KR20050095167A - Composition for whitening of skin comprising candida parapsilosis as an effect gradient - Google Patents
Composition for whitening of skin comprising candida parapsilosis as an effect gradient Download PDFInfo
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- KR20050095167A KR20050095167A KR1020040020376A KR20040020376A KR20050095167A KR 20050095167 A KR20050095167 A KR 20050095167A KR 1020040020376 A KR1020040020376 A KR 1020040020376A KR 20040020376 A KR20040020376 A KR 20040020376A KR 20050095167 A KR20050095167 A KR 20050095167A
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- candida parapsilosis
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- tyrosinase
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- 241000222173 Candida parapsilosis Species 0.000 title claims abstract description 42
- 229940055022 candida parapsilosis Drugs 0.000 title claims abstract description 41
- 230000002087 whitening effect Effects 0.000 title claims abstract description 27
- 239000000203 mixture Substances 0.000 title claims abstract description 11
- 230000000694 effects Effects 0.000 title abstract description 10
- 239000001963 growth medium Substances 0.000 claims abstract description 7
- 239000004480 active ingredient Substances 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 claims abstract description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 150000002632 lipids Chemical class 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000004006 olive oil Substances 0.000 claims description 6
- 235000008390 olive oil Nutrition 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 108090000637 alpha-Amylases Proteins 0.000 claims description 4
- 102000004139 alpha-Amylases Human genes 0.000 claims description 4
- 229940024171 alpha-amylase Drugs 0.000 claims description 4
- 235000020429 malt syrup Nutrition 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 3
- 102100022624 Glucoamylase Human genes 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims 1
- 102000003425 Tyrosinase Human genes 0.000 abstract description 19
- 108060008724 Tyrosinase Proteins 0.000 abstract description 19
- 239000013543 active substance Substances 0.000 abstract description 15
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 abstract description 14
- 230000002401 inhibitory effect Effects 0.000 abstract description 12
- 239000000126 substance Substances 0.000 abstract description 7
- 208000017520 skin disease Diseases 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 10
- 239000002537 cosmetic Substances 0.000 description 7
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000011149 active material Substances 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 239000011777 magnesium Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 206010014970 Ephelides Diseases 0.000 description 3
- 208000003351 Melanosis Diseases 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 229910052500 inorganic mineral Inorganic materials 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000011707 mineral Substances 0.000 description 3
- 235000010755 mineral Nutrition 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 235000006408 oxalic acid Nutrition 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- HQBKFSFXKKNIDP-GRHHLOCNSA-N (2s)-2-azanyl-3-(4-hydroxyphenyl)propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1.OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 HQBKFSFXKKNIDP-GRHHLOCNSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 241000271309 Aquilaria crassna Species 0.000 description 1
- 208000012641 Pigmentation disease Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 208000002474 Tinea Diseases 0.000 description 1
- 241000893966 Trichophyton verrucosum Species 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000004694 pigment cell Anatomy 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 108010075550 termamyl Proteins 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- E—FIXED CONSTRUCTIONS
