KR102663013B1 - Composition for Skin Moisturizing, Skin Barrier, Elasticity and Improving Skin Itching Comprising Lactobacillus Complex Strain as Active Ingredient - Google Patents
Composition for Skin Moisturizing, Skin Barrier, Elasticity and Improving Skin Itching Comprising Lactobacillus Complex Strain as Active Ingredient Download PDFInfo
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
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- A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
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- A—HUMAN NECESSITIES
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- 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
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Abstract
본 발명은 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 2종 복합 균주, 그 배양액, 또는 이들의 추출물을 유효성분으로 포함하고, 피부 보습, 장벽 강화, 탄력 개선 및 가려움증 개선의 활성을 나타내는 조성물에 관한 기술이다.
본 발명의 조성물은 세포 독성이 없고, 피부 보습에 우수한 효과가 있으며, 피부 장벽 및 피부 탄력을 개선하고, 피부 가려움증 개선에 우수한 효과가 있으므로 화장품 및 식품 분야에서 유용하게 이용 가능하다. 또한, 본 발명의 조성물은 인체에 매우 안전할 뿐만 아니라, 안정성도 매우 탁월하다.The present invention includes two complex strains of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain, their cultures, or extracts thereof as active ingredients, and has the activities of moisturizing the skin, strengthening the barrier, improving elasticity, and improving itching. This is a description of a composition that represents.
The composition of the present invention has no cytotoxicity, has an excellent effect on skin moisturizing, improves skin barrier and skin elasticity, and has an excellent effect on improving skin itching, so it can be usefully used in the cosmetics and food fields. In addition, the composition of the present invention is not only very safe for the human body, but also has excellent stability.
Description
본 발명은 유산균 복합물을 유효성분으로 포함하는 조성물에 관한 것으로, 더욱 구체적으로 락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 2종 복합 균주, 그 배양액, 또는 이들의 복합추출물을 유효성분으로 포함하고, 피부 보습, 장벽 강화, 탄력 개선 및 가려움증 개선 활성을 나타내는 조성물에 관한 것이다. The present invention relates to a composition containing a lactic acid bacteria complex as an active ingredient, and more specifically, to two types of complex strains: Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP). , its culture, or a complex extract thereof as an active ingredient, and exhibits skin moisturizing, barrier strengthening, elasticity improvement, and itching improvement activities.
생물체의 항상성(homeostasis)을 유지하는 과정에서 중요한 역할을 담당하고 있는 물질 중에서 일부는 각종 생물체 유래의 생리 활성 물질이다. 지금까지 수많은 생리 활성 물질에 관해 많은 연구가 진행되고 있으며, 그 중, 미생물, 식물, 또는 동물체 등에서 분리된 생리 활성을 갖는 물질에 관한 연구는 생명과학 및 의학 분야에서 매우 중요한 영역이다.Among the substances that play an important role in the process of maintaining homeostasis of living organisms, some are physiologically active substances derived from various living organisms. To date, much research has been conducted on numerous biologically active substances, and among them, research on substances with biological activity isolated from microorganisms, plants, or animals is a very important area in the fields of life science and medicine.
피부 주름 개선, 미백, 항산화 등에 효과가 있는 것으로 알려진 물질로, 종래 개발된 비타민C, 레티노익산, 형질전환생장인자(transforming growth factor, TGF) 등이 있다. 그러나, 비타민C, 레티노익산, 형질전환 생장인자(transforming growth factor, TGF), 동물태반 유래의 단백질(JP8-231370), 베툴린산(betulinic acid, JP8-208424), 클로렐라 추출물(JP9-40523, JP10-36283, 섬유아세포 증식 촉진작용) 등의 소재들은 피부에 적용할 때 발생하는 자극과 발적 등의 안전성 문제로 사용량에 제한이 있거나, 그 효과가 미미하여 실질적으로 피부 탄력 및 주름, 미백 등의 개선에 효과를 기대할 수 없다는 문제점이 있었다.Substances known to be effective in improving skin wrinkles, whitening, and anti-oxidation include previously developed vitamin C, retinoic acid, and transforming growth factor (TGF). However, vitamin C, retinoic acid, transforming growth factor (TGF), protein derived from animal placenta (JP8-231370), betulinic acid (JP8-208424), chlorella extract (JP9-40523, JP10) Materials such as -36283, which promotes fibroblast proliferation) have limited usage due to safety issues such as irritation and redness that occur when applied to the skin, or their effects are minimal, so they are not effective in improving skin elasticity, wrinkles, and whitening. There was a problem that no effect could be expected.
또한, 최근에는 천연물 등으로부터 얻은 생리 활성 물질을 화장료에 도입하여 피부 자체의 고유 방어 기능을 더욱 강화함으로써 피부에 작용하는 기능성을 증가시키는 화장품의 개발이 활발하게 진행되는 추세이며, 생체에 안전하고, 유효성분이 안정하며, 무엇보다도 기존의 피부 주름 개선, 보습, 및 피부 재생 효과가 있는 물질보다 효과가 더욱 우수한 천연소재의 개발이 절실히 요망되고 있다.In addition, in recent years, there has been an active trend in the development of cosmetics that increase the functionality of the skin by incorporating physiologically active substances obtained from natural products into cosmetics to further strengthen the skin's own defense function, and are safe for the living body. There is an urgent need to develop natural materials that have stable active ingredients and, above all, are more effective than existing materials that improve skin wrinkles, moisturize, and regenerate the skin.
