KR20020036225A - Biomembrane Dressing for Healing Dermal Wound and Infection - Google Patents

Biomembrane Dressing for Healing Dermal Wound and Infection Download PDF

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KR20020036225A
KR20020036225A KR1020000066305A KR20000066305A KR20020036225A KR 20020036225 A KR20020036225 A KR 20020036225A KR 1020000066305 A KR1020000066305 A KR 1020000066305A KR 20000066305 A KR20000066305 A KR 20000066305A KR 20020036225 A KR20020036225 A KR 20020036225A
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이종윤
여인호
김은애
신혜윤
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이종윤
(주) 하판바이오텍
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K35/60—Fish, e.g. seahorses; Fish eggs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein

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Abstract

PURPOSE: A biomembrane containing an antimicrobial material obtained by dissolving a fish having mucous membranes or fish skin and then centrifuging is provided. The biomembrane can be used as a food conservative, a skin-protecting and treating agent or the like because it exhibits an antimicrobial action against various microorganism. CONSTITUTION: A fish having mucous membranes is poured into an aqueous solution containing organic acid and solids are removed therefrom. Thereafter, a fat component is removed therefrom with chloroform or diethylether, the extract is spreaded on a flat plate and then dried. The fish is one of an eel and a loach and the organic acid is one of acetic acid, formic acid and lactic acid.

Description

피부질환 보호-치료용 생체막{Biomembrane Dressing for Healing Dermal Wound and Infection}Biomembrane Dressing for Healing Dermal Wound and Infection}

본 발명은 점액성 표피를 갖는 물고기 전체 또는 껍질을 유기산으로 용해시킨 후 원심분리하여 얻은 항균성물질을 함유한 생체막에 관한 것이다.The present invention relates to a biofilm containing an antimicrobial substance obtained by dissolving whole fish or shells having a mucus epidermis with organic acids and then centrifuging.

항생물질이 함유된 생체막은 피부상처 치료용 반창고 및 붕대를 만드는 산업적 이용이 가능하다. 현재, 잘 알려진 시판품은 죤슨 앤 죤슨(Johnson & Johnson)사의 얇은 스트립(strip), 패치(patch), 스포트(spot)와 콜게이트-팔몰리브 컴퍼니(Colgate-Palmolive Co.)의 큐리티 큐러드(curity curad) 오우취리스(ouchless) 반창고이다. 이들은 합성 폴리머(polymer)로 형성되어 있고, 합성 바이오폴리머(biopolymer)의 기질과 하이알루로닉 산(hyaluronic acid), 더마탄 설페이트(dermatan sulfate), 알기닉 산(alginic acid), 키토산(chitosan)등과 같은 생물체에서 추출한 고분자 유기물을 혼합하여 제조한것(미국특허 5,876,744), 멤브레인/겔 기질에 생리활성 물질을 첨가한 것(미국특허 5,705,485; 5,833,078)등이 있다.Biofilms containing antibiotics can be used industrially to create bandages and bandages to treat skin wounds. Currently, well-known commercial products are thin strips, patches, spots and the Curity Cured by Colgate-Palmolive Co., Ltd. of Johnson & Johnson. curity curad) Ouchless bandage. They are formed of synthetic polymers, which are the substrates of synthetic biopolymers, hyaluronic acid, dermatan sulfate, alginic acid, chitosan. It is prepared by mixing a polymer organic material extracted from an organism such as (US Pat. No. 5,876,744), and adding a bioactive material to the membrane / gel substrate (US Pat. No. 5,705,485; 5,833,078).

이 외에도, 생물체에서 추출한 고분자 물질을 이용하여 박막 조성 기질로 사용한 대표적인 예는 콜라겐(collagen), 젤라틴(gelatin), 셀룰로스(cellulose), 키토산(chitosan)등이 있는데, 이 중 콜라겐과 키토산이 보편적으로 사용되고 있다(미국특허 5,883,078; 5,897,821; 4,452,906; 4,572,906).In addition, representative examples of using a high-molecular substance extracted from a living organism as a thin film composition substrate include collagen, gelatin, cellulose, chitosan, and the like. US Patent 5,883,078; 5,897,821; 4,452,906; 4,572,906.