- E02—HYDRAULIC ENGINEERING; FOUNDATIONS; SOIL SHIFTING
- E02B—HYDRAULIC ENGINEERING
- E02B3/00—Engineering works in connection with control or use of streams, rivers, coasts, or other marine sites; Sealings or joints for engineering works in general
- E02B3/04—Structures or apparatus for, or methods of, protecting banks, coasts, or harbours
- E02B3/046—Artificial reefs
Landscapes
- Engineering & Computer Science (AREA)
- General Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Ocean & Marine Engineering (AREA)
- Mechanical Engineering (AREA)
- Civil Engineering (AREA)
- Structural Engineering (AREA)
- Cosmetics (AREA)
Abstract
본 발명은 칸디다 파라프실로시스 배양액을 유효성분으로 함유하는 피부 미백용 조성물에 관한 것으로, 칸디다 파라프실로시스(Candida parapsilosis)(기탁번호: KCCM 50271T)을 배양한 뒤 피부미백효과가 있는 유효물질을 포함하는 배양액을 분리 및 정제한 다음 상기에서 얻은 칸디다 파라프실로시스(Candida parapsilosis) 배양액의 타이로시나아제(Tyrosinase) 억제효과를 조사하고 칸디다 파라프실로시스(Candida parapsilosis)의 배양조건에 따른 유효물질 생산성을 조사함으로써 피부 발색색소인 멜라닌을 형성하는 인자로서의 타이로시나아제(Tyrosinase)의 활성을 효과적으로 억제하여 피부 미백효과 및 검버섯같은 피부 장애요소에 대한 개선 및 치료효과를 가지는 효과가 있으므로 화장품산업상 매우 유용한 발명인 것이다.The present invention relates to a composition for skin whitening containing the candida parapsilosis culture as an active ingredient, Candida parapsilosis (Accession No .: KCCM 50271T) after culturing the effective substance having a skin whitening effect After the separation and purification of the culture medium containing Candida parapsilosis ( Candida parapsilosis ) obtained above to investigate the tyrosinase (Tyrosinase) inhibitory effect and the culture conditions of Candida parapsilosis ( Candida parapsilosis ) By investigating the productivity of active substances, it effectively inhibits the activity of tyrosinase as a factor that forms melanin, a skin chromophore, and thus has the effect of improving skin whitening and skin disorders such as blotch and treating effect. It is an industrially useful invention.
Description
본 발명은 칸디다 파라프실로시스(Candida parapsilosis) 배양액를 유효성분으로 함유하는 피부 미백용 조성물에 관한 것으로, 칸디다 파라프실로시스(Candida parapsilosis)가 배양중 생성하는 유효물질이 피부의 발색색소인 멜라닌 색소를 형성하는 효소를 억제함으로써 피부 미백효과를 갖는 조성물에 관한 것이다.The invention Candida parapsilosis (Candida parapsilosis) relates to a composition for skin whitening comprising the baeyangaekreul active ingredient, Candida parapsilosis (Candida parapsilosis) is an effective substance is coloring pigment in the skin to generate in the culture melanin It relates to a composition having a skin whitening effect by inhibiting the enzyme forming a.
일반적으로 말하는 미백화장품이란 피부의 발색정도를 가름하는 색소가 침착되어 생기는 기미나 주근깨를 교정 또는 회복시키는 기능을 가진 화장품이다. 이때 기미나 주근깨를 생성하는 요인 중 멜라닌 색소는 주 요인이며 피부 멜라닌의 함량이 증가하면 기미나 주근깨가 생기기 쉬운 조건이 되어 피부색이 갈색으로 발색된다. 또한 자외선에 장시간 노출되었을 경우에도 광반응에 의한 멜라닌 색소 세포의 증가가 초래되고 그 결과로 색소 침착이 늘어난다. Generally speaking, whitening cosmetics are cosmetics that have the function of correcting or restoring blemishes and freckles caused by the deposition of pigments that color the skin. At this time, melanin pigment is one of the main factors that produce blemishes and freckles. When the content of skin melanin increases, it becomes a condition that tends to produce blemishes and freckles. In addition, exposure to ultraviolet rays for a long time causes melanin pigment cells to increase due to the photoreaction, resulting in increased pigmentation.
현대 여성들은 과거로부터 하얗고 맑은 얼굴은 한국 고유의 미인상을 나타내는 대표적인 표현으로 모든 여성들이 갈구하고 희망하는 이상향이다. 그러므로 예로부터 민간적인 방법으로 미백효과를 내는 갖은 물질들을 찾아 화장품 업계 또한 소비자 심리에 맞추어 여러 가지 미백화장품 제품을 개발하고 출시하기도 하였으며 현재는 미백화장품이 기능성 제품으로 자리를 확고히 굳히는 동시에비약적인 발전을 가져오고 있다. Modern women have a white and clear face from the past, which is a representative expression of Korean unique beauty. Therefore, the cosmetics industry has also developed and launched a variety of whitening cosmetics products in line with consumer sentiment in search of various substances that have a whitening effect in the civilian way. At present, whitening cosmetics have firmly established itself as a functional product and have made rapid progress. Coming.
종래의 미백효과를 나타내는 물질 개발에 대한 연구로는 다음과 같은 방법들이 이용되고 있었다.The following methods have been used as a study on the development of a material having a conventional whitening effect.
대한민국 공개특허 특2003-0071893호에서는 수용성 미백 조성물인 왈더리안 인디카 잎 추출물이 갖는 미백효과에 대해 공지한바 있었고,In Korean Patent Laid-Open Publication No. 2003-0071893, the whitening effect of the water-soluble whitening composition Walderian Indica leaf extract was known.
대한민국 공개특허 특개평 4-9320호에서는 아스콜빈산이 갖는 미백효과에 대하여 공지했었고 특개평 6-192062호에서는 하이드로퀴논, 특개평 56-7710호 에서는 코직산, 특개평 4-9315호에서는 알부틴이 갖는 미백효과에 대하여 공지된바 있었다.In Korean Patent Laid-Open Publication No. 4-9320, it is known about the whitening effect of ascorbic acid. It has been known about the effect.
대한민국 공개특허 특2003-0090320호에서는 방풍, 백선, 무환자나무, 피나무 각각의 추출물을 함유하는 피부 미백 효과를 갖는 화장료 조성물에 대한 공지가 있었다.Korean Patent Laid-Open Publication No. 2003-0090320 has been known for a cosmetic composition having a skin whitening effect containing an extract of windproof, ringworm, agar wood, bark.
그러나, 상기와 같은 종래의 방법은 주로 식물추출물에서 추출되는것에 의한 것으로 생산성이 뒤떨어지는 것에 비하여 미생물을 배양하여 그 배양산물을 이용하는 것은 보다 새롭고 획기적인 방법이라 할 것이다.However, the conventional method as described above is mainly by extracting from plant extracts, and cultivating microorganisms and using the culture products will be called a new and groundbreaking method as compared to poor productivity.
이에 본 발명은 상기와 같은 문제점을 개선 또는 해결하고자 안출된 것으로, 곰팡이류인 칸디다 파라프실로시스(Candida parapsilosis)가 생장하면서 생성하는 유효물질 중 피부미백효과를 갖는 유효물질의 효과를 규명하는데 그 목적이 있다.Accordingly, the present invention has been made to improve or solve the above problems, to identify the effect of the active substance having a skin whitening effect among the active substances produced during the growth of fungi Candida parapsilosis ( Candyda parapsilosis ) There is this.
본 발명의 상기 목적은 칸디다 파라프실로시스(Candida parapsilosis)(기탁번호: KCCM 50271T)을 배양한 뒤 피부미백효과가 있는 유효물질을 포함하는 배양액을 분리 및 정제한 다음 상기에서 얻은 칸디다 파라프실로시스(Candida parapsilosis) 배양액의 타이로시나아제(Tyrosinase) 억제효과를 조사하고 칸디다 파라프실로시스(Candida parapsilosis)의 배양조건에 따른 유효물질 생산성을 조사함으로써 달성하였다.The object of the present invention is to cultivate Candida parapsilosis (Accession No .: KCCM 50271T), and then to isolate and purify the culture medium containing the active substance having a skin whitening effect and then to the Candida parapsil obtained above This study was performed by investigating the inhibitory effect of tyrosinase on the culture of Candida parapsilosis and by examining the productivity of the active substances according to the culture conditions of Candida parapsilosis .
이하 본 발명의 구성을 설명한다.Hereinafter, the configuration of the present invention.
본 발명은 칸디다 파라프실로시스(Candida parapsilosis)(기탁번호: KCCM 50271T)을 배양한 뒤 피부미백효과가 있는 유효물질을 포함하는 배양액을 분리 및 정제하는 단계; 상기 단계에서 얻은 칸디다 파라프실로시스(Candida parapsilosis) 배양액의 타이로시나아제(Tyrosinase) 억제효과를 조사하는 단계; 및 칸디다 파라프실로시스(Candida parapsilosis)의 배양조건에 따른 유효물질 생산성을 조사하는 단계로 구성된다.The present invention comprises the steps of culturing Candida parapsilosis (Candam para: KCCM 50271T) and then separating and purifying the culture medium containing the active substance having a skin whitening effect; Investigating the tyrosinase inhibitory effect of Candida parapsilosis culture obtained in the above step; And Candida parapsilosis ( Candida parapsilosis ) consists of a step of examining the effective material productivity according to the culture conditions.
본 발명은 칸디다 파라프실로시스(Candida parapsilosis)(기탁번호: KCCM 50271T)의 배양액을 유효성분으로 함유하는 피부 미백용 조성물을 제공한다.The present invention provides a composition for skin whitening containing a culture solution of Candida parapsilosis (Accession Number: KCCM 50271T) as an active ingredient.
본 발명 칸디다 파라프실로시스(Candida parapsilosis) 배양액은 총 용량 1L를 기준으로 맥아물엿 50g, 효모 추출물 6g, 황산 마그네슘 1.5g, 올리브 오일 10g, 트윈(Tween) 40 20g을 포함하는 조성물에 α-아밀라제 및 글루코-아밀라제를 이용하여 효소처리한 배지에 칸디다 파라프실로시스(Candida parapsilosis)(기탁번호: KCCM 50271T)를 접종하여 배양한 후 얻은 배양액을 원심분리하여 상등액을 수거한 뒤 상기 상등액에 클로로포름을 첨가하여 지질층을 분리한 후 상기 지질층을 컬럼크로마토그래피로 분리하여 얻은 극성지질층이다. Candida parapsilosis culture solution of the present invention is α-amylase in a composition comprising 50 g malted starch syrup, 6 g yeast extract, 1.5 g magnesium sulfate, 10 g olive oil, 20 g Tween 40 based on a total volume of 1 L And inoculating Candida parapsilosis (Accession No .: KCCM 50271T) to the enzymatically treated medium using gluco-amylase. After separating the lipid layer by addition is a polar lipid layer obtained by separating the lipid layer by column chromatography.
이하, 본 발명의 구성을 실시 예를 들어 상세히 설명하지만, 본 발명의 권리 범위가 하기 실시예에만 한정되는 것은 아니다. Hereinafter, the configuration of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.
실시예 1: 칸디다 파라프실로시스(Example 1 Candida parapsissis Candida parapsilosisCandida parapsilosis )의 배양 및 유효물질 분리 정제Culture and separation of active substance
칸디다 파라프실로시스(Candida parapsilosis)(기탁번호: KCCM 50271T)를 배양하기 위해서 조제배지를 사용하였다.Preparation medium was used to culture Candida parapsilosis (Accession No .: KCCM 50271T).
상기 조제배지를 제조하기 위하여 먼저 총 용량 1L당 맥아물엿 50g, 트윈(Tween) 40 20g, 효모 추출물 6g, 올리브 오일 10g, 황산 마그네슘 1.5g을 혼합한 후 용해가 잘되도록 교반하며 가열하였다. 그 후 배지의 온도가 70℃에 이르면 이를 유지토록 조정하며, 여기에 α-아밀라제 및 글루코-아밀라제를 1L당 각각 1%되도록 첨가한 후, 효소들의 반응시간을 2시간으로 설정하였다. 반응이 끝난 배지는 멸균한 후 산소공급을 시켜주면서 균주를 접종하여 37℃에서 4일간 배양하여 초기 균체를 생성시킨 후 멸균된 1.7% 옥살산(oxalic acid) 100mL를 더하여 Mg2+이온을 침전시켰다. 상기 배약액을 다시 산소공급하에 30℃에서 3일간 배양하였다. 배양 완료시에는 배양액을 10,000×g에서 20분간 원심분리를 하여 상등액을 수거하고, 상등액을 클로로포름(chloroform)으로 지질층만 분리한 다음 상기 지질층을 컬럼크로마토그래피에 의거하여 분리하여 극성지질층만 얻었다.To prepare the prepared medium, first, 50 g of malt syrup, 20 g of Tween 40, 6 g of yeast extract, 10 g of olive oil, and 1.5 g of magnesium sulfate were mixed and heated with stirring to facilitate dissolution. Thereafter, when the temperature of the medium reached 70 ° C., it was adjusted to maintain it. After adding α-amylase and gluco-amylase to 1% per 1 L, the reaction time of the enzymes was set to 2 hours. After completion of the reaction, the culture medium was sterilized, inoculated with oxygen, and inoculated with the strain, followed by incubation at 37 ° C. for 4 days to generate initial cells, and 100 mL of sterilized 1.7% oxalic acid was added to precipitate Mg 2+ ions. The solution was incubated at 30 ° C. for 3 days again under oxygen supply. At the completion of the culture, the supernatant was collected by centrifuging the culture broth at 10,000 × g for 20 minutes, and the supernatant was separated only from the lipid layer by chloroform, and the lipid layer was separated by column chromatography to obtain only the polar lipid layer.
본 발명에서 사용된 맥아물엿은 시중에서 구입한 (주)오뚜기의 맥아물엿을 사용하였으며, 효모 추출물은 디프코(Difco)사에서 제조한 박토TM 효모 추출물(BactoTM yeast extract)을, 황산 마그네슘(magnesium sulfate)은 (주)대정화학의 1급시약을 사용하였다. 또한 α-아밀라제 및 글루코-아밀라제는 노보자임스(NOVOZYMES)사에서 제조하여 (주)노브노르디스코리아에서 공급받은 에이엠지®300L(AMG®300L)과 터마밀®120L(Termamyl®120L)을 각각 사용하였다.The malt syrup used in the present invention was used for malt syrup of Co. Ottogi a commercially available yeast extract is a deep nose stripping TM yeast extract (Bacto TM yeast extract) prepared in (Difco) four, magnesium sulfate ( Magnesium sulfate was used as the first grade reagent of Daejung Chemical Co., Ltd. Also α- amylase and glucosidase-amylase is used to let the furnace Eames prepared from (NOVOZYMES) Co. Ltd. Korea knob Nord discharge eyiemji ® 300L (AMG ® 300L) and ground mamil ® 120L (Termamyl ® 120L) that received in the respective It was.
실험예 1: 칸디다 파라프실로시스(Experimental Example 1: Candida parapsissis ( Candida parapsilosisCandida parapsilosis ) 배양액의 타이로시나아제(Tyrosinase) 억제효과 조사Investigation of Tyrosinase Inhibition Effect of Culture Medium
칸디다 파라프실로시스(Candida parapsilosis) 배양액의 미백효과를 입증하기 위하여 피부 발색색소인 멜라닌을 형성하는 타이로시나아제(Tyrosinase)에 대한 억제효과를 측정하였다.In order to demonstrate the whitening effect of Candida parapsilosis culture, the inhibitory effect on tyrosinase forming melanin, a skin chromophore, was measured.
먼저 타이로시나아제(Tyrosinase)를 칼륨 완충용액에 용해하여 100unit/mL가 되도록 조정하고, 상기에서 얻은 타이로시나아제 용액 1mL를 상기 실시예 1에서 얻은 극성지질층 0.05mL와 혼합하여 37℃에서 10분간 전반응을 시킨다. 그리고 다시 0.01mL의 200mM L-티로신(L-Tyrosine) 용액을 더하여, 분광흡광광도계를 사용하여 437nm에서 10분동안의 흡광도 감소치를 측정하였다(표 1).First, Tyrosinase was dissolved in potassium buffer and adjusted to 100 units / mL, and 1 mL of the tyrosinase solution obtained above was mixed with 0.05 mL of the polar lipid layer obtained in Example 1, at 10 ° C at 37 ° C. Allow to react for a minute. Then, 0.01 mL of 200 mM L-tyrosine (L-Tyrosine) solution was further added, and the absorbance decrease for 10 minutes was measured at 437 nm using a spectrophotometer (Table 1).
그 결과 올리브 오일 단독으로도 약간의 타이로시나아제(tyrosinase) 억제효과를 기대할 수 있었지만, 칸디다 파라프실로시스(Candida parapsilosis)를 배양한 후의 얻은 배양액에서 월등히 우수한 타이로시나아제(tyrosinase) 억제효과를 얻을 수 있었다. 따라서, 칸디다 파라프실로시스(Candida parapsilosis) 배양액에 피부미백효과가 있는 유효물질이 포함되어 있다고 판단할 수 있었다.As a result, even a little tyrosinase inhibitory effect could be expected with olive oil alone, but the tyrosinase inhibitory effect was excellent in the culture obtained after the culture of Candida parapsilosis . Could get Therefore, it was determined that Candida parapsilosis culture contained an active substance having a skin whitening effect.
실험예 2: 칸디다 파라프실로시스(Experimental Example 2: Candida parapsissis ( Candida parapsilosisCandida parapsilosis )의 배양조건에 따른 유효물질 생산성 조사Investigation of Effective Material Productivity According to Culture Conditions
칸디다 파라프실로시스(Candida parapsilosis) 배양의 최적 조건 및 유효물질의 획득량과 유효성을 최대한 확보할 수 있는 범위를 정립하기 위하여 배양의 조건으로 사료되는 인자들 중 온도, pH, 미네랄의 종류 및 농도에 따른 유효물질의 획득률, 유효성 및 균체 회수량과의 관계를 실험하였다(표 3). 이때 균체의 생성량은 분광흡광광도계 파장 620nm 수준에서의 흡광도치를 미루어 탁도가 클수록 균체 생성이 많았다고 판정하였으며, 유효물질의 획득량은 HPLC(Agillent co. 1100 series, vwd detector, 190nm, μBONDAPACK C18, 1.5mL/min using acrtonitrile with water)에서 측정하였다(도 1 및 표 2 참조).In order to establish the optimum conditions of Candida parapsilosis culture and the range to obtain the maximum amount and the effective amount of the active substance, the temperature, pH, and the type and concentration of the factors among the factors considered as the culture conditions The relationship between the acquisition rate, effectiveness, and cell recovery of the active substance was tested (Table 3). At this time, the amount of cell production was determined by the absorbance value at the wavelength of 620 nm spectrophotometer, and the higher the turbidity was, the more the cell was produced.The amount of active material was obtained by HPLC (Agillent co. mL / min using acrtonitrile with water) (see FIG. 1 and Table 2).
상기 표 2의 결과를 미루어 판단하면, HPLC 분석을 통한 유효물질 중 미백효과를 기대하여 볼때, 분취물 5~7분대에서 타이로시나아제(Tyrosinase)의 억제효과가 뚜렷이 나타났음을 알 수 있었다. 따라서, 이후 유효물질 획득량의 판단의 기준으로 5~6분대에서의 HPLC 피크 영역(peak area)의 크기를 설정하였다.Judging from the results of Table 2, when the expectation of the whitening effect of the effective substance through HPLC analysis, it was found that the inhibitory effect of tyrosinase (Tyrosinase) in the aliquots 5 ~ 7 minutes appeared. Therefore, the size of the HPLC peak area in 5 to 6 minutes was set as a reference for determining the amount of active substance obtained.
상기 표 3의 결과에 미루어 보아, 배양온도는 균체 생성기는 37℃가 적합하나, 유효물질 생성에는 30℃가 적합하여 균체 생성기 4일간은 37℃에서의 배양을 하였고, 이후 유효물질 생성기 3일간은 30℃에서 배양하였다. 미네랄과의 관계는 Ca2+ 이온보다 Mg2+ 이온이 균체 생성 및 유효물질 획득에 있어서 유리하였으며, Mg2+ 이온의 량이 많으면 많을수록 균체생성량은 증가하나, 유효물질 획득량은 감소함을 알수 있었다. 따라서 유효물질 생성기에서는 옥살산(Oxalic acid)을 사용하여 Mg2+를 침전시킴으로서 개선의 효과를 기대하였다. 배양 pH는 중성범위에서 가장 큰 균체회수량 및 유효물질 생성을 가져왔으나, 각 군당 유의차는 보이지 않았다.In view of the results of Table 3, the culture temperature is suitable for the cell generator 37 ℃, 30 ℃ is suitable for the production of the active material 4 days of the cell generator was incubated at 37 ℃, then the active material generator 3 days Incubated at 30 ° C. Relationship with the mineral was unknown that the Ca 2+ ion than Mg 2+ ions are produced and the cells were advantageous to obtain effective substances, Mg 2+ ions the more the amount of large cell production is increased by one, the effective amount of reduced material obtained . Therefore, the effective substance generator was expected to improve by precipitating Mg 2+ using oxalic acid. The culture pH resulted in the largest cell recovery and effective substance generation in the neutral range, but no significant difference was found in each group.
이상 상기에서 살펴본 바와 같이 본 발명 칸디다 파라프실로시스 배양액을 유효성분으로 함유하는 피부 미백용 조성물은 칸디다 파라프실로시스(Candida parapsilosis)(기탁번호: KCCM 50271T)를 배양한 뒤 피부미백효과가 있는 유효물질을 포함하는 배양액을 분리 및 정제한 다음 상기에서 얻은 칸디다 파라프실로시스(Candida parapsilosis) 배양액의 타이로시나아제(Tyrosinase) 억제효과를 조사하고 칸디다 파라프실로시스(Candida parapsilosis)의 배양조건에 따른 유효물질 생산성을 조사함으로써 피부 발색색소인 멜라닌을 형성하는 인자로서의 타이로시나아제(Tyrosinase)의 활성을 효과적으로 억제하여 피부 미백효과 및 검버섯같은 피부 장애요소에 대한 개선 및 치료효과를 가지는 효과가 있으므로 화장품산업상 매우 유용한 발명인 것이다.As described above, the composition for skin whitening containing the present invention Candida parapsisylsis culture solution as an active ingredient has a skin whitening effect after culturing Candida parapsilosis (Accession No .: KCCM 50271T). After separating and purifying the culture medium containing the active substance, the Tyrosinase inhibitory effect of Candida parapsilosis culture obtained above was investigated and the culture conditions of Candida parapsilosis were examined. Investigate the productivity of active substances according to the effect of effectively inhibiting the activity of tyrosinase (Tyrosinase) as a factor to form melanin, a skin chromophore, thereby improving the skin whitening effect and the effect of having a therapeutic effect on skin disorders such as blotch Therefore, it is a very useful invention in the cosmetic industry.
도 1은 HPLC 분석을 통한 칸디다 파라프실로시스(Candida parapsilosis) 배양 유효물질의 분석 및 검정 결과를 나타내는 그래프이다.1 is a graph showing the results of analysis and assay of Candida parapsilosis culture active material through HPLC analysis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014163896A1 (en) | 2013-03-12 | 2014-10-09 | Avon Products, Inc | A topical lightening composition and methods of use thereof |
| CN115725428A (en) * | 2022-11-17 | 2023-03-03 | 陈盈如 | Candida parapsilosis strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014163896A1 (en) | 2013-03-12 | 2014-10-09 | Avon Products, Inc | A topical lightening composition and methods of use thereof |
| CN115725428A (en) * | 2022-11-17 | 2023-03-03 | 陈盈如 | Candida parapsilosis strain and application thereof |
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