한편, 화장품 소재는 안전성, 효능, 물성 등이 균형을 이루어야 한다. 또한, 경제성 확보도 중요한 요인으로서 화장품용 소재는 다양한 개발 방법, 천연물 추출과 미생물 등에 의한 발효를 이용하여 피부 투과, 노화 억제, 생리활성, 안전성 및 물성을 최적화하여 개발하게 된다. 화장품과 식품 및 의약품의 경계가 무너지면서 화장품의 안정성과 의약품, 건강기능식품 등의 효과성을 겸비한 복합 기능성 원료 및 바이오 소재를 통해 다양한 코스메슈티컬 제품들이 높은 성장세를 이어가고 있다.Meanwhile, cosmetic materials must have a balance of safety, efficacy, and physical properties. In addition, securing economic efficiency is an important factor, and cosmetic materials are developed by optimizing skin penetration, anti-aging, physiological activity, safety, and physical properties using various development methods, natural product extraction, and fermentation by microorganisms. As the boundaries between cosmetics, food, and pharmaceuticals are collapsing, various cosmeceutical products are continuing to grow rapidly through complex functional raw materials and biomaterials that combine the safety of cosmetics and the effectiveness of pharmaceuticals and health functional foods.
이러한 배경 아래에서, 본 발명자들은 피부 주름 개선, 보습 등 피부 상태 개선에 우수한 효과를 나타내는 유산균 소재들의 조합에 관하여 연구를 수행하였으며, 다양한 피부 상태 개선 효과를 나타내는 유산균의 조합을 찾기 위해 예의 연구노력한 결과, 전통 장류에서 분리 동정된 2종 균주의 조합이 피부 주름 개선, 보습, 피부 장벽 강화 및 피부 상처 재생에 우수한 활성을 나타내어 피부 상태 개선의 목적으로도 유용하게 활용될 수 있음을 확인하고, 본 발명을 완성하게 되었다.Under this background, the present inventors conducted research on the combination of lactic acid bacteria materials that show excellent effects in improving skin conditions, such as improving skin wrinkles and moisturizing, and as a result of diligent research efforts to find combinations of lactic acid bacteria that show various skin condition improvement effects. , it was confirmed that the combination of two strains isolated and identified from traditional soybeans showed excellent activity in improving skin wrinkles, moisturizing, strengthening the skin barrier, and regenerating skin wounds, and could be usefully used for the purpose of improving skin condition, and the present invention was completed.
따라서, 본 발명의 주된 목적은 피부 보습, 장벽 강화, 탄력 개선 및 가려움증 개선 활성을 나타내는 락토코커스 락티스 Nabysol07(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07(기탁번호 KCTC 15001BP)의 2종 균주, 그 배양액, 또는 이들의 추출물을 유효성분으로 포함하는 조성물을 제공하는 데 있다.Therefore, the main object of the present invention is to produce two strains of Lactococcus lactis Nabysol07 (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 (accession number KCTC 15001BP), which exhibit skin moisturizing, barrier strengthening, elasticity improvement, and itching-improving activities. , the culture medium, or extracts thereof as active ingredients.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.
본 발명의 한 양태에 따르면, 본 발명은 락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 2종 복합 균주, 그 배양액, 또는 이들의 복합추출물을 유효성분으로 포함하는 화장료 조성물을 제공한다.According to one aspect of the present invention, the present invention provides two complex strains of Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP), a culture medium thereof, or a combination thereof. Provided is a cosmetic composition containing an extract as an active ingredient.
본 발명자들은 여러 가지 천연물질들을 대상으로 피부상태 개선의 효과를 탐색하였고, 유산균 소재들의 피부 상태 개선 효과에 주목하여 우수한 유산균 소재들의 조합을 발굴하고자 연구노력하였다. 그 결과, 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 2종 복합 균주 배양액 및 이의 추출물이 피부 보습 및 피부 탄력 개선에 시너지 효과를 나타내는 동시에 피부 장벽 강화 및 피부 가려움증 개선에도 우수한 효과가 있음을 입증하였으며, 피부에 적용 시에도 안전성에 문제가 없음을 확인하고 본 발명을 완성하였다.The present inventors explored the effect of various natural substances on improving skin condition, and focused on the effect of lactic acid bacteria materials on improving skin condition, and made research efforts to discover combinations of excellent lactic acid bacteria materials. As a result, the two complex strain cultures of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain and their extracts showed a synergistic effect in moisturizing the skin and improving skin elasticity, while also having excellent effects in strengthening the skin barrier and improving skin itching. It was confirmed that there was no safety problem even when applied to the skin, and the present invention was completed.
또한, 상기 락토코커스 락티스(Lactococcus lactis) Nabysol07 균주는 부다페스트 조약에 따른 국제기탁기관인 한국생명공학연구원(KCTC)에 국제기탁하여 수탁번호 KCTC 15002BP를 부여받았고, 상기 스트렙토코커스 써모필러스(Streptococcus thermophilus) Nabysol07 균주는 부다페스트 조약 하의 국제기탁기관인 한국생명공학연구원(KCTC)에 국제기탁하여 수탁번호 KCTC 15001BP를 부여받았다. In addition, the Lactococcus lactis Nabysol07 strain was internationally deposited with the Korea Research Institute of Bioscience and Biotechnology (KCTC), an international depositary organization under the Budapest Treaty, and was given the accession number KCTC 15002BP, and the Streptococcus thermophilus The Nabysol07 strain was internationally deposited at the Korea Research Institute of Bioscience and Biotechnology (KCTC), an international depository under the Budapest Treaty, and was given the accession number KCTC 15001BP.
본 발명에서 사용되는 용어 "추출물"은 유산균 배양액의 추출 처리에 따라 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. 본 발명의 상기 추출물은 바람직하게 추출 후 건조 분말 형태로 제조되어 사용될 수 있다. 상기 2종 복합 균주의 배양액 추출에 있어서, 상기 추출물을 추출하는 방법은 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 상기 추출방법의 비제한적인 예로는, 열수 추출법, 초음파 추출법, 여과법, 환류 추출법 등을 들 수 있으며, 이들은 단독으로 수행되거나 2종 이상의 추출방법을 병용하여 수행될 수 있다.The term "extract" used in the present invention refers to an extract obtained by extraction treatment of a lactic acid bacteria culture, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, or a mixture thereof, etc. This includes the extract itself and all formulations of the extract that can be formed using the extract. The extract of the present invention can preferably be prepared and used in the form of a dry powder after extraction. In extracting the culture fluid of the two complex strains, the method of extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, which may be performed alone or by combining two or more extraction methods.
본 발명에서 상기 2종 복합 균주의 배양액을 추출하는데 사용되는 추출 용매의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 추출 용매의 비제한적인 예로는 물; 메탄올, 에탄올, 프로필알코올, 부틸알코올 등의 C1 내지 C4의 저급 알코올; 글리세린, 부틸렌글리콜, 프로필렌글리콜 등의 다가 알코올; 및 메틸아세테이트, 에틸아세테이트, 아세톤, 벤젠, 헥산, 디에틸에테르, 디클로로메탄 등의 탄화수소계 용매; 또는 이들의 혼합물을 사용할 수 있으며, 바람직하게, 물, 저급알코올, 1,3-부틸렌글리콜, 에틸아세테이트를 단독으로 사용하거나 2종 이상 혼합하여 사용할 수 있다.In the present invention, the type of extraction solvent used to extract the culture medium of the two complex strains is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include water; C 1 to C 4 lower alcohols such as methanol, ethanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof can be used, and preferably, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate can be used alone or in a mixture of two or more.
본 발명에서 열수 추출 또는 냉침 추출한 추출물은 부유하는 고체 입자를 제거하기 위해 여과, 예를 들어 나일론 등을 이용해 입자를 걸러내거나 냉동여과법 등을 이용해 여과한 후, 그대로 사용하거나 이를 동결건조, 열풍건조, 분무건조 등을 이용해 건조하여 사용할 수 있다.In the present invention, the extract obtained by hot water extraction or cold immersion extraction is filtered to remove floating solid particles, for example, by filtering the particles using nylon, etc., or by cryofiltration, and then used as is or freeze-dried, hot-air dried, etc. It can be dried and used using spray drying, etc.
또한, 본 발명의 화장료 조성물은 히알루론산 합성을 촉진하여 피부 보습 활성을 나타내는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 상기 본 발명의 2종 유산균 배양액 추출물이 히알루론산(Hyaluronan)의 생성량을 농도 의존적으로 증가시키는 양상을 확인할 수 있었고, 각각의 단일 균주의 배양액 추출물에 비해 본 발명의 2종 유산균 배양액의 복합추출물이 피부 보습에 매우 우수한 시너지 효과가 있음을 확인할 수 있었다(도 2 참조). In addition, the cosmetic composition of the present invention is characterized by promoting hyaluronic acid synthesis and exhibiting skin moisturizing activity. In a preferred embodiment of the present invention, it was confirmed that the two types of lactic acid bacteria culture extracts of the present invention increase the amount of hyaluronic acid (Hyaluronan) produced in a concentration-dependent manner, and compared to the culture extracts of each single strain, the two types of lactic acid bacteria culture medium extracts of the present invention It was confirmed that the complex extract of the lactic acid bacteria culture medium had an excellent synergistic effect on skin moisturization (see Figure 2).
또한, 본 발명의 화장료 조성물은 필라그린 유전자 발현을 증가시켜 피부 장벽 강화 활성을 갖는 것을 특징으로 한다. 필라그린은 표피 과립층에서 케라토히알린과립(keratohyalin granule)을 형성하는 단백질인 프로필라그린(profilaggrin)의 형태로 존재하다가, 각질형성세포의 최종분화과정에서 필라그린으로 분해된 후에 각질세포막을 형성할 때 케라틴 필라멘트를 응집하여 각질세포의 단단하고 편평한 구조를 형성함으로써 피부 장벽에서 벽돌(brick)의 역할을 한다. 본 발명의 바람직한 실시예에서는 본 발명의 2종 균주 배양액 추출물의 처리에 따라 필라그린(filaggrin)의 mRNA 발현량이 현저하게 증가하는 것을 확인할 수 있었다(도 3 참조).In addition, the cosmetic composition of the present invention is characterized by having skin barrier strengthening activity by increasing filaggrin gene expression. Filaggrin exists in the form of profilaggrin, a protein that forms keratohyalin granules in the granular layer of the epidermis, and is decomposed into filaggrin during the final differentiation of keratinocytes to form keratinocyte membranes. When keratin filaments are aggregated to form a hard, flat structure of keratinocytes, they play the role of a brick in the skin barrier. In a preferred embodiment of the present invention, it was confirmed that the mRNA expression level of filaggrin significantly increased upon treatment of the culture extract of the two strains of the present invention (see Figure 3).
본 발명의 화장료 조성물은 MMP-1(matrix metalloproteinase-1) 유전자의 발현을 억제하고, 타입 1 프로콜라겐 유전자의 발현을 증가시켜 피부 탄력 개선 활성을 나타내는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 본 발명의 2종 균주 배양액 추출물 처리에 의해서 MMP-1 단백질의 발현량이 농도 의존적으로 감소하는 것을 확인할 수 있었고(도 4 참조) 타입 1 프로콜라겐 유전자의 발현량이 농도 의존적으로 증가하는 것을 확인할 수 있었다(도 5 참조).The cosmetic composition of the present invention is characterized by exhibiting skin elasticity improvement activity by suppressing the expression of the MMP-1 (matrix metalloproteinase-1) gene and increasing the expression of the type 1 procollagen gene. In a preferred embodiment of the present invention, it was confirmed that the expression level of MMP-1 protein decreased in a concentration-dependent manner by treatment with the culture medium extract of the two strains of the present invention (see Figure 4), and the expression level of the type 1 procollagen gene decreased in a concentration-dependent manner. An increase was confirmed (see Figure 5).
또한, 본 발명의 화장료 조성물은 TSLP(Thymic stromal lymphopoietin) 생성을 억제하여 아토피 피부염 또는 피부 가려움증 개선 활성을 나타내는 것을 특징으로 한다. 본 발명에서 용어, "아토피 피부염"은 만성적이고 재발성의 염증성 피부질환으로 가려움증(가려움증)과 피부건조증, 특징적인 홍반 또는 습진을 동반하는 질병을 의미한다. 아토피성 피부염의 급성 병소에서는 혈청 면역글로불린 E(IgE)의 유의한 증가 현상이 특징으로 나타나며, 이에 부가하여 병소의 조직병리학적인 변화 및 피부염 병변에 대한 평가 등을 행하여 아토피성 피부염의 진단 및 심각도를 판정하기도 한다. 아토피성 피부염의 정확한 원인은 아직까지 완전히 이해되고 있지 않지만 유전적 소인과 함께 면역학적, 비면역학적 기전이 관여한다고 보고 있다. In addition, the cosmetic composition of the present invention is characterized by suppressing the production of TSLP (Thymic stromal lymphopoietin), thereby improving atopic dermatitis or skin itching. In the present invention, the term "atopic dermatitis" refers to a chronic, recurrent inflammatory skin disease accompanied by itching, dry skin, and characteristic erythema or eczema. Acute lesions of atopic dermatitis are characterized by a significant increase in serum immunoglobulin E (IgE), and in addition, histopathological changes in the lesion and evaluation of dermatitis lesions are performed to diagnose and severity of atopic dermatitis. It is also judged. The exact cause of atopic dermatitis is not yet fully understood, but it is believed that immunological and non-immunological mechanisms, along with genetic predisposition, are involved.
또한, 용어 "피부 가려움증"은 피부를 긁거나 문지르고 싶은 욕망을 일으키는 불쾌한 감각을 의미하며, 하나의 예시로서, 피부건조에 의한 가려움증; 두드러기에 의한 가려움증; 벌레물림에 의한 가려움증; 땀띠, 여드름, 짓무름, 동상, 접촉성 피부염, 지루성 피부염 또는 건선 등으로 유발되는 가려움증; 각종 피부질환에 의한 가려움증; 두피 가려움증 등을 들 수 있다. 그러나, 본 발명에서는 이상에서 열거한 피부 가려움증 이외의 다양한 원인의 피부 가려움증을 배제하는 것이 아님은 물론이다. 본 발명은 당업계에 알려진 모든 가려움증, 예를 들어 피부과적 원인 가려움증, 전신성 원인 가려움증, 신경병증성 원인 가려움증 및 심인성 원인 가려움증 등을 포함한다.Additionally, the term "skin itching" refers to an unpleasant sensation that causes a desire to scratch or rub the skin, and examples include itching due to dry skin; Itching caused by hives; Itching caused by insect bites; Itching caused by heat rash, acne, ulcers, frostbite, contact dermatitis, seborrheic dermatitis, or psoriasis; Itching due to various skin diseases; Examples include scalp itching. However, it goes without saying that the present invention does not exclude various causes of skin itching other than the skin itching listed above. The present invention includes all itch known in the art, such as itch of dermatological etiology, itch of systemic etiology, itch of neuropathic etiology and itch of psychogenic etiology.
현재까지의 연구 결과에 따르면, 가려움증 매개물질이 여러 피부질환에서 가려움증을 유발하는 것으로 알려져 있는데, 면역세포(T 세포, 대식세포 등)로부터 분비된 여러 사이토카인 및 히스타민이 가려움증을 일으키는 것으로 알려져 있다. 또한, 최근에는 TSLP(Thymic stromal lymphopoietin)가 피부염의 중증도와 연관되어 있고, 가려움 증세의 원인이라고 알려져 있다(Wilson SR et al., The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell. 2013;155(2):285-95).According to research results to date, itching mediators are known to cause itching in various skin diseases, and various cytokines and histamines secreted from immune cells (T cells, macrophages, etc.) are known to cause itching. Additionally, it has recently been known that TSLP (Thymic stromal lymphopoietin) is associated with the severity of dermatitis and is the cause of itching symptoms (Wilson SR et al., The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell. 2013;155(2):285-95).
본 발명의 바람직한 실시예에서는 본 발명의 2종 균주 배양액 추출물 처리군은 시료 무처리군에 비해 TSLP mRNA 발현량이 각각 18%(50μg/mL), 33%(100μg/mL) 및 51%(250μg/mL) 감소한 것으로 관찰되어 단일추출물에 비해 TLSP 발현이 현저하게 감소된 것을 확인할 수 있었고, 2종 균주 조합에 따른 시너지 효과를 확인할 수 있었다(도 6 참조).In a preferred embodiment of the present invention, the group treated with the culture fluid extract of the two strains of the present invention had TSLP mRNA expression levels of 18% (50 μg/mL), 33% (100 μg/mL), and 51% (250 μg/mL), respectively, compared to the untreated sample group. mL) was observed to decrease, confirming that TLSP expression was significantly reduced compared to the single extract, and a synergistic effect according to the combination of the two strains was confirmed (see Figure 6).
본 발명의 화장료 조성물은 식용 가능 유산균 소재에서 유래하여 인체에 해가 없으며, 그 첨가로 인해 화장품의 물질의 품질을 손상하지 않으므로 화장료 조성물에 포함되어 사용되는데 적합하다. 특히, 피부 보습, 장벽 강화, 탄력 개선 및 가려움증 개선과 관련된 기능이 있으므로, 기능성 화장료 조성물에 포함되어 사용되는 것이 바람직하다.The cosmetic composition of the present invention is derived from edible lactic acid bacteria material, is not harmful to the human body, and its addition does not damage the quality of the cosmetic material, so it is suitable for use in a cosmetic composition. In particular, since it has functions related to skin moisturizing, strengthening the barrier, improving elasticity, and improving itching, it is preferable to be included and used in a functional cosmetic composition.
본 발명의 화장료 조성물은 스킨로션, 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐 로션, 영양로션, 맛사지크림, 영양크림, 모이스처크림, 핸드크림, 파운데이션, 에센스, 영양에센스, 마스크팩, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션 및 바디클린저에서 선택된 어느 하나의 제형으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, foundation, essence, nutritional essence, mask pack, It can be manufactured in any one formulation selected from soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto.
본 발명의 상기 화장료 조성물은 일반 피부 화장료에 배합되는 화장품학적으로 허용 가능한 담체를 1종 이상 추가로 포함할 수 있으며, 통상의 성분으로 예를 들면 유분, 물, 계면 활성제, 보습제, 저급 알코올, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 적절히 배합할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers that are blended with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, and thickener. , chelating agents, pigments, preservatives, fragrances, etc. may be appropriately mixed, but are not limited thereto.
본 발명의 화장료 조성물에 포함되는 화장품학적으로 허용 가능한 담체는 화장료 조성물의 제형에 따라 다양하다.Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.
본 발명의 제형이 연고, 페이스트, 크림 또는 젤인 경우에는, 담체 성분으로서 동물성 유, 식물성 유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화아연 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier ingredients. may be used, but is not limited thereto. These may be used alone or in combination of two or more types.
본 발명의 제형이 파우더 또는 스프레이인 경우에는, 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록사이드, 칼슘 실케이트, 폴리아미드 파우더 등이 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로하드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진제를 포함할 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, etc. may be used as carrier ingredients, and especially in the case of a spray, chlorofluorohydride may be additionally used. It may include, but is not limited to, propellants such as low carbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more types.
본 발명의 제형이 용액 또는 유탁액인 경우에는, 담체 성분으로서 용매, 용해화제 또는 유탁화제 등이 이용될 수 있으며, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일 등이 이용될 수 있고, 특히, 목화씨 오일, 땅콩 오일, 옥수수 배종 오일, 올리브 오일, 피마자 오일 및 참깨 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent may be 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, etc. can be used, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan. may be used, but is not limited thereto. These may be used alone or in combination of two or more types.
본 발명의 제형이 현탁액인 경우에는, 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타하이드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a suspension, the carrier components include water, liquid diluents such as ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used, but are not limited thereto. These may be used alone or in combination of two or more types.
본 발명의 제형이 비누인 경우에는, 담체 성분으로서 지방산의 알칼리 금속 염, 지방산 헤미에스테르 염, 지방산 단백질 히드롤리제이트, 이세티오네이트, 라놀린 유도체, 지방족 알코올, 식물성 유, 글리세롤, 당 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is soap, alkali metal salts of fatty acids, hemiester salts of fatty acids, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. are used as carrier components. It may be, but is not limited to this. These may be used alone or in combination of two or more types.
본 발명의 화장료 조성물에서, 상기 락토코커스 락티스(Lactococcus lactis) Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스(Streptococcus thermophilus) Nabysol07 균주(기탁번호 KCTC 15001BP)의 2종 균주, 배양액, 또는 이의 복합추출물은 구체적으로는 건조 중량으로, 화장료 조성물 총 중량의 0.0001 중량% 내지 50 중량%로 포함될 수 있으며, 보다 구체적으로는 0.0005 중량% 내지 10 중량%로 포함될 수 있다. 상기 범위 내에서 피부 주름 개선, 보습, 피부 장벽 강화 및 피부 상처 재생 촉진에 대한 우수한 효능을 나타내는 이점이 있으며, 조성물의 제형이 안정화되는 이점이 있다.In the cosmetic composition of the present invention, two strains of the Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP), culture medium, or Specifically, the complex extract may be included in a dry weight of 0.0001% to 50% by weight of the total weight of the cosmetic composition, and more specifically, may be included in an amount of 0.0005% to 10% by weight. Within the above range, it has the advantage of showing excellent efficacy in improving skin wrinkles, moisturizing, strengthening the skin barrier, and promoting skin wound regeneration, and has the advantage of stabilizing the formulation of the composition.
본 발명의 다른 양태에 따르면, 본 발명은 락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 2종 복합 균주, 그 배양액, 또는 이들의 복합추출물을 유효성분으로 포함하는 식품 조성물을 제공한다.According to another aspect of the present invention, the present invention provides two complex strains of Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP), a culture medium thereof, or a combination thereof. Provided is a food composition containing an extract as an active ingredient.
본 발명의 락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 2종 복합 균주, 그 배양액, 또는 이들의 추출물은 피부 보습, 장벽 강화, 탄력 개선 및 가려움증 개선에 우수한 효과를 나타내어 피부 상태 개선을 위한 식품 조성물로 유용하게 사용될 수 있다. 본 발명의 2종 복합 균주와 피부 보습, 장벽 강화, 탄력 개선 및 가려움증 개선에 대한 효과는 상기에서 설명한 바와 같다. 상기 식품 조성물은 건강기능식품의 형태로 사용될 수 있으나, 이에 제한되는 것은 아니다. The two complex strains of Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP) of the present invention, their cultures, or their extracts have skin moisturizing, barrier strengthening, and elasticity properties. It has excellent effects on improving skin condition and itching, so it can be usefully used as a food composition for improving skin condition. The effects of the two complex strains of the present invention on skin moisturization, barrier strengthening, elasticity improvement, and itching improvement are as described above. The food composition may be used in the form of a health functional food, but is not limited thereto.
본 발명의 식품 조성물은 상기 2종 복합 균주, 배양액, 또는 그 추출물과 이들의 가공물의 형태로 포함될 수 있다. 또한, 상기 조성물은 유효성분 이외에 식품학적으로 허용 가능한 식품 보조첨가제를 포함할 수 있다.The food composition of the present invention may be included in the form of the two types of complex strains, cultures, or extracts thereof and processed products thereof. Additionally, the composition may include food additives that are foodologically acceptable in addition to the active ingredients.
본 발명에 있어서, "식품 보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품 보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품 보조첨가제의 종류가 제한되는 것은 아니다.In the present invention, "food auxiliary additive" refers to a component that can be added to food as an auxiliary ingredient, and can be appropriately selected and used by a person skilled in the art as it is added to manufacture each type of health functional food. Examples of food auxiliary additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplements of the present invention are not limited to the above examples.
본 발명의 식품 조성물에는 건강기능식품이 포함될 수 있다. 본 발명에 있어서, "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법으로 제조 가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. The food composition of the present invention may include health functional foods. In the present invention, “health functional food” refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functional properties useful to the human body. Here, “functionality” means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and components commonly added in the art.
또한, 상기 건강기능식품의 제형 또한 건강기능식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 본 발명의 식품 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식용 유산균을 원료로 하여 식품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나며, 피부 주름 개선, 보습, 피부 장벽 강화 및 피부 상처 재생 효과를 증진시키기 위한 보조제로 섭취할 수 있다.Additionally, the formulation of the health functional food can also be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various types of formulations, and unlike general drugs, it is made of edible lactic acid bacteria as a raw material, so it has the advantage of having no side effects that may occur when taking food for a long time, is excellent in portability, and reduces skin wrinkles. It can be taken as a supplement to improve skin texture, moisturize, strengthen the skin barrier, and improve skin wound regeneration.
본 발명의 건강기능식품이 취할 수 있는 형태에는 제한이 없으며, 통상적인 의미의 식품을 모두 포함할 수 있고, 기능성 식품 등 당업계에 알려진 용어와 혼용하여 사용할 수 있다. 아울러 본 발명의 건강기능식품은 당업자의 선택에 따라 식품에 포함될 수 있는 적절한 기타 보조 성분과 공지의 첨가제를 혼합하여 제조할 수 있다. 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 본 발명에 따른 2종 복합 균주, 그 배양액, 또는 이들의 추출물을 주성분으로 하여 제조한 즙, 차, 젤리 및 주스 등에 첨가하여 제조할 수 있다. 또한, 동물을 위한 사료로 이용되는 식품도 포함된다.There is no limit to the form that the health functional food of the present invention can take, and it can include all foods in the conventional sense, and can be used interchangeably with terms known in the art, such as functional food. In addition, the health functional food of the present invention can be manufactured by mixing known additives with other appropriate auxiliary ingredients that can be included in the food according to the selection of a person skilled in the art. Examples of foods that can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes, etc., and they can be prepared by adding the two complex strains according to the present invention, their cultures, or their extracts to juices, teas, jellies, juices, etc. prepared as main ingredients. It also includes foods used as feed for animals.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(1) 본 발명은 피부 보습, 장벽 강화, 탄력 개선 및 가려움증 개선의 활성을 나타내는 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 2종 복합 균주, 그 배양액, 또는 이들의 추출물을 유효성분으로 포함하는 조성물을 제공한다.(1) The present invention uses two complex strains of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain, cultures thereof, or extracts thereof, which exhibit activities of skin moisturizing, barrier strengthening, elasticity improvement, and itching improvement. Provided is a composition comprising the ingredients.
(2) 본 발명의 조성물은 세포 독성이 없고, 피부 보습에 우수한 효과가 있으며, 피부 장벽을 강화하고, 피부 탄력을 개선하고, 피부 가려움증 개선에 우수한 효과가 있으므로 화장품 및 식품 분야에서 유용하게 이용 가능하다.(2) The composition of the present invention has no cytotoxicity, has an excellent effect on skin moisturizing, strengthens the skin barrier, improves skin elasticity, and has an excellent effect on improving skin itching, so it can be usefully used in the cosmetics and food fields. do.
(3) 또한, 본 발명의 조성물은 인체에 매우 안전할 뿐만 아니라, 안정성도 매우 탁월하다.(3) In addition, the composition of the present invention is not only very safe for the human body, but also has excellent stability.
도 1은 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주 배양액의 단일 또는 이들의 복합 추출물(Dr. Navy) 처리에 따른 인간피부섬유아세포(NHDF)의 세포 생존율을 측정하여 나타낸 그래프이다.
도 2는 인간각질형성세포(HaCaT)에 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주 배양액의 단일 또는 이들의 복합 추출물(Dr. Navy)을 처리한 후, 히알루론산(Hyaluronan) 합성량 변화를 측정하여 나타낸 그래프이다.
도 3은 인간각질형성세포(HaCaT)에 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주 배양액의 단일 또는 이들의 복합 추출물(Dr. Navy)을 처리한 후, 프로필라그린 mRNA 발현 변화를 측정하여 나타낸 그래프이다.
도 4는 인간피부섬유아세포(NHDF)에 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주 배양액의 단일 또는 이들의 복합 추출물(Dr. Navy)을 처리한 후, MMP-1(matrix metalloproteinase-1) 유전자의 발현 변화를 측정하여 나타낸 그래프이다.
도 5는 인간피부섬유아세포(NHDF)에 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주 배양액의 단일 또는 이들의 복합 추출물(Dr. Navy)을 처리한 후, 타입 1 프로콜라겐 유전자의 발현 변화를 측정하여 나타낸 그래프이다.
도 6은 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주 배양액의 단일 또는 이들의 복합 추출물(Dr. Navy) 처리에 따른 TSLP 생성량 변화를 측정하여 나타낸 그래프이다.Figure 1 is a graph showing the cell survival rate of human dermal fibroblasts (NHDF) measured according to treatment with single or combined extracts (Dr. Navy) of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain culture medium.
Figure 2 shows the amount of hyaluronic acid (Hyaluronan) synthesized after treating human keratinocytes (HaCaT) with single or combined extracts (Dr. Navy) of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain culture medium. This is a graph measuring changes.
Figure 3 shows changes in profilaggrin mRNA expression after treating human keratinocytes (HaCaT) with single or combined extracts (Dr. Navy) of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain culture medium. This is a graph shown by measurement.
Figure 4 shows that after treating human skin fibroblasts (NHDF) with single or combined extracts (Dr. Navy) of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain culture medium, MMP-1 (matrix metalloproteinase- 1) This is a graph showing changes in gene expression.
Figure 5 shows the expression of type 1 procollagen gene after treating human dermal fibroblasts (NHDF) with single or combined extracts (Dr. Navy) of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain culture medium. This is a graph measuring changes.
Figure 6 is a graph showing the change in TSLP production according to treatment of single or combined extracts (Dr. Navy) of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain culture medium.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.
실시예 1. 균주 선발 및 추출Example 1. Strain selection and extraction
1-1. 균주 선발1-1. Strain selection
전통 장류에서 균주를 채취하고, 멸균 식염수를 사용하여 희석한 후, BCP 아가(agar)에 도말하여 배지가 노란색으로 변한 콜로니를 채취하고, MRS 배지에 단독 배양하였다. 분리된 균주의 동정을 위해 16S rRNA 염기서열 분석을 통해 분리된 균주들이 공시균주인 락토코커스 락티스(Lactococcus lactis) NBRC 100933, 스트렙토코커스 써모필러스(Streptococcus thermophilus) PNGR-K13 균주와 99% 이상의 상동성을 나타내는 것을 확인하였다. 상기 분리된 2종 균주를 각각 락토코커스 락티스(Lactococcus lactis) Nabysol07 및 스트렙토코커스 써모필러스(Streptococcus thermophilus) Nabysol07 균주로 명명하였고, 한국생명공학연구원 생물자원센터(KCTC)에 수탁하여 각각 KCTC 15002BP 및 KCTC 15001BP의 기탁번호를 얻었다.Strains were collected from traditional soybean paste, diluted using sterile saline solution, and then spread on BCP agar. Colonies that turned yellow were collected and cultured alone on MRS medium. To identify the isolated strains, 16S rRNA base sequence analysis showed that the isolated strains were more than 99% identical to the reference strains Lactococcus lactis NBRC 100933 and Streptococcus thermophilus PNGR-K13. It was confirmed that it shows the same sex. The two isolated strains were named Lactococcus lactis Nabysol07 and Streptococcus thermophilus Nabysol07 strains, respectively, and were deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC), respectively, as KCTC 15002BP and The deposit number of KCTC 15001BP was obtained.
1-2. 균주 추출물의 제조1-2. Preparation of strain extract
MRS 브로스(Difco) 100mL에 락토코커스 락티스(Lactococcus lactis) Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스(Streptococcus thermophilus) Nabysol07 균주(기탁번호 KCTC 15001BP)를 각각 접종하고, 37℃ 항온기에서 OD 600nm에서 1.0이 될 때까지 배양하여 배양물을 획득하였다. 배양액 10㎖를 4℃에서 4,000rpm으로 20분간 원심분리하여 상층액만을 모았다. 이렇게 얻은 각각의 균주 배양액을 동결건조하여 분말 형태의 락토코커스 락티스(Lactococcus lactis) Nabysol07 균주 배양액 추출물(제조예 1) 및 스트렙토코커스 써모필러스(Streptococcus thermophilus) Nabysol07 균주 배양액 추출물(제조예 2)을 수득하였다. Inoculate 100 mL of MRS broth (Difco) with Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP), respectively, in a thermostat at 37°C. The culture was obtained by culturing at OD 600nm until it reached 1.0. 10 ml of culture medium was centrifuged at 4,000 rpm for 20 minutes at 4°C, and only the supernatant was collected. Each strain culture thus obtained was freeze-dried to produce powdered Lactococcus lactis Nabysol07 strain culture extract (Preparation Example 1) and Streptococcus thermophilus Nabysol07 strain culture extract (Preparation Example 2). Obtained.
또한, 상기 2종 균주의 추출물을 동량으로 혼합하여 2종 복합 균주 추출물을 제조하였다(제조예 3).In addition, extracts of the two types of complex strains were prepared by mixing equal amounts of extracts of the two types of strains (Preparation Example 3).
실시예 2. 세포독성 시험(MTT Assay)을 통한 세포 보호 효과 확인Example 2. Confirmation of cell protection effect through cytotoxicity test (MTT Assay)
실시예 1에서 준비한 락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 단일추출물과 이들의 복합추출물이 갖는 인간피부섬유아세포(human dermal fibroblasts, NHDFs)에 대한 세포 독성을 시험하였다. Single extracts of the Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP) prepared in Example 1 and their complex extracts contained human dermal fibroblasts. The cytotoxicity of NHDFs was tested.
세포독성은 MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] 비색정량분석법(colorimetric assay)을 사용하여 측정하였다. MTT(Methyl thiazol tetrazolium) 분석은 세포 생존 능력을 측정하기 위해 사용되는 것으로, MTT가 보라색을 생성하는 포마잔 염색으로 바꾸는 것이다. 인큐베이션 72시간 후, 배지의 부피는 1㎖로 감소하였고, 1㎎/㎖ MTT의 100㎕는 각 벽에 첨가되었다. 이후, 인간피부섬유아세포(human dermal fibroblasts, NHDFs)를 37℃에서 2시간 동안 5%의 CO2 및 95%의 O2 존재 하에 배양하였다. Cytotoxