Korean J Environ Biol 10, 92-99 (1992)) 및 해수의 생물학적 평가(Yu and Cho, J Korean Environ Sci Soc 8, 160-164 (1999))에 관한 연구가 있으나, 성게, 특히 성게알에 대한 연구는 거의 알려져 있지 않다.In this regard, studies on sea urchins are mostly about ingredients, production, and processing of sea urchins, such as Nam (J. Korea Oil Chemists'Soc 3. 33-37 (1986)) and De la Cruz-Garcia (J. Sci Food Agric 80, 1189-1192.(2000)) reported on the protein, amino acid and fatty acid composition of sea urchin and canned products, and Yoo et al. (Bull. Korean Fish Soc 15, 345-358 (1982)) reported Spawning and growth of sea urchins have been reported. In addition, a low-quality contamination test in the southwestern sea using sea urchins (Wui et al., Korean J Environ Biol 10, 92-99 (1992)) and biological evaluation of seawater (Yu and Cho, J Korean Environ Sci Soc 8, 160-164 (1999) )), but little is known about sea urchins, especially sea urchin roe.
또한 지구 온난화와 함께 바다의 사막화라고 불리는 백화 현상이 심해지면서 성게가 한 원인으로 지목되고 있다. 성게가 해조류를 먹어치움에 따라 해중림백화현상이 더 심해지기 때문이다. 이에, 도미 등의 성게의 천적들을 풀어 잡아먹게 하거나 직접 잠수부들이 바다에서 성게를 제거하기도 한다. 식용 성게는 보라성게, 말똥성게, 분홍성게 등으로 몇 종류 되지 않고 백화현상이 발생한 어장의 성게는 상품성이 없어 식용으로의 활용도 용이하지 않은 실정이다. In addition, sea urchins are being pointed out as one of the causes as the bleaching phenomenon called sea desertification intensifies with global warming. This is because as sea urchins eat seaweed, the phenomenon of sea forest bleaching gets worse. As a result, natural enemies of sea urchins such as sea bream are released and eaten, or divers directly remove sea urchins from the sea. There are only a few types of edible sea urchins, such as purple sea urchins, horse dung sea urchins, and pink sea urchins.
이에, 성게를 활용하는 기술의 개발이 이루어지고 있다. 예를 들면, 성게는 단백질, 아미노산 및 지방산 함량이 높고 성게껍질에서 분리된 칼슘은 칼슘보조제로 개발되어 의약품으로 시판되고 있다. 일부 양계장에서는 산란계에 성게껍질 투여 시 칼슘성분이 높고 불포화지방산이 풍부한 계란이 생산 가능하다는 연구보고도 있다. 다른 많은 해양 무척추 동물과 마찬가지로 성게는 생의학 응용 분야의 생물학적 활성물질이 연구되고 있다. 그러나 전반적인 성게의 생체기능성에 대한 연구나 보고는 국내외적으로 미약한 상황이다.Accordingly, the development of technology utilizing sea urchins is being made. For example, sea urchins have high protein, amino acid and fatty acid content, and calcium isolated from sea urchin shells has been developed as a calcium supplement and is marketed as a medicine. In some poultry farms, there is also a research report that it is possible to produce eggs high in calcium and rich in unsaturated fatty acids when sea urchin shells are administered to laying hens. Like many other marine invertebrates, sea urchins are being studied for biologically active substances for biomedical applications. However, studies or reports on the overall biofunctionality of sea urchins are weak domestically and internationally.
이에, 본 발명자들은 상기와 같은 기술적 요구에 착안하여, 화장품 원료로서 특히 성게알의 기능성을 확인하고, 본 발명을 완성하였다. Accordingly, the present inventors focused on the above technical requirements, confirmed the functionality of sea urchin, in particular, as a cosmetic raw material, and completed the present invention.
따라서 본 발명에서는 성게알 가수분해 발효물을 포함하는, 주름개선 및 피부재생 기능성을 갖는 화장료 조성물을 제공하는 것을 기술적 해결과제로 한다. Therefore, in the present invention, it is a technical problem to provide a cosmetic composition containing a hydrolyzed fermented sea urchin roe and having wrinkle-improving and skin-regenerating functions.
또한 본 발명은 성게알의 가수분해 발효물을 유효성분으로 포함하는 화장품 조성물을 제공하는 것을 다른 기술적 해결과제로 한다. Another technical problem of the present invention is to provide a cosmetic composition comprising fermented sea urchin hydrolyzate as an active ingredient.
또한 본 발명은 성게알의 가수분해 발효물을 제조하는 방법을 제공하는 것을 또다른 기술적 해결과제로 한다. In addition, another technical problem of the present invention is to provide a method for producing a fermented hydrolyzate of sea urchin.
상기 과제를 해결하기 위하여 본 발명은, 성게알의 가수분해물 또는 성게알의 가수분해발효물을 유효성분으로 포함하여, 주름개선 및 피부재생 기능성을 갖는 것을 특징으로 하는, 화장료 조성물을 제공한다. In order to solve the above problems, the present invention provides a cosmetic composition characterized in that it has wrinkle-improving and skin regenerating functions, including a hydrolyzate of sea urchin or a hydrolyzed fermentation product of sea urchin as an active ingredient.
또한 본 발명에 있어서, 상기 조성물은 항산화 기능성을 더 포함하는 것을 특징으로 한다. In addition, in the present invention, the composition is characterized in that it further comprises an antioxidant function.
또한 본 발명에 있어서, 상기 조성물은, 엘라스타제 활성을 저해하고 피부세포의 증식 활성을 나타내어 주름개선 및 피부 재생 기능성을 갖는 것을 특징으로 한다. In addition, in the present invention, the composition is characterized by inhibiting elastase activity and exhibiting skin cell proliferation activity to have wrinkle improvement and skin regeneration functions.
또한 본 발명에 있어서, 상기 성게알 가수분해물은 성게알 동결건조물을 단백질 분해효소로 가수분해하여 얻어지는 것을 특징으로 한다. In addition, in the present invention, the sea urchin hydrolyzate is characterized in that it is obtained by hydrolyzing the sea urchin freeze-dried product with a proteolytic enzyme.
또한 본 발명에 있어서, 상기 성게알의 가수분해 발효물은, 성게알 가수분해물에 유산균, 고초균 또는 효모를 접종하여 배양발효함으로써 얻어지는 것을 특징으로 한다. In the present invention, the fermented hydrolyzate of sea urchin is characterized in that it is obtained by culturing and fermenting the hydrolyzate of sea urchin by inoculating lactic acid bacteria, Bacillus subtilis, or yeast into the hydrolyzate of sea urchin.
또한 상기 다른 기술적 과제를 해결하기 위하여 본 발명은, 성게알의 가수분해물 또는 성게알의 가수분해발효물을 유효성분으로 포함하는 화장품 조성물을 제공한다. In addition, in order to solve the above other technical problem, the present invention provides a cosmetic composition containing a hydrolyzate of sea urchin or a hydrolyzed fermentation product of sea urchin as an active ingredient.
상기 본 발명에 있어서, 상기 성게알의 가수분해물 또는 성게알의 가수분해발효물은 전체 조성물 총 중량 대비 0.00001 중량% 내지 10 중량%로 포함되는 것을 특징으로 한다. In the present invention, the hydrolyzate of sea urchin or the fermented hydrolyzate of sea urchin is characterized in that it is included in 0.00001% to 10% by weight based on the total weight of the total composition.
또한 상기 또다른 기술적 과제를 해결하기 위하여 본 발명은,In addition, in order to solve the above another technical problem, the present invention,
성게알 가수분해물을 배지로 제조하는 단계;preparing a sea urchin hydrolyzate as a medium;
상기 배지를 멸균하는 단계;sterilizing the medium;
상기 멸균된 배지에 균주를 접종하는 단계;Inoculating a strain into the sterilized medium;
상기 균주가 접종된 배지를 정치배양하여 발효하는 단계; 및 Fermenting the culture medium inoculated with the strain; and
상기 배양발효된 발효물을 멸균하고 여과하는 단계;를 포함하여 이루어지는 것을 특징으로 하는 성게알 가수분해 발효물을 제조하는 방법을 제공한다. It provides a method for producing a fermented sea urchin hydrolyzed product comprising the steps of sterilizing and filtering the cultured fermented product.
상기 본 발명에 있어서, 상기 성게알 가수분해물은 성게알 동결건조물을 단백질 분해효소로 가수분해하여 얻어지는 것을 특징으로 한다. In the present invention, the sea urchin hydrolyzate is characterized in that it is obtained by hydrolyzing the sea urchin freeze-dried product with a proteolytic enzyme.
상술한 본 발명에 따르면, 성게알의 가수분해물 또는 성게알의 가수분해발효물은 ABTS 라디칼 소거활성, DPPH 라디칼 소거활성의 항산화 기능성, 엘라스타제 저해 활성에 의한 주름 개선 기능성, 피부세포 증식 활성에 의한 피부재생 기능성을 갖는 효과가 있다. 따라서 본 발명에 따라 성게알의 가수분해물 또는 가수분해발효물을 유효성분으로 포함하는 기능성 화장료와, 이러한 화장품 원료를 이용한 주름 개선 및 피부재생 기능성 화장품을 제공할 수 있는 효과가 있다. According to the present invention described above, the hydrolyzate of sea urchin or the hydrolyzed fermented product of sea urchin has the ABTS radical scavenging activity, the antioxidant function of DPPH radical scavenging activity, the anti-wrinkle function by elastase inhibitory activity, and the skin cell proliferation activity. It has the effect of having a skin regeneration function by Therefore, according to the present invention, it is possible to provide functional cosmetics containing hydrolyzate or fermented hydrolyzate of sea urchin as an active ingredient, and functional cosmetics for wrinkle improvement and skin regeneration using such cosmetic raw materials.
도 1은 본 발명의 일 실시예에 따른 성게알의 가수분해발효물의 제조공정을 순서도로 나타낸 것이다.
도 2는 본 발명의 일 실시예에 따른 성게알의 가수분해물 제조공정을 순서도로 나타낸 것이다.
도 3는 본 발명의 일 실시예에 따른 성게알의 동결건조분말을 제조공정을 순서도로 나타낸 것이다.
도 4는 본 발명의 실시예 1에 따른 성게알의 에탄올 추출물, 가수분해물 및 가수분해발효물의 제조공정을 나타낸 것이다.
도 5는 본 발명의 일 실시예에 따른 성게알의 에탄올 추출물, 가수분해물 및 가수분해발효물의 활성 그래프를 나타낸 것이다.
도 6은 본 발명의 일 실시예에 따른 성게알 가수분해 발효물의 발효균주에 따른 항산화 활성 그래프를 나타낸 것이다.
도 7은 본 발명의 일 실시예에 따른 성게알 가수분해발효물의 당함량 및 발효시간에 따른 활성 그래프를 나타낸 것이다. 1 is a flow chart showing a manufacturing process of hydrolysis and fermentation of sea urchin according to an embodiment of the present invention.
2 is a flow chart showing a process for producing a hydrolyzate of sea urchin according to an embodiment of the present invention.
3 is a flow chart showing a manufacturing process for the freeze-dried powder of sea urchin according to an embodiment of the present invention.
Figure 4 shows the manufacturing process of the ethanol extract, hydrolyzate and hydrolysis fermentation product of sea urchin according to Example 1 of the present invention.
5 is a graph showing activity of an ethanol extract, a hydrolyzate, and a hydrolysis fermentation product of sea urchin according to an embodiment of the present invention.
Figure 6 shows a graph of antioxidant activity according to fermented strains of fermented sea urchin hydrolysis according to an embodiment of the present invention.
7 shows a graph of activity according to sugar content and fermentation time of hydrolyzed fermented sea urchin roe according to an embodiment of the present invention.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 개념에 따른 실시예는 다양한 변경을 가할 수 있고 여러가지 형태를 가질 수 있으므로 특정 실시예들은 본 명세서에 상세하게 설명하고자 한다. 그러나, 이는 본 발명의 개념에 따른 실시예들을 특정한 개시 형태에 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변경, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다.Embodiments according to the concept of the present invention can apply various changes and can have various forms, so specific embodiments will be described in detail herein. However, this is not intended to limit the embodiments according to the concept of the present invention to a specific form disclosed, and should be understood to include all modifications, equivalents, or substitutes included in the spirit and scope of the present invention.
본 명세서에서 사용하는 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로 본 발명을 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한 복수의 표현을 포함한다.Terms used in this specification are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise.
명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 갖는 것으로 해석되어야 하며, 본 명세서에서 명백하게 정의하지 않는 한 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and are not interpreted in an ideal or excessively formal sense unless explicitly defined herein. .
일 양태로서 본 발명은, 성게알의 가수분해물 또는 성게알의 가수분해발효물을 유효성분으로 포함하여, 주름개선 및 피부재생 기능성을 갖는 것을 특징으로 하는, 화장료 조성물에 관한 것이다.In one aspect, the present invention relates to a cosmetic composition comprising a hydrolyzate of sea urchin or a hydrolyzed fermentation product of sea urchin as an active ingredient, characterized in that it has wrinkle-improving and skin-regenerating functions.
이 때, 상기 조성물은 성게알의 가수분해물 또는 가수분해발효물에 의해 엘라스타제 활성을 저해하고 피부세포의 증식활성을 나타냄으로써 주름개선 기능성 및 피부재생 기능성을 갖는 것이 특징이다. 또한 본 발명의 화장료 조성물은 성게알의 가수분해물 또는 가수분해발효물에 의해 ABTS 라디칼 소거활성, DPPH 소거활성을 나타내어 항산화 기능성을 갖는 특징이 있다. At this time, the composition inhibits elastase activity by hydrolyzate or fermented hydrolysis of sea urchin and exhibits skin cell proliferation activity, so it is characterized by having wrinkle improvement and skin regeneration functions. In addition, the cosmetic composition of the present invention exhibits ABTS radical scavenging activity and DPPH scavenging activity by hydrolyzate or fermented hydrolyzate of sea urchin, and thus has antioxidant functionality.
이러한 성게알을 가수분해물은 성게알에 포함된 단백질의 기능적 특성을 수식하거나 향상시키고, 2차 구조가 다른 분자량이 적은 펩타이드를 생성할 수 있게 된다. 즉, 화장품 효능 물질이 피부 깊숙이 흡수되기 위해서는 피부 표피층에 투과하여야 하는 바 본 발명의 성게알 가수분해물은 저분자화 되어 있어 성게알의 생리활성물질의 피부 속 투과가 가능해지고, 가수분해에 의한 펩타이드의 생성 등에 의해 항산화 활성, 엘라스타제 저해활성 및 각질형성세포의 증식활성을 나타낼 수 있게 되는 것이다. The hydrolyzate of sea urchin can modify or improve the functional properties of the protein contained in sea urchin, and produce low molecular weight peptides with different secondary structures. That is, in order for cosmetic efficacy substances to be absorbed deep into the skin, they must penetrate the epidermal layer of the skin. The sea urchin hydrolyzate of the present invention is low-molecular, so that the physiologically active substances of sea urchin can penetrate into the skin, and the hydrolysis of peptides Antioxidant activity, elastase inhibitory activity, and proliferation activity of keratinocytes can be exhibited by production, etc.
특히, 발효는 미생물 특히, 유용미생물인 프로바이오틱스(probiotics)가 당질을 이용하여 산물로서 알코올, 유기산, CO2 등을 생성하는 것으로, 발효를 통해 독성물질 파괴, 소화성 증진, 비타민 생성, 가용성 성분의 함량 증가 및 다양한 생리활성 물질을 생성할 뿐만 아니라 탄수화물, 단백질, 지질 등을 분해하는 가수분해 효소 및 기타 효소를 생성하여 영양학적 가치가 높고 건강기능성을 증진시키는 효과가 있다. 본 발명에서는 상기 성게알 가수분해물을 다시 발효시킨 가수분해 발효물을 유효성분으로 포함할 수 있는 바, 상기 가수분해발효물은 가수분해에 의한 저분자화 및 활성 증가의 효과에 더하여 발효공정에서 기능성을 보다 증진시킬 수 있게 된다. 본 발명의 바람직한 실시예에서는 특히 가수분해발효물에서 기존 성게알 에탄올 추출물 대비 2.5배의 ABTS 라디칼 소거활성, 4배의 DPPH 라디칼 소거활성, 6배 이상의 엘라스타제 저해활성을 나타내었고 에탄올 추출물에서는 보이지 않는 피부세포 증식활성을 나타냄을 확인할 수 있었고, 가수분해물의 활성에 비해서도 보다 향상된 활성을 나타냄을 확인할 수 있었다. In particular, fermentation is the production of alcohol, organic acids, CO 2 , etc. as products by microorganisms, especially useful microorganisms, probiotics, using carbohydrates. It not only increases and produces various physiologically active substances, but also creates hydrolytic enzymes and other enzymes that decompose carbohydrates, proteins, lipids, etc., which have high nutritional value and enhance health functionality. In the present invention, a fermented hydrolysis product obtained by further fermenting the sea urchin hydrolyzate may be included as an active ingredient. can be further improved. In a preferred embodiment of the present invention, in particular, the hydrolysis fermentation product showed 2.5 times more ABTS radical scavenging activity, 4 times more DPPH radical scavenging activity, and 6 times more elastase inhibitory activity compared to the existing sea urchin ethanol extract, and was not found in the ethanol extract. It was confirmed that the skin cell proliferation activity was not shown, and it was confirmed that the activity was more improved than that of the hydrolyzate.
따라서 본 발명의 화장료 조성물은 성게알의 가수분해물 또는 성게알의 가수분해발효물을 포함함으로써 ABTS 라디칼 소거활성, DPPH 라디칼 소거활성의 항산화 기능성, 엘라스타제 저해 활성에 의한 주름 개선 기능성, 피부세포 증식 활성에 의한 피부재생 기능성을 갖게 되는 것이다. Therefore, the cosmetic composition of the present invention contains the hydrolyzate of sea urchin or the hydrolyzed fermented product of sea urchin, thereby exhibiting ABTS radical scavenging activity, antioxidant function of DPPH radical scavenging activity, wrinkle improvement function by elastase inhibitory activity, and skin cell proliferation. It will have skin regeneration function by activation.
다른 양태로서 본 발명은, 성게알의 가수분해물을 배지로 제조하는 단계; 상기 배지를 멸균하는 단계; 상기 멸균된 배지에 균주를 접종하는 단계; 상기 균주가 접종된 배지를 정치배양하여 발효하는 단계; 및 상기 배양발효된 발효물을 멸균하고 여과하는 단계;를 포함하여 이루어지는 것을 특징으로 하는 성게알 가수분해 발효물을 제조하는 방법을 제공한다. In another aspect, the present invention includes the steps of preparing a hydrolyzate of sea urchin as a medium; sterilizing the medium; Inoculating a strain into the sterilized medium; Fermenting the culture medium inoculated with the strain; and sterilizing and filtering the cultured fermented product.
관련하여 도 1 내지 도 3은 성게알의 가수분해발효물, 가수분해물 및 동결건조물의 제조공정을 나타낸 것이다.1 to 3 show manufacturing processes of fermented hydrolysis products, hydrolyzates and freeze-dried products of sea urchin.
먼저 도 1을 참고하면, 성게알 가수분해물을 제조한 다음 가수분해물 배지를 멸균하여 균주를 접종하고 배양발효한 다음, 멸균, 여과하여 성게알 가수분해발효물을 제조할 수 있다. First, referring to FIG. 1 , sea urchin hydrolyzate is prepared, and then the hydrolyzate medium is sterilized to inoculate the strain, cultured and fermented, and then sterilized and filtered to prepare sea urchin hydrolyzed and fermented product.
이 때, 상기 균주로는 유산균, 고초균 또는 효모 중에서 어느 하나 이상을 선택할 수 있다. 바람직하게는 유산균을 이용할 수 있다. At this time, as the strain, any one or more of lactic acid bacteria, Bacillus subtilis, or yeast may be selected. Preferably, lactic acid bacteria may be used.
또한 상기 배양 발효시, 덱스트로스, 수크로오스, 말토오스 등의 탄소원, 식물의 세포벽 성분인 셀룰로오스(Cellulose), 수용성 식이섬유(다당류) 및 기타 당류(예를 들어, 엿당, 설탕, 포도당, 과당, 및 프락토올리고당으로 이루어지는 군에서 선택되는 하나 이상의 당류)로 이루어지는 군에서 선택되는 1종 이상의 첨가물을 추가로 포함하는 배양배지에서 이루어질 수 있다. 상기 기타 당류는 유산균에 영양을 공급하고 미생물 대사과정에 있어 단쇄지방산(short chain lipid, SCL)을 생산할 수 있도록 한다. 본 발명의 바람직한 실시예에 따르면, 덱스트로스를 첨가하여 발효원료로 이용하여 발효물을 제조한 결과, 유산균의 생장이 증가하여, ABTS 라디칼 소거활성, DPPH 라디칼 소거활성, 엘라스타제 저해 활성이 더 증가되는 것을 확인할 수 있었다. 따라서 상기 배양발효시 전체 배양배지 100 중량부 기준할 때, 탄소원 등의 상기 첨가물을 0.5 내지 5 중량부 포함하도록 할 수 있다. 다만 상기 범위에 제한되는 것은 아니며, 탄소원의 종류와 활성 향상 목적 범위 내에서 적절히 변형하여 사용될 수 있다. In addition, during the culture fermentation, carbon sources such as dextrose, sucrose, maltose, cellulose, which is a cell wall component of plants, water-soluble dietary fiber (polysaccharide) and other sugars (eg, maltose, sucrose, glucose, fructose, and fructose) At least one saccharide selected from the group consisting of lacto-oligosaccharide) may be made in a culture medium further comprising one or more additives selected from the group consisting of. The other saccharides supply nutrients to lactic acid bacteria and enable the production of short chain fatty acids (SCL) in the metabolic process of microorganisms. According to a preferred embodiment of the present invention, as a result of preparing a fermented product by adding dextrose and using it as a fermentation raw material, the growth of lactic acid bacteria increases, and the ABTS radical scavenging activity, DPPH radical scavenging activity, and elastase inhibitory activity are further increased. increase was observed. Therefore, based on 100 parts by weight of the entire culture medium during the culture fermentation, 0.5 to 5 parts by weight of the additives such as carbon sources may be included. However, it is not limited to the above range, and may be appropriately modified and used within the type of carbon source and the purpose of improving activity.
또한 도 2를 참고하면 상기 성게알 가수분해물은 성게알 동결건조물을 가열한 후 냉각시킨 다음, 단백질 분해효소로 가수분해하여 얻어진다. 이 때, 상기 단백질 분해 효소는, 플라보자임(Flavourzyme), 프로타맥스(Protamex), 뉴트라제(Neutrase), 파파인(Papain), 펩신(Pepsin), 트립신(Trypsin), 알파-키모트립신(α-Chymotrypsin), 알칼라제(Alcalase), 수미자임(Sumizyme), 코지자임(Kojizyme), 판크레아틴(pancreatin), 칼리크레인(kallikrein), 카텝신(Cathepsin), 써모리신(thermolisin), 서브틸리신(Subtilisin), 브로멜라인(Bromelain), 피신(Ficin), 액티니딘(Actinidin), 펩티다제(Peptidase) 및 트랜스글루타미나제(Transglutaminase)로 이루어진 군에서 선택되는 것일 수 있고, 더욱 구체적으로 플라보자임(Flavourzyme), 프로타맥스(Protamex), 뉴트라제(Neutrase) 또는 파파인(Papain)일 수 있으나, 이에 제한되지 않는다.Also, referring to FIG. 2 , the sea urchin hydrolyzate is obtained by heating the sea urchin freeze-dried product, cooling it, and then hydrolyzing it with a proteolytic enzyme. At this time, the proteolytic enzyme, Flavorzyme, Protamex, Neutrase, Papain, Pepsin, Trypsin, Alpha-chymotrypsin (α -Chymotrypsin, Alcalase, Sumizyme, Kojizyme, pancreatin, kallikrein, cathepsin, thermolisin, subtilisin It may be selected from the group consisting of (Subtilisin), Bromelain, Ficin, Actinidin, Peptidase and Transglutaminase, and more specifically It may be Flavorzyme, Protamex, Neutrase or Papain, but is not limited thereto.
상기 단백질 분해 효소로 가수분해시 효소의 활성 범위에 따라 pH와 온도를 조절할 수 있다. 예를 들어 펩신의 경우 pH 2.0 ~ 4.0, 트립신의 경우 pH 7.0 ~ 9.0, 프로타맥스의 경우 pH 5.5 ~ 7.5, 플라보자임은 pH 5.0 ~ 7.0 로 조절할 수 있다. 또한 pH의 조절을 위해서 유기산이 사용될 수 있다. 상기 유기산은 상기 pH를 조절할 수 있는 유기산이라면 무방하며, 예컨대 구연산, 젖산, 아세트산, 포름산, 옥살산, 요산 등에서 선택되는 어느 하나 또는 둘 이상을 포함할 수 있다.During hydrolysis with the proteolytic enzyme, pH and temperature can be adjusted according to the activity range of the enzyme. For example, pH 2.0 ~ 4.0 for pepsin, pH 7.0 ~ 9.0 for trypsin, pH 5.5 ~ 7.5 for Protamax, and pH 5.0 ~ 7.0 for flavozyme. Organic acids can also be used to adjust the pH. Any organic acid capable of adjusting the pH may be used as the organic acid, and may include, for example, one or two or more selected from among citric acid, lactic acid, acetic acid, formic acid, oxalic acid, and uric acid.
또한, 가수분해 온도의 경우 단백질 분해 효소가 높은 활성을 갖는 온도일 수 있으며, 효소의 종류에 따라 적절히 조절될 수 있다. 예컨대 가수분해 온도는 플라보자임(Flavourzyme), 프로타맥스(Protamex) 또는 뉴트라제(Neutrase)의 경우 45℃ 내지 60℃, 트립신의 경우 35℃ 내지 40℃, 파파인의 경우 35℃ 내지 40℃ 범위일 수 있다.In addition, the hydrolysis temperature may be a temperature at which proteolytic enzymes have high activity, and may be appropriately adjusted according to the type of enzyme. For example, the hydrolysis temperature ranges from 45°C to 60°C for Flavorzyme, Protamex or Neutrase, from 35°C to 40°C for trypsin, and from 35°C to 40°C for papain. can be
가수분해 시간의 경우 1시간에서 10시간, 더욱 구체적으로 2시간에서 9시간 동안 교반시키면서 반응을 진행시킬 수 있다.In the case of hydrolysis time, the reaction may proceed while stirring for 1 hour to 10 hours, more specifically, 2 hours to 9 hours.
상기 가수분해를 수행한 후, 70℃ 내지 120℃, 구체적으로 80℃ 내지 100℃에서 1분 내지 20분간 가열하여 효소를 불활성화할 수 있다.After performing the hydrolysis, the enzyme may be inactivated by heating at 70 ° C to 120 ° C, specifically 80 ° C to 100 ° C for 1 minute to 20 minutes.
또한 도 3에 도시한 바와 같이 성게알 동결건조물은, 준비한 성게알을 세척하고 동결건조시킨 다음 분쇄하여 분말형태로 제조될 수 있다. 이는 가수분해 및 발효공정 진행시 추출을 용이하게 하기 위함이다. In addition, as shown in FIG. 3, the sea urchin lyophilized product may be prepared in the form of a powder by washing, freeze-drying, and pulverizing the prepared sea urchin. This is to facilitate extraction during the hydrolysis and fermentation process.
또한 본 발명은 또다른 양태로서 성게알의 가수분해물 또는 성게알의 가수분해발효물을 유효성분으로 포함하는 화장품 조성물을 제공한다. In another aspect, the present invention provides a cosmetic composition comprising a hydrolyzate of sea urchin or a hydrolyzed fermentation product of sea urchin as an active ingredient.
바람직하게는 상기 유효성분은 전체 화장품 조성물 총 중량 대비 0.00001 중량% 내지 10 중량%로 포함될 수 있다.Preferably, the active ingredient may be included in an amount of 0.00001% to 10% by weight based on the total weight of the total cosmetic composition.
본 발명에 있어서, '유효 성분'이라 함은 주름을 개선하거나, 항산화 효과 또는 피부 재생 효과를 나타낼 수 있는 화합물의 양을 의미한다. 본 발명의 조성물에 포함되는 상기 유효성분의 함량은 조성물이 제품화되는 형태, 상기 화합물이 피부에 적용되는 방법 및 피부에 머무르는 시간 등에 따라 달라질 것이다.In the present invention, the term 'active ingredient' means the amount of a compound capable of improving wrinkles, or exhibiting an antioxidant effect or a skin regeneration effect. The content of the active ingredient included in the composition of the present invention will vary depending on the form in which the composition is commercialized, how the compound is applied to the skin, and how long it stays on the skin.
화장품으로 제품화되는 경우에 유효성분이 단기간 내에 피부에 머무르게 되는 메이크업 제거제, 세정제 등과 같은 워쉬-오프(wash-off) 타입의 화장품의 경우에는 비교적 높은 농도의 상기 유효성분을 포함할 수 있을 것이다.In the case of cosmetics of the wash-off type, such as make-up removers and cleansers, in which active ingredients stay on the skin for a short period of time when commercialized as cosmetics, a relatively high concentration of the active ingredient may be included.
반면, 유효성분이 장기간 동안 피부에 머무르게 되는 화장수, 유액, 크림, 에센스 등의 리브-온(leave-on) 타입의 화장품의 경우에는 워쉬-오프 타입의 화장품에 비해 낮은 농도의 상기 유효성분을 포함해도 무방할 것이다. 이에 제한되는 것은 아니나, 구체적으로 상기 조성물은 상기 유효성분을 전체 조성물 중량에 대하여 0.00001 중량% 내지 10 중량%, 더욱 구체적으로는 0.0001 중량% 내지 1 중량%로 포함할 수 있다. 본 발명의 조성물이 상기 유효성분을 0.00001 중량% 미만으로 포함할 경우에는 충분한 피부 재생, 주름 개선 및 항산화 효과를 기대할 수 없고, 10 중량%를 초과하여 포함할 경우에는 알러지 등 원치 않는 반응이 발생하거나 피부 안전성에 문제가 있을 수 있으므로 이를 방지하기 위한 것이다.On the other hand, in the case of leave-on type cosmetics such as lotion, lotion, cream, essence, etc., in which active ingredients stay on the skin for a long time, even if they contain the active ingredient at a lower concentration than wash-off type cosmetics It will be free. Although not limited thereto, specifically, the composition may include the active ingredient in an amount of 0.00001 wt% to 10 wt%, more specifically, 0.0001 wt% to 1 wt%, based on the total weight of the composition. When the composition of the present invention contains less than 0.00001% by weight of the active ingredient, sufficient skin regeneration, wrinkle improvement and antioxidant effects cannot be expected, and when it contains more than 10% by weight, unwanted reactions such as allergies occur or This is to prevent problems with skin safety.
또한 본 발명의 화장품 조성물에는 상기 유효성분 외에 화장품 조성물에 통상적으로 첨가되는 성분, 예컨대 항산화제, 안정화제, 가용화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 및 담체를 추가로 첨가할 수 있다.In addition, to the cosmetic composition of the present invention, in addition to the above active ingredients, ingredients commonly added to cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers may be further added. .
본 발명의 화장품 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 영양 크림, 수렴 화장수, 유연 화장수, 로션, 에센스, 영양젤 또는 마사지 크림의 제형으로 제조될 수 있다.The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, astringent lotion, softening lotion, lotion, essence, nutritional gel or massage cream.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트 검, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. It can be.
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 토스, 탈크, 실리카, 알루미늄히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or spray, toss, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 가용화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산에스테르가 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌소르비톨에스테르 및 폴리옥시에틸렌소르비탄에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄메타히드록시드, 벤토나이트, 아가 또는 트라칸트 검등이 이용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component, liquid diluents such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, suspending agents such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Acidic cellulose, aluminum metahydroxide, bentonite, agar or tracanth gum and the like may be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산글리세리드, 지방산디에탄올아미드, 식물성유, 라놀린 유도체 또는 에톡실화글리세롤지방산에스테르 등이 이용될 수 있다.When the formulation of the present invention is a surfactant-containing cleanser, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.
이하, 실시예 및 시험예를 통해서 본 발명을 더욱 구체적으로 설명하기로 하되, 하기 예는 본 발명의 이해를 돕기 위한 것일 뿐, 본 발명의 범위를 제한하는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples and test examples, but the following examples are only for helping understanding of the present invention, and do not limit the scope of the present invention.
<실시예 1> 성게알 추출물, 가수분해물 및 가수분해발효물의 제조<Example 1> Preparation of sea urchin extract, hydrolyzate and hydrolysis fermentation product
1) 성게알 에탄올 추출물1) Sea urchin ethanol extract
성게알 동결건조분말 10g에 95% 에탄올을 50 ml 가하여 5시간 실온에서 교반하며 추출 및 여과하여 활성에 사용하였다.50 ml of 95% ethanol was added to 10 g of sea urchin lyophilized powder, stirred at room temperature for 5 hours, extracted and filtered, and used for activation.
2) 성게알 가수분해물2) Sea urchin hydrolyzate
성게알 동결건조분말 10g에 증류수를 50 ml 가하여 0.1g의 효소(neutrase)를 첨가하였다. 3시간 동안 50~60℃에서 교반하며 가수분해 후 효소불활성화를 위해 121℃에서 15분 고압멸균하였다. 여과 후 활성에 사용하였다.50 ml of distilled water was added to 10 g of freeze-dried sea urchin powder, and 0.1 g of neutrase was added. It was stirred at 50-60 ° C for 3 hours and sterilized under high pressure at 121 ° C for 15 minutes to inactivate the enzyme after hydrolysis. After filtration, it was used for activity.
3) 성게알 가수분해발효물3) Sea urchin hydrolysis fermented product
성게알 동결건조분말 10g에 증류수를 50 ml 가하여 0.1g의 효소(neutrase)를 첨가하였다. 3시간 동안 50~60℃에서 교반하며 가수분해하였다. 0.3g Dextrose를 첨가한 후 효소불활성화를 위해 100℃에서 15분 간 가열하였다. 활성화된 유산균(Lactobacillus plantarum, OD600nm: 1.0~1.5)을 0.5ml 접종하여 37℃에서 48 시간 정치배양하였다. 균주의 불활성화를 위해 121℃에서 10분간 고압멸균을 한 후 여과하여 활성에 사용하였다.50 ml of distilled water was added to 10 g of freeze-dried sea urchin powder, and 0.1 g of neutrase was added. It was hydrolyzed while stirring at 50-60 ° C. for 3 hours. After adding 0.3g Dextrose, the mixture was heated at 100°C for 15 minutes to inactivate the enzyme. 0.5ml of activated lactic acid bacteria ( Lactobacillus plantarum, OD 600nm : 1.0-1.5) was inoculated and cultured at 37°C for 48 hours. For inactivation of the strain, high-pressure sterilization was performed at 121 ° C. for 10 minutes, and then filtered and used for activity.
본 실시예에 따른 성게알의 에탄올 추출물, 가수분해물 및 가수분해발효물의 제조공정은 도 4에 나타내었다. The manufacturing process of the ethanol extract, hydrolyzate, and hydrolysis fermentation product of sea urchin according to this embodiment is shown in FIG. 4.
<시험예 1> 기능성의 측정<Test Example 1> Measurement of functionality
1) 항산화 기능성 - ABTS 라디칼 소거 활성1) Antioxidant functionality - ABTS radical scavenging activity
ABTS radical 소거 활성은 Re et al. (1999)의 방법을 변형하여 측정하였다. 7 mM ABTS 5 ㎖과 140 mM potassium persulfate 88 ㎕를 섞은 후 상온에서 16 시간 빛을 차단하여 ABTS 양이온을 형성시켰다. 이 용액을 734 nm에서 흡광도 값이 0.7이 되도록 PBS로 희석하였다. ABTS 용액 190 ㎕와 시료 10 ㎕를 혼합한 후 상온에서 6 분간 반응시킨 후 734 nm에서 흡광도를 측정하였다. 용매 대조군으로 하여 대조군에 대한 라디칼 소거능을 백분율로 나타내었다. ABTS radical scavenging activity was investigated by Re et al. (1999) was modified and measured. After mixing 5 ml of 7 mM ABTS and 88 μl of 140 mM potassium persulfate, ABTS cations were formed by blocking light at room temperature for 16 hours. This solution was diluted with PBS to obtain an absorbance value of 0.7 at 734 nm. After mixing 190 μl of the ABTS solution and 10 μl of the sample, the mixture was reacted at room temperature for 6 minutes, and the absorbance was measured at 734 nm. As a solvent control group, the radical scavenging ability of the control group was expressed as a percentage.
2) 항산화 기능성 - DPPH 라디칼 소거 활성2) Antioxidant functionality - DPPH radical scavenging activity
DPPH assay 방법은 Yoshida et al. (1989)이 사용한 방법에 따라 측정하였다. 96well plate에 control에는 추출물을 추출한 용매를 12 ㎕, 시험군에는 시료를 12 ㎕ 씩 각각 넣는다. 모든 well에 Ethanol 6 ㎕씩 넣고 222 ㎕의 DPPH (0.15 mM 1,1-diphenyl-2-picrylhydrazyl(DPPH)/ethanol) 용액을 가하여 혼합한다. 실온에서 30 분 동안 반응시킨 후 517 nm에서 흡광도를 측정한다. 용매만을 첨가한 대조군에 대한 추출물의 라디칼 소거 활성을 백분율로 나타내었다.The DPPH assay method was described by Yoshida et al. (1989). In a 96-well plate, add 12 μl of the extract extract solvent to the control group and 12 μl of each sample to the test group. Add 6 μl of ethanol to each well, add 222 μl of DPPH (0.15
3) 주름개선 기능성 - 엘라스타제(Elastase) 저해활성3) Anti-wrinkle functionality - Elastase inhibitory activity
엘라스타제 저해활성은 Invitrogen사의 EnzChek® Elastase Assay Kit (600 Assays) 시약을 구입하여 사용하였다. 시료를 96well plate에 50 ㎕씩 triple로 준비 후 DQ elastin 50 ㎕를 첨가한다. Sample blank에는 시료 대신 reaction buffer 50 ㎕를 주입하여 공시료액으로 사용하였다. 이 후 sample에는 elastase working solution 50 ㎕를 시료에 가하고, 공시료액에는 elastase working solution 대신 reaction buffer를 50 ㎕ 첨가하였다. 빛을 차단한 상태로 실온에서 1시간 동안 방치한 후 형광 강도 excitation wavelength 485 nm 및 emission wavelength 535 nm에서 ELISA plate reader (VICTOR X3™, PerkinElmer, US)로 형광강도를 측정하였다.For the elastase inhibitory activity, Invitrogen's EnzChek ® Elastase Assay Kit (600 Assays) reagent was purchased and used. Samples are prepared in triplicates by 50 μl in a 96-well plate, and then 50 μl of DQ elastin is added. In the sample blank, 50 μl of reaction buffer was injected instead of the sample and used as a blank sample solution. After that, 50 μl of elastase working solution was added to the sample, and 50 μl of reaction buffer was added to the blank sample instead of the elastase working solution. After being allowed to stand at room temperature for 1 hour in a light-blocking state, fluorescence intensity was measured with an ELISA plate reader (VICTOR X3™, PerkinElmer, US) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
(단, a: 공시료액의 반응 후의 흡광도, b: 시료액의 반응 후의 흡광도(However, a: absorbance after reaction of blank sample solution, b: absorbance after reaction of sample solution
a',b': 엘라스타제 대신 완충액으로 대체하여 측정한 흡광도)a',b': absorbance measured by replacing elastase with buffer)
4) 피부재생 기능성 - 인간피부표피세포(HaCaT) 증식 활성4) Skin regeneration function - human skin epidermal cell (HaCaT) proliferation activity
- HaCaT 세포(인간피부표피세포) 독성시험- HaCaT cell (human epidermal cell) toxicity test
세포주 선택 및 세포배양Cell line selection and cell culture
배지는 10% Fetal Bovine Serum (FBS, Lonza, Valais, Switzerland) medium을 함유한 DMEM (21063-029, fee phenol red, GIBCO, Grand Island, NY) 배지를 이용하였다. 세포주는 습도 95%, 5% CO2, 37℃로 조절된 배양기에서 배양하며, 이때 미생물의 오염이나 증식을 억제하기 위해 배지용 항생제(Penicillin streptomycin, Gibco, CA, USA)를 사용하였다. 세포가 80% 정도 dish를 덮으면 phosphated-buffered saline (PBS)로 세척한 후 0.05% Trypsin EDTA를 1 ml 처리하여 dish 바닥에 부착되어있는 세포를 떼어내 계대 배양하였다. 배지는 48 시간마다 교환하여 세포를 배양하였다. As the medium, DMEM (21063-029, fee phenol red, GIBCO, Grand Island, NY) medium containing 10% Fetal Bovine Serum (FBS, Lonza, Valais, Switzerland) medium was used. The cell line was cultured in an incubator controlled at 95% humidity, 5% CO 2 , and 37° C. At this time, an antibiotic for culture medium (Penicillin streptomycin, Gibco, CA, USA) was used to inhibit contamination or proliferation of microorganisms. When the cells covered the dish by about 80%, they were washed with phosphated-buffered saline (PBS) and treated with 1 ml of 0.05% Trypsin EDTA to remove the cells attached to the bottom of the dish and subcultured. The medium was exchanged every 48 hours to culture the cells.
HaCaT 세포독성HaCaT cytotoxicity
세포주의 생존율을 측정은 MTS (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, USA)시약을 이용하여 세포의 생존율을 측정하였다. 96 well plate에 세포를 1×104 cells/well의 농도로 분주 후 습도 95%, 5% CO2, 37℃로 조절된 배양기에서 24 시간 안정화하였다. 이 후 시료를 농도별로 처리하여 습도 95%, 5% CO2, 37℃로 조절된 배양기에서 24 시간 배양하였다. 배지를 제거한 후 시약 1 ml에 배지(DMEM low, free FBS) 9 ml을 넣어 희석한 후 well 당 100 ㎕씩 처리하였다. 37℃에서 60 분간 반응 후 microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA)로 490 nm에서 흡광도를 측정하였다. 세포의 생존율은 시료를 처리하지 않은 대조군에 대비한 시료 처리군의 흡광도로 표시히였다. The viability of the cell line was measured using MTS (CellTiter 96 ® AQueous One Solution Cell Proliferation Assay, Promega, USA) reagent. After dispensing the cells in a 96 well plate at a concentration of 1×10 4 cells/well, the cells were stabilized for 24 hours in an incubator controlled at 95% humidity, 5% CO 2 , and 37°C. Thereafter, the samples were treated by concentration and incubated for 24 hours in an incubator controlled at 95% humidity, 5% CO 2 , and 37°C. After removing the medium, 9 ml of medium (DMEM low, free FBS) was added to 1 ml of the reagent, diluted, and treated at 100 μl per well. After reaction at 37° C. for 60 minutes, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). The viability of the cells was expressed as the absorbance of the sample treated group compared to the control group not treated with the sample.
- HaCaT 세포(인간피부표피세포)를 이용한 세포증식활성 확인시험- Cell proliferation activity confirmation test using HaCaT cells (human skin epidermal cells)
HaCaT 세포주를 5×103 cells/well의 농도로 96 well plate에 분주하고, 24시간 동안 37℃, 습도 95%, CO2 5%로 조절된 CO2 배양기에서 배양하였다. 새로운 배지에 각 시료를 세포독성이 나타나지 않는 알맞은 농도 범위로 세포에 처리한 후 1, 2일 동안 배양하였다. 세포주의 생존율을 측정하기 위해 Cell Counting Kit-8 (CCK-8, Dojindo Mol. Tech., Rockville., MD, USA)를 이용하여 microplate reader (Molecular Devices, Versa Max ELISA Microplate Reader, USA)로 450 nm에서 흡광도를 측정하였다. 세포의 재생효능은 시료를 처리하지 않은 대조군에 대비한 시료 처리군의 흡광도로 표시하였다.The HaCaT cell line was dispensed into a 96 well plate at a concentration of 5×10 3 cells/well, and cultured for 24 hours in a CO 2 incubator controlled at 37° C., 95% humidity, and 5% CO 2 . Each sample was treated with cells in an appropriate concentration range that does not show cytotoxicity in a new medium, and then cultured for 1 or 2 days. To measure the viability of cell lines, Cell Counting Kit-8 (CCK-8, Dojindo Mol. Tech., Rockville., MD, USA) was used and a microplate reader (Molecular Devices, Versa Max ELISA Microplate Reader, USA) was used to measure 450 nm. The absorbance was measured at. The cell regeneration efficiency was expressed as the absorbance of the sample-treated group compared to the control group not treated with the sample.
본 시험예에 따른 기능성 시험 결과는 하기 표 1 및 도 5에 나타내었다. The functional test results according to this test example are shown in Table 1 and FIG. 5 below.
에탄올 추출물ethanol extract
가수분해물 hydrolysate
가수분해 발효물 hydrolysis fermented product
표 1 및 도 5를 참고하면, 항산화기능성과 관련해서 1% 농도에서 성게알 에탄올 추출물은 29.05%, 성게알 가수분해물은 67.33% 성게알 가수분해발효물은 80.61%로 ABTS 라디칼 소거활성을 확인할 수 있는 바, 가수분해 발효물에서 가장 우수한 활성을 나타내고 가수분해물과 성게알 에탄올 추출물에 비해 2배 이상의 우수한 활성을 나타냄을 확인할 수 있다. Referring to Table 1 and FIG. 5, in relation to antioxidant functionality, at 1% concentration, the sea urchin ethanol extract was 29.05%, the sea urchin hydrolyzate was 67.33%, and the sea urchin hydrolyzed fermentation product was 80.61%, confirming the ABTS radical scavenging activity. As a result, it can be confirmed that it shows the best activity in the hydrolyzed fermented product and exhibits more than twice the activity compared to the hydrolyzate and the sea urchin ethanol extract.
또한 1% 농도에서 성게알 에탄올 추출물은 16.44%, 성게알 가수분해물은 15.21% 성게알 가수분해발효물은 59.56%로 DPPH 라디칼 소거활성을 확인할 수 있다. 즉, 가수분해 발효물에서 가장 우수한 활성을 나타낼 수 있다. In addition, at 1% concentration, sea urchin ethanol extract was 16.44%, sea urchin hydrolyzate was 15.21%, and sea urchin hydrolyzed fermentation product was 59.56%, confirming the DPPH radical scavenging activity. That is, it can exhibit the best activity in the hydrolyzed fermented product.
이를 통해 성게알의 에탄올 추출물에 비해서, 가수분해물이 ABTS 라디칼 소거활성을 더 나타내고, 가수분해발효물은 ABTS 라디칼 소거활성 및 DPPH 라디칼 소거활성을 더 나타내면서 현저하게 우수한 활성을 나타내는 것을 확인할 수 있다. Through this, it can be confirmed that, compared to the ethanol extract of sea urchin, the hydrolyzate exhibits more ABTS radical scavenging activity, and the fermented hydrolysis product exhibits significantly better activity while exhibiting more ABTS radical scavenging activity and DPPH radical scavenging activity.
또한 주름개선 기능성과 관련하여, 20% 농도에서 성게알 에탄올 추출물은 8.99%, 성게알 가수분해물은 40.13% 성게알 가수분해발효물은 58.17%로 엘라스타제 저해 활성을 확인하였다. 즉, 가수분해 발효물에서 에탄올 추출물의 6배 이상의 활성으로 가장 우수한 활성을 나타내고, 가수분해물도 에탄올 추출물에 비해 5배 이상의 우수한 활성을 나타냄을 확인할 수 있다. In addition, in relation to the wrinkle improvement function, at 20% concentration, the elastase inhibitory activity was confirmed at 8.99% for sea urchin ethanol extract, 40.13% for sea urchin hydrolysate, and 58.17% for sea urchin hydrolyzed fermented product. That is, it can be confirmed that the hydrolyzate exhibits the most excellent activity with 6 times or more activity of the ethanol extract in the fermented hydrolyzate, and the hydrolyzate also exhibits 5 times or more excellent activity compared to the ethanol extract.
또한 피부재생 기능성과 관련하여, 3종 추출물 전 농도에서 독성은 나타나지 않았는 바, 0.1~1% 농도로 세포 증식 활성을 진행하였다. 그 결과, 성게알 에탄올 추출물은 증식 2일 차에서 0.05~0.2% 농도에서는 증식하는 경향을 보였으나 1%에서는 감소하는 세포가 감소하는 것을 확인하였다. 반면, 성게알 가수분해물 및 가수분해발효물은 증식 2일 차에서 농도에 따라 유의적으로 증가하는 것으로 나타났다. In addition, in relation to skin regeneration functionality, toxicity was not observed at all concentrations of the three extracts, and cell proliferation activity was performed at a concentration of 0.1 to 1%. As a result, it was confirmed that the sea urchin ethanol extract showed a tendency to proliferate at a concentration of 0.05 to 0.2% on the second day of proliferation, but decreased cells at a concentration of 1%. On the other hand, sea urchin hydrolysates and fermented hydrolysates showed a significant increase according to the concentration on the second day of proliferation.
이러한 결과들을 종합해볼 때 성게알 가수분해물 또는 성게알 가수분해물은 항산화 기능성, 주름개선 기능성 및 피부재생 기능성을 가질 수 있음을 확인할 수 있고, 성게알 가수분해발효물이 가장 우수한 기능성을 나타낼 수 있는 것으로 확인된다. Taking these results together, it can be confirmed that sea urchin hydrolyzate or sea urchin hydrolyzate can have antioxidant, wrinkle-improving, and skin regenerating functions, and sea urchin hydrolyzed and fermented product has the best functionality. confirmed
<실시예 2> 성게알 가수분해발효물의 제조 및 활성 시험<Example 2> Production and activity test of fermented sea urchin hydrolysis
1) 성게알 최적 발효균의 확립1) Establishment of optimal fermentation bacteria for sea urchin
성게알의 최적 발효조건을 확인하기 위하여 고초균(Bacillus subtilis), 유산균(Lactobacillus plantarum), 효모(Saccharomyces cerevisiae)를 발효균으로 선정하고 3종 균을 이용하여 성게알 가수분해물 발효 최적 조건을 탐색하였다. In order to confirm the optimal fermentation conditions for sea urchin roe, Bacillus subtilis , Lactobacillus plantarum , and yeast (S accharomyces cerevisiae ) were selected as fermenting bacteria, and the optimal conditions for fermentation of sea urchin hydrolyzate were explored using three types of bacteria.
(1) 균 배양 (1) Bacterial culture
① 유산균(Lactobacillus plantarum) 발효① Lactic acid bacteria ( Lactobacillus plantarum ) Fermentation
37℃에서 액체배양한 유산균을 새로운 배지(MRS broth)에 1% 접종한다. 37℃에서 24 시간 동안 배양 후 발효에 사용한다. 이 때 탁도를 0.6~0.7로 조정하여 사용한다(600 nm).Lactic acid bacteria cultured in liquid at 37 ° C are inoculated into a new medium (MRS broth) at 1%. After incubation at 37°C for 24 hours, it is used for fermentation. At this time, adjust the turbidity to 0.6 ~ 0.7 (600 nm).
② 고초균(Bacillus subtilis) 발효② Bacillus subtilis fermentation
30℃에서 액체배양한 유산균을 새로운 배지(Plate Count Broth)에 1% 접종한다. 30℃에서 150 rpm으로 24 시간 동안 배양 후 발효에 사용한다. 이 때 탁도를 0.6~0.7로 조정하여 사용한다(600 nm).
③ 효모(Saccharomyces cerevisiae) 발효③ Yeast (S accharomyces cerevisiae ) fermentation
30℃에서 액체배양한 유산균을 새로운 배지(Potato Dextrose Broth)에 1% 접종한다. 30℃에서 150 rpm으로 24 시간 동안 배양 후 발효에 사용한다. 이 때 탁도를 0.6~0.7로 조정하여 사용한다(600 nm).Lactic acid bacteria cultured in liquid at 30 ° C are inoculated into a new medium (Potato Dextrose Broth) at 1%. After culturing for 24 hours at 30 ° C. at 150 rpm, it is used for fermentation. At this time, adjust the turbidity to 0.6 ~ 0.7 (600 nm).