그러나, 시중에 유통되고 있는 전형적인 붕대는 섬유질의 가제이며, 반창고의 경우 일회용으로서 플라스틱 테이프(plastic tape)에 접착제를 처리한 부분과 환부와 접촉하는 부분이 섬유질 막으로 형성되어 있다. 또한, 물과의 접촉시 쉽게 떨어지며 장시간 부착시에는 피부 표면에 자국을 남기거나, 알레르기 현상이 발생하는 문제점이 있다. 이의 단점을 보완하기 위해 섬유질 막에 항생작용을 발현할 수 있는 물질을 처리한 막도 시판되고 있으나, 사용하기 쉬운 반면 지혈효과가 없고, 물의 침투시 감염가능성이 높으며, 투명하지 않아 환부의 진행을 관찰할 수 없다는 단점이 있다. 따라서, 이런 단점을 보완할 수 있는 박막의 필요성이 대두되어 왔다.However, typical bandages in the market are fibrous gauze, and in the case of a band-aid, the adhesive treated portion and the contact portion of the plastic tape are formed of the fibrous membrane as disposable. In addition, there is a problem that easily fall off contact with water and leave a mark on the surface of the skin, or allergic phenomenon when attached for a long time. In order to make up for the shortcomings, membranes treated with substances capable of expressing antibiotics on the fibrous membranes are commercially available, but they are easy to use but have no hemostatic effect, are highly susceptible to water penetration, and are not transparent. The disadvantage is that it can not be observed. Therefore, there is a need for a thin film that can compensate for this disadvantage.

본 발명은 점액성 표피를 갖는 물고기 전체 또는 껍질을 유기산으로 용해시킨 후 원심분리하여 얻은 항균성물질을 함유한 생체막을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a biofilm containing an antimicrobial substance obtained by dissolving whole fish or shells having a mucus epidermis with organic acids and then centrifuging.

도 1은 미꾸라지표피 추출물의 크로마토그래피1 Chromatography of Loach Cuticle Extract

도 2a는 미꾸라지표피 추출물의 대장균에 대한 항균성Figure 2a is the antimicrobial activity of E. coli of the loach epidermis extract

도 2b는 미꾸라지표피 추출물의 마이크로코커스 류테우스에 대한 항균성Figure 2b shows the antimicrobial activity of micrococcus luteus of the loach epidermis extract

도 2c는 미꾸라지표피 추출물의 칸디다 트로피칼리스에 대한 항균성FIG. 2C shows the antimicrobial activity of Candida Tropicalis of Loach Cuticle Extract

도 3은 미꾸라지표피 추출물로 제조된 생체막의 마이크로코커스 류테우스에 대한 항균성3 is an antimicrobial activity against micrococcus luteus of a biofilm prepared with loach epidermis extract

도 4는 장어표피 추출물의 크로마토그래피Figure 4 Chromatography of the eel epidermis extract

도 5a는 장어표피 추출물의 대장균에 대한 항균성Figure 5a is the antimicrobial activity of E. coli of the eel epidermis extract

도 5b는 장어표피 추출물의 마이크로코커스 류테우스에 대한 항균성Figure 5b is the antimicrobial activity of micrococcus luteus of the eel epidermis extract

도 5c는 장어표피 추출물의 칸디다 트로피칼리스에 대한 항균성Figure 5c shows the antimicrobial activity of Candida tropicalis of eel epidermis extract

도 6a는 장어표피 추출물의 제조된 생체막의 대장균에 대한 항균성Figure 6a is the antimicrobial activity of E. coli of the prepared biofilm of eel epidermis extract

도 6b는 장어표피 추출물의 제조된 생체막의 마이크로코커스 류테우스에 대한 항균성Figure 6b is an antimicrobial activity against micrococcus luteus of the prepared biofilm of eel epidermis extract

도 6c는 장어표피 추출물의 제조된 생체막의 칸디다 트로피칼리스에 대한 항균성Figure 6c is an antimicrobial activity against Candida Tropicalis of the prepared biofilm of eel epidermis extract

앞에서 기술한 목적을 달성하기 위한 본 발명은, 점액성 표피의 어류를 유기산과 물로 구성된 분해액 또는 유기산, 물 및 유기용매로 구성된 분해액으로 분해한 후 고형물을 제거하여 수득되는 추출물로 제조되는 항균성 생체막에 관한 것이다.The present invention for achieving the above object, the antimicrobial activity prepared by extracting the fish obtained by decomposing the fish of the mucous epidermis with a decomposition solution consisting of organic acids and water or a decomposition solution consisting of organic acids, water and organic solvents and then removing the solids It relates to a biological membrane.

보다 구체적으로는, 상기 어류 또는 상기 어류의 표피를 세척하여 불순물을 제거한 다음 유기산이나 유기용매가 함유된 유기산에 담가 교반하여 표피를 완전히 용해시킨다. 교반시간은 원재료의 상태나 처리온도 등에 따라 변화될 것이지만 대략 15 ~ 60시간인 것이 바람직하다. 이때 작업의 편의와 효율을 위해 상기 어류 또는 그 표피를 적절한 크기로 분쇄하여 용해시키는 것이 바람직하다. 원재료의 저장보관이나 분쇄를 위해 원재료를 동결하여 두는 것도 가능하다. 표피가 완전히 용해되면 원심분리하여 고형물을 제거함으로써 본 발명에 의한 항균성 물질이 함유된 생체막형성 물질을 얻게 된다.More specifically, the fish or the skin of the fish are washed to remove impurities, and then immersed in an organic acid or organic acid containing an organic solvent and stirred to completely dissolve the skin. The stirring time will vary depending on the state of the raw materials, the processing temperature and the like, but is preferably about 15 to 60 hours. At this time, it is preferable to dissolve the fish or its skin to an appropriate size for convenience and efficiency of operation. It is also possible to freeze the raw materials for storage or grinding of the raw materials. When the epidermis is completely dissolved, it is centrifuged to remove the solids, thereby obtaining a biofilm-forming material containing the antimicrobial material according to the present invention.

본 발명에서 점액성 표피를 갖는 어류란 담수어, 해수어를 막론하고 비늘이 없으면서(메기) 표피가 점액성 물질로 싸여 있거나, 비늘이 체표면에 깊이 파묻혀있는 원린(元鱗: 둥근비늘: cycloid)을 가지고 있으면서 표피가 점액성 물질로 싸여있는 어류를 말한다. 여기에는 바다장어, 뱀장어, 미꾸라지, 미꾸리, 메기 등 다양한 어류가 해당되겠지만, 원재료 입수의 편의성을 고려할 때, 상기 어류는 장어, 또는 미꾸라지인 것이 좋다. 또한 상기 어류는 물고기 전체를 사용할 수도 있지만, 작업의 편의와 효율을 위해서 표피만을 분리하여 사용하는 것이 바람직하다.In the present invention, a fish having a mucous epidermis is a freshwater fish or a sea fish, but without the scales (catfish), the epidermis is covered with a mucous substance, or the scales are buried deep in the body surface. A fish that has a skin covered with a mucous substance. This may include a variety of fish such as sea eel, eel, loach, loach, catfish, etc., considering the convenience of obtaining raw materials, the fish is preferably an eel or loach. In addition, the fish may use the entire fish, but it is preferable to use only the epidermis separated for convenience and efficiency of operation.

본 발명에서 상기 분해액은 유기산이 1 ~ 10%(v/v)인 수용액이며, 필요에 따라 분해효율을 높이기 위해서 유기용매를 첨가할 수도 있다. 첨가되는 유기용매는 20 ~ 80%(v/v) 함량인 것이 좋다. 상기 유기산은 초산, 개미산 또는 젖산이고, 상기 유기용매는 친수성 유기용매로서 에틸알콜 또는 아세토나이트릴인 것이 바람직하다. 분해액의 사용량은 어류 생체량에 대하여 5 ~ 15배(w/v) 사용하는 것이 바람직하다.In the present invention, the decomposition solution is an aqueous solution having an organic acid of 1 to 10% (v / v), and if necessary, an organic solvent may be added to increase the decomposition efficiency. The organic solvent to be added is preferably 20 to 80% (v / v) content. The organic acid may be acetic acid, formic acid, or lactic acid, and the organic solvent may be ethyl alcohol or acetonitrile as a hydrophilic organic solvent. The amount of the decomposition solution is preferably used 5 to 15 times (w / v) with respect to the fish biomass.

한편, 표피를 분해하고 원심분리하여 고형물을 제거하더라도 분해액 내에 지방성분이 용해되어 있을 수가 있다. 지방 함유량이 많아 아래에서 설명될 생체막 제조시에 지방 미립자에 의해 생체막의 물리적 특성이 저하되는 경우가 발생한다. 따라서, 분해액에 클로로포름 또는 다이에틸에테르를 가하여 현탁시킨 후 일정시간 방치하여 분해액 및 클로로포름 또는 다이에틸에테르가 상분리되면 클로로포름 또는 다이에틸에테르 분획을 분리제거함으로써 분해액 내의 지방성분을 제거하는 것이 바람직하다.On the other hand, even when the epidermis is decomposed and centrifuged to remove the solids, the fat component may be dissolved in the decomposition solution. When the fat content is high, the physical properties of the biofilm may be degraded by the fat microparticles during the production of the biofilm described below. Therefore, if chloroform or diethyl ether is added to the digestion solution and suspended, it is left to stand for a certain time, and when the digestion solution and chloroform or diethyl ether are phase separated, it is preferable to remove the fatty component in the digestion solution by separating and removing the chloroform or diethyl ether fraction. Do.

항균성 검증결과, 이러한 방식으로 얻은 추출물은 유독성 미생물인 대장균(E. coli), 마이크로코커스 류테우스(Micrococcus luteus) 및 칸디다 트로피칼리스(Candida tropicalis)에 대하여 항균성을 보유하고 있음을 알 수 있었다.As a result of the antimicrobial test, the extract obtained in this manner was found to have antimicrobial activity against the toxic microorganisms E. coli , Micrococcus luteus and Candida tropicalis .

따라서, 본 발명에 의한 항균성 추출물을 동결건조하고 분쇄하여 식품보존제, 항생제, 연고제 등의 소재로 활용하는 것이 가능할 것이다. 또한, 본 발명에 의한 항균성 추출물을 적절히 농축하면 점성이 높은 액상형태가 되므로 이를 항균성 접착제, 무좀치료용 항균성 도포제, 상처 보호용 항균성 도포제 등으로도 활용할 수 있다.Therefore, it will be possible to freeze-dried and pulverized the antimicrobial extract according to the present invention to be used as a food preservative, antibiotic, ointment and the like. In addition, if the antimicrobial extract according to the present invention is properly concentrated, it becomes a highly viscous liquid form, so it can also be used as an antimicrobial adhesive, antibacterial coating for athlete's foot, antibacterial coating for wound protection and the like.

앞에서 기술한 목적을 달성하기 위하여 본 발명은, 상기 항균성 추출물로 제조되는 항균성 생체막을 제공한다.In order to achieve the above object, the present invention provides an antimicrobial biofilm prepared from the antimicrobial extract.

즉, 상기 항균성 추출물을 유리 또는 플라스틱 평판에 도포한 후 건조하여 항균성 생체막을 얻게 된다. 제조된 생체막의 탈리가 용이하도록 디메칠실래인(dichlorodimethylsilane)으로 처리한 프라스틱이나 아크릴용기에 부어 무균상태의 클린벤치 내에서 건조시키는 것이 바람직하다. 이때 제조되는 생체막의 물리적·생물학적 성질을 개선하기 위하여 0.5 ~ 2.0%(v/v)의 글리세롤, 0.2 ~ 2.0%(w/v)의 젤라틴 또는 PVA(polyvinyl alcohol), 0.03 ~ 0.14%(w/v)의 키토산을 첨가할 수 있다. 글리세롤은 생체막의 유연성을 증가시키며, 젤라틴 또는 PVA는 생체막의 강도를 증대시키는 역할을 한다. 키토산은 생체막의 물리적 특성을 다소 저하시키지만 키토산에 의한 효과를 얻을 수 있게 한다.That is, the antimicrobial extract is applied to a glass or plastic plate and then dried to obtain an antimicrobial biofilm. In order to facilitate the detachment of the prepared biofilm, it is preferable to pour it in a plastic or acrylic container treated with dichlorodimethylsilane and to dry it in a sterile clean bench. At this time, 0.5 ~ 2.0% (v / v) glycerol, 0.2 ~ 2.0% (w / v) gelatin or PVA (polyvinyl alcohol), 0.03 ~ 0.14% (w / chitosan of v) may be added. Glycerol increases the flexibility of the biofilm, and gelatin or PVA serves to increase the strength of the biofilm. Chitosan slightly lowers the physical properties of the biofilm, but allows the effect of chitosan to be obtained.

항균성 검증결과, 이러한 방식으로 얻은 생체막은 유독성 미생물인 대장균(E. coli), 마이크로코커스 류테우스(Micrococcus luteus) 및 칸디다 트로피칼리스(Candida tropicalis)에 대하여 항균성을 보유하고 있음을 알 수 있었다.As a result of the antimicrobial test, the biofilm obtained in this way was found to be antimicrobial against the toxic microorganisms E. coli , Micrococcus luteus and Candida tropicalis .

상기와 같은 방법으로 얻어진 본 발명에 의한 생체막은 천연물질이며 항균성이 있을 뿐 아니라 아주 투명한 형태의 것이다. 따라서, 이를 피부질환 치료 및 보호용 커버제, 화상 치료용 보호용 커버제, 무좀 치료용 밴드 등의 형태로 활용하는 것이 가능하다.The biofilm according to the present invention obtained by the above method is a natural substance and has antimicrobial properties as well as a very transparent form. Therefore, it is possible to utilize this in the form of a skin disease treatment and protective cover, a burn treatment protective cover, athlete's foot treatment band.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

실시예 1Example 1 : 미꾸라지 표피추출물의 항균성분석: Antimicrobial Analysis of Loach Epidermis Extract

미꾸라지를 구입하여 24시간 천연수에 방치하여 오물질 및 미꾸라지의 성장수질에 포함되어 있을 가능성이 있는 항생물질을 제거하였다. 50 g의 생 미꾸라지를 동결시킨 다음 직경이 약 1 ~ 3cm 정도 되도록 분쇄하여 추출용 원재료로 하여, 초산이 5%(v/v)(0.88M) 함유된 수용액을 분해액으로 하였다. 원재료에 대하여 10배(w/v)인 500ml의 분해액을 가하고 분쇄한 원재료 조직이 완전히 분해될 때까지 상온에서 교반하였다. 분해가 완료되면 209 ×g에서 30분동안 원심분리하여 고형물을 제거하였다. 이때 얻어진 상층액은 450ml이었으며, 건물량은 0.78g/100ml 이었다. 이렇게 수집한 상층액은 4℃에 보관하면서 필요한 경우에 꺼내서 실험에 사용하였다.Loach was purchased and left for 24 hours in natural water to remove dirt and antibiotics that might be included in the quality of the loach. 50 g of raw loach was frozen and ground to a diameter of about 1 to 3 cm to prepare an extraction material. An aqueous solution containing 5% (v / v) (0.88 M) of acetic acid was used as a decomposition solution. 10 times (w / v) of 500 ml of decomposition solution was added to the raw materials, and the mixture was stirred at room temperature until the pulverized raw material structure was completely decomposed. Once the digestion was complete, the solids were removed by centrifugation at 209 × g for 30 minutes. The supernatant obtained at this time was 450ml, dry weight was 0.78g / 100ml. The supernatant thus collected was taken out if necessary while being stored at 4 ° C. and used for experiments.

라이시스 존 에쎄이(lysis zone assay) 방법으로 상기 방법에서 수득한 추출물의 항균활성을 확인하였다.The antimicrobial activity of the extract obtained in the above method was confirmed by a lysis zone assay.

먼저 상기 미꾸라지 추출물을 세파덱스(Sephadex) LH-20으로 크로마토그래피하여 각 분획에 대해 항균성 작용을 검증하였다. 대장균과 마이크로 코커스 류테우스는 LB-agarose 배지에 칸디다 트로피칼리스는 potato dextrose 배지에 최종균수가 2×105cfu/ml 되도록 섞어 만든다. 굳힌 배지에 0.3㎝크기의 구멍을 뚫고 상기 분획을 4㎕씩 주입하여 물질이 배지에 스며들도록 실온에서 1시간 방치 후 30℃에서 24시간 배양하여 항균성물질에 의해 균의 성장이 저해되어 생성된 클리어 존을 관찰하였다.First, the loach extract was chromatographed with Sephadex LH-20 to verify the antimicrobial activity of each fraction. Escherichia coli and Micrococcus luteus are mixed in LB-agarose medium and Candida Tropicalis in potato dextrose medium so that the final bacterial count is 2 × 10 5 cfu / ml. A 0.3 cm-sized hole was made in the hardened medium, and 4 µl of the fractions were injected. The resultant was allowed to stand at room temperature for 1 hour to infiltrate the medium, followed by incubation at 30 ° C for 24 hours to inhibit the growth of bacteria by antimicrobial substances. John was observed.

도 1에 미꾸라지 추출물을 세파덱스 LH-20으로 크로마토그래피한 결과 그래프를 도시하였으며, 도 2에 라이시스 존 어쎄이 결과 사진을 도시하였다.In Fig. 1, the loach extract was chromatographed with Sephadex LH-20, and in Fig. 2, the Lysis zone assay result photograph is shown.

도 2a ~2c에서 볼 수 있듯이 수개 ~ 수십개의 구멍 주위로 커다란 클리어존이 형성된 것으로 보아 본 발명에 의한 미꾸라지 추출물(의 분획물)은 상기 균주들에 대해 강력한 항균력을 보유하고 있음을 알 수 있다.As can be seen in Figures 2a to 2c, a large clear zone is formed around several to several tens of pores, so it can be seen that the loach extract (fraction of) according to the present invention possesses strong antibacterial activity against the strains.

또한, 도 3에서 미꾸라지표피 추출물을 재료로 제조된 생체막이 마이크로코커스 류테우스에 대해 항균성을 나타냄을 알 수 있다.In addition, it can be seen that the biofilm prepared from the loach epidermis extract in FIG. 3 exhibits antimicrobial activity against micrococcus luteus.

실시예 2Example 2 : 장어표피추출물의 항균성 분석: Antimicrobial Analysis of Eel Cuticle Extracts

장어의 껍질을 직경이 약 1 ~ 3cm 정도 되도록 분쇄하여 추출용 원재료로 하였다. 50g의 원재료에 초산이 5%(v/v)(0.88M) 함유된 수용액으로 된 분해액 400ml를 가하고 상온에서 24시간 동안 교반하면서 분해하였다. 분해가 완료되면 209 ×g에서 30분동안 원심분리하여 고형물을 제거하였다. 상층액에 함유된 지방성분 및 현탁물을 제거하기 위하여 80ml의 크로로포름(chloroform)을 가하고 흔들어 주어 용액을 완전히 섞은 다음, 30분간 원심분리(209×g)한 후 두 용매를 분리하여 상등액만을 취한다. 장어추출물층의 잔류 크로로포름을 제거하기 위해 60℃에서 감압증발시켜 350㎖의 장어추출물을 얻는다. 이때, 건물량은 2g/100㎖이었다. 이렇게 수집한 상층액은 4℃에 보관하면서 필요한 경우에 꺼내서 실험에 사용하였다.The shell of the eel was pulverized to a diameter of about 1 to 3 cm to prepare a raw material for extraction. To 50 g of raw material, 400 ml of a decomposition solution of an aqueous solution containing 5% (v / v) (0.88 M) of acetic acid was added and decomposed with stirring at room temperature for 24 hours. Once the digestion was complete, the solids were removed by centrifugation at 209 × g for 30 minutes. To remove fatty components and suspensions in the supernatant, add 80 ml of chloroform and shake to thoroughly mix the solution. Centrifuge for 30 minutes (209 × g) and separate the two solvents Take it. To remove residual chromoform in the eel extract layer, 350 ml of eel extract was obtained by evaporation under reduced pressure at 60 ° C. At this time, the dry weight was 2 g / 100 ml. The supernatant thus collected was taken out if necessary while being stored at 4 ° C. and used for experiments.

라이시스 존 에쎄이(lysis zone assay) 방법으로 상기 방법에서 수득한 추출물의 항균활성을 확인하였다.The antimicrobial activity of the extract obtained in the above method was confirmed by a lysis zone assay.

먼저 상기 장어 추출물을 세파덱스(Sephadex) G-25로 크로마토그래피하여 각분획을 분리하였다(도시 생략). 대장균과 마이크로코커스 류테우스는 LB-agarose 배지에, 칸디다 트로피칼리스는 potato dextrose 배지에 최종균수가 2×105cfu/ml 되도록 섞어 만든다. 굳힌 배지에 0.3㎝크기의 구멍을 뚫고 상기 분획을 4㎕씩 주입하여 물질이 배지에 스며들도록 실온에서 1시간 방치 후 30℃에서 24시간 배양하여 항균성물질에 의해 균의 성장이 저해되어 생성된 클리어 존을 관찰하였다.First, the eel extract was chromatographed with Sephadex G-25 to separate each fraction (not shown). E. coli and Micrococcus luteus are mixed in LB-agarose medium and Candida tropicalis in potato dextrose medium with a final bacterial count of 2 × 10 5 cfu / ml. A 0.3 cm-sized hole was made in the hardened medium, and 4 µl of the fractions were injected. The resultant was allowed to stand at room temperature for 1 hour to infiltrate the medium, followed by incubation at 30 ° C for 24 hours to inhibit the growth of bacteria by antimicrobial substances. John was observed.

도 4에 장어추출물을 세파덱스 G-25로 크로마토그래피한 결과 그래프를 도시하였고, 도 5에 라이시스 존 에쎄이 결과 사진을 도시하였다.In FIG. 4, the result of the eel extract chromatographed with Sephadex G-25 is shown, and FIG. 5 is a photograph of the results of Lysis zone assay.

도 5a ~ 5c에서 볼 수 있듯이 수개 ~ 수십개의 구멍 주위로 커다란 클리어존이 형성된 것으로 보아 본 발명에 의한 장어 추출물(의 분획물)은 상기 균주들에 대해 강력한 항균력을 보유하고 있음을 알 수 있다.As can be seen in Figures 5a to 5c, a large clear zone is formed around several to several tens of pores, it can be seen that the extract of the eel according to the present invention has a strong antimicrobial activity against the strains.

실시예 3Example 3 : 생체막의 제조 및 생체막의 항균특성 분석: Preparation of Biofilm and Analysis of Antimicrobial Properties of Biofilm

실시예 1 ~ 2에서 수득된 추출물에 글리세롤 0.5%(v/v)를 첨가하여, 디메틸실레인(dichlorodimethylsilane)으로 처리한 플라스틱 용기에 부은 다음, 무균상태를 유지하는 클린벤치내에서 건조시켰다. 시간 경과에 따라 수분과 유기산이 증발하면 합성수지막과 유사한 형태의 투명막이 형성되었다. 생체막의 물리적 성질을 개선시키기 위해 젤라틴(gelatin) 및 PVA 각각 0.2%(v/v), 키토산(chitosan)0.03%(v/v)를 첨가할 수 있다. 제조된 생체막의 항균활성을 확인하기 위하여 라이시스 존 에쎄이(lysis zone assay) 방법을 적용하였다. 대장균과 마이크로코커스 류테우스는 LB-agarose 배지에, 칸디다 트로피칼리스는 potato dextrose 배지에 최종균수가 2×105cfu/ml 되도록 섞어 만든다. 굳힌 배지에 제조된 생체막이 0.6㎝되도록 원형으로 절취 후 상기 배지에 일정간격으로 얹고, 30℃에서 24시간배양 후 생체막의 항균성 물질에 의해 균들의 성장이 저해되어 생성된 클리어존을 관찰하였다. 첨부된 도 5와 도 6에 각각 실시예1 ~ 2에 의한 추출물로 제조된 생체막을 사용하여 라이시스 존 에쎄이 실험한 결과를 보여주는 사진을 첨부하였다.Glycerol 0.5% (v / v) was added to the extract obtained in Examples 1 and 2, poured into a plastic container treated with dimethylsilane (dichlorodimethylsilane), and dried in a clean bench maintained aseptically. As time elapsed, moisture and organic acid evaporated to form a transparent film similar to a synthetic resin film. In order to improve the physical properties of the biofilm, gelatin and PVA, respectively, 0.2% (v / v) and chitosan (chitosan) 0.03% (v / v) may be added. In order to confirm the antimicrobial activity of the prepared biofilm, a lysis zone assay method was applied. E. coli and Micrococcus luteus are mixed in LB-agarose medium and Candida tropicalis in potato dextrose medium with a final bacterial count of 2 × 10 5 cfu / ml. The biofilm prepared in the hardened medium was cut into a circle so as to be 0.6 cm, placed on the medium at regular intervals, and cultured at 30 ° C. for 24 hours, and the growth of the bacteria was inhibited by the antimicrobial material of the biofilm to observe the clear zone. 5 and 6 are attached to the photographs showing the results of experiments with the Lysis zone assay using the biofilm prepared by the extract according to Examples 1 and 2, respectively.

도 6a∼6c에서 장어표피추출물로 제조된 생체막이 상기 균주들에 대해 항균력을 보임을 나타내었다. 도에서 볼 수 있듯이, 각 추출물과 마찬가지로 각 추출물을 재료로 하여 제조된 생체막이 상기 균주들에 대해 강력한 항균력을 보유하고 있음을 알 수 있다.6a to 6c showed that the biofilm prepared with the eel epidermal extract showed antimicrobial activity against the strains. As can be seen in the figure, it can be seen that the biofilm prepared from each extract as a material like each extract has a strong antimicrobial activity against the strains.

이상에서 상세히 설명하고 입증하였듯이, 본 발명에 의하면 점액성 표피를 갖는 어류전체나 어류껍질을 유기산이 함유된 유기용매에서 분해시킨 후 원심분리하여 항균성 추출물과 이 추출물을 함유하는 항균성 생체막을 얻을 수 있다.As described and demonstrated in detail above, according to the present invention, the whole fish or the fish shell having the mucous epidermis is decomposed in an organic solvent containing an organic acid, and then centrifuged to obtain an antimicrobial extract and an antimicrobial biofilm containing the extract. .

본 발명에 의한 추출물 및 생체막은 천연의 성분이며, 대량생산이 가능하며 각종의 미생물에 대해 항균성을 나타내기 때문에 식품보존제, 피부보호-치료제 등으로 활용이 가능할 것이다.Extracts and biofilms according to the present invention are natural ingredients, can be mass-produced and exhibit antimicrobial properties against various microorganisms, and thus may be used as food preservatives, skin protection-treatments, and the like.

Claims (6)

점액성 표피의 어류를 유기산이 함유된 수용액으로 분해한 후 고형물을 제거하여 수득된 추출물을 평판에 도포한 후 건조하여 제조하는 것을 특징으로 하는 항균성 생체막.An antimicrobial biomembrane, characterized in that the fish obtained by decomposing the fish of the mucus epidermis into an aqueous solution containing an organic acid and then removing the solids is applied to a plate and dried. 제 1 항에 있어서,The method of claim 1, 상기 어류는 장어, 미꾸라지 중의 어느 하나인 것을 특징으로 하는 항균성 생체막.The fish is an antimicrobial biofilm, characterized in that any one of eel, loach. 제 1 항에 있어서,The method of claim 1, 상기 유기산은 초산, 개미산, 젖산 중의 어느 하나인 것을 특징으로 하는 항균성 생체막.The organic acid is antimicrobial biofilm, characterized in that any one of acetic acid, formic acid, lactic acid. 제 1 항에 있어서,The method of claim 1, 상기 유기용매는 에틸알콜 또는 아세토나이트릴인 것을 특징으로 하는 항균성 생체막.The organic solvent is an antimicrobial biofilm, characterized in that ethyl alcohol or acetonitrile. 제 1 항에 있어서,The method of claim 1, 상기 고형물 제거 후 클로로포름 또는 다이에틸에테르로 지방성분을 제거하는 것을 특징으로 하는 항균성 생체막.Antimicrobial biofilm, characterized in that to remove the fat component with chloroform or diethyl ether after removing the solids. 제 1항에 있어서,The method of claim 1, 그리세롤, 키토산, 젤라틴 또는 PVA를 추가로 포함하는 것을 특징으로 하는 항균성 생체막.An antimicrobial biomembrane further comprising glycerol, chitosan, gelatin or PVA.
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KR101460669B1 (en) * 2012-11-23 2014-11-11 주식회사 뉴메디온 Cosmetic composition with mucus from fish
WO2017069365A1 (en) 2015-10-21 2017-04-27 Republic Of Korea(Management : Rural Development Administration) Artificial biomembrane using silk matrix and method of manufacturing the same

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KR200453763Y1 (en) * 2008-09-24 2011-05-30 백동호 sleeping disturbance control machinery
KR101460669B1 (en) * 2012-11-23 2014-11-11 주식회사 뉴메디온 Cosmetic composition with mucus from fish
WO2017069365A1 (en) 2015-10-21 2017-04-27 Republic Of Korea(Management : Rural Development Administration) Artificial biomembrane using silk matrix and method of manufacturing the same

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