(2) ABTS 라디칼 소거 활성 시험(2) ABTS radical scavenging activity test
상기 균 3 종에 따른 성게알 가수분해 발효물의 ABTS 라디칼 소거 활성은 시험예 1과 동일한 방법으로 수행하였고, 그 결과 배양시간과 균의 종류에는 상관없이 활성이 비슷하게 측정되었다(도 6 참고)The ABTS radical scavenging activity of the fermented sea urchin hydrolyzed fermented product according to the three strains was performed in the same manner as in Test Example 1, and as a result, the activity was similarly measured regardless of the incubation time and the type of bacteria (see FIG. 6).
<실시예 3> 유산균을 이용한 성게알 가수분해발효물의 제조 및 활성 시험<Example 3> Production and activity test of fermented sea urchin hydrolysis using lactic acid bacteria
이취 등을 고려하여 발효 균주로 유산균을 선택하고 당 첨가에 따른 최적 발효 조건을 시험하였다. Lactic acid bacteria were selected as fermentation strains in consideration of off-flavors, etc., and optimal fermentation conditions according to sugar addition were tested.
성게알 가수분해 추출물에 덱스트로스(dextrose)를 각각 1, 3, 5%씩 첨가한 후 121℃에서 15 분 고압 멸균하여 배지를 멸균한 다음, 37℃에서 액체배양한 유산균을 각 배지에 1% 접종하였다. 이 때 탁도를 0.6~0.7로 조정하여 사용하였다 (O.D. 600nm). 이후 37℃에서 24, 48, 72시간 동안 정치 배양하여 발효시킨 다음 121℃에서 15 분 고압 멸균하였다. 이후 1,700 rpm에서 20 분 동안 원심분리 후 상등액을 취하여 활성을 측정하였다. After adding 1, 3, and 5% of dextrose to the hydrolyzed sea urchin extract, the medium was sterilized by high-pressure sterilization at 121 ° C for 15 minutes, and then 1% of lactic acid bacteria cultured at 37 ° C was added to each medium. Inoculated. At this time, the turbidity was adjusted to 0.6 ~ 0.7 (OD 600nm ). Then, it was fermented by stationary culture at 37°C for 24, 48, and 72 hours, followed by high-pressure sterilization at 121°C for 15 minutes. Then, the activity was measured by taking the supernatant after centrifugation at 1,700 rpm for 20 minutes.
그 결과는 하기 표 2 및 도 7에 나타내었다. The results are shown in Table 2 and FIG. 7 below.
가수분해
발효액sea urchin
hydrolysis
fermentation broth
이를 참고하면, ABTS 라디칼 소거 활성은 덱스트로스 함량과 배양시간에 유의적이지 않았고(p>0.05), DPPH 라디칼 소거 활성은 덱스트로스 함량에 따라 유의적으로 나타났다(p<0.05). 또한 엘라스타제 저해 활성은 배양시간 및 덱스트로스 함량에 따라 유의적으로 나타났다(p<0.05).Referring to this, ABTS radical scavenging activity was not significant according to dextrose content and incubation time ( p> 0.05), and DPPH radical scavenging activity was significantly dependent on dextrose content ( p <0.05). In addition, the elastase inhibitory activity was significantly dependent on the incubation time and dextrose content ( p <0.05).
이러한 결과로부터 당의 첨가 및 배양시간에 따라서 활성을 조절할 수 있으며, 최적 발효 조건은 당 3% 첨가 및 48 시간 배양인 것으로 확인되었다. 이 조건에서 기대할 수 있는 효능 결과는 ABTS 라디칼 소거 활성 44.1%(1% 시료농도), DPPH 라디칼 소거 활성 84.3%(20% 시료농도), Elastase 저해 활성 53.6%(100% 시료농도)로 예상된다.From these results, it was confirmed that the activity could be adjusted according to the addition of sugar and the incubation time, and the optimum fermentation conditions were the addition of 3% sugar and incubation for 48 hours. The efficacy results that can be expected under these conditions are expected to be ABTS radical scavenging activity of 44.1% (1% sample concentration), DPPH radical scavenging activity of 84.3% (20% sample concentration), and Elastase inhibitory activity of 53.6% (100% sample concentration).
이와같이 성게알의 가수분해물 또는 성게알의 가수분해발효물은 ABTS 라디칼 소거활성, DPPH 라디칼 소거활성의 항산화 기능성, 엘라스타제 저해 활성에 의한 주름 개선 기능성, 피부세포 증식 활성에 의한 피부재생 기능성을 가진다. 따라서 본 발명에 따라 성게알의 가수분해물 또는 가수분해발효물을 유효성분으로 포함하는 기능성 화장료와, 이러한 화장품 원료를 이용한 화장품의 제조가 가능하다. As described above, the hydrolyzate of sea urchin or the hydrolyzed fermented product of sea urchin has ABTS radical scavenging activity, antioxidant function of DPPH radical scavenging activity, wrinkle improvement function by elastase inhibitory activity, and skin regeneration function by skin cell proliferation activity. . Therefore, according to the present invention, it is possible to manufacture functional cosmetics containing hydrolyzate or fermented hydrolysis of sea urchin as an active ingredient, and cosmetics using such cosmetic raw materials.
Claims (9)
상기 조성물은 항산화 기능성을 더 포함하는 것을 특징으로 하는, 화장료 조성물.According to claim 1,
The composition is characterized in that it further comprises an antioxidant functionality, a cosmetic composition.
상기 조성물은, 엘라스타제 활성을 저해하고 피부세포의 증식 활성을 나타내어 주름개선 및 피부 재생 기능성을 갖는 것을 특징으로 하는, 화장료 조성물. According to claim 1,
The composition inhibits elastase activity and exhibits skin cell proliferation activity, characterized in that it has wrinkle improvement and skin regeneration functionality, cosmetic composition.
상기 성게알 가수분해물은 성게알 동결건조물을 단백질 분해효소로 가수분해하여 얻어지는 것을 특징으로 하는, 화장료 조성물. According to claim 1,
The cosmetic composition, characterized in that the sea urchin hydrolyzate is obtained by hydrolyzing the sea urchin lyophilisate with a proteolytic enzyme.
상기 성게알의 가수분해발효물은,
성게알 가수분해물에 유산균, 고초균 또는 효모를 접종하여 배양발효함으로써 얻어지는 것을 특징으로 하는, 화장료 조성물. According to claim 1,
The hydrolysis and fermentation of the sea urchin,
A cosmetic composition characterized in that it is obtained by culturing and fermenting sea urchin hydrolyzate by inoculating lactic acid bacteria, Bacillus subtilis, or yeast.
상기 배지를 멸균하는 단계;
상기 멸균된 배지에 균주를 접종하는 단계;
상기 균주가 접종된 배지를 정치배양하여 발효하는 단계; 및
상기 배양발효된 발효물을 멸균하고 여과하는 단계;를 포함하여 이루어지는 것을 특징으로 하는 성게알 가수분해 발효물을 제조하는 방법. preparing a hydrolyzate of sea urchin as a medium;
sterilizing the medium;
Inoculating a strain into the sterilized medium;
Fermenting the culture medium inoculated with the strain; and
A method for producing a fermented sea urchin hydrolyzed product comprising the steps of sterilizing and filtering the cultured fermented product.
상기 성게알의 가수분해물은 성게알 동결건조물을 단백질 분해효소로 가수분해하여 얻어지는 것을 특징으로 하는, 성게알 가수분해 발효물을 제조하는 방법. According to claim 8,
The method for producing fermented sea urchin hydrolyzed product, characterized in that the hydrolyzate of sea urchin is obtained by hydrolyzing the freeze-dried product of sea urchin with a proteolytic enzyme.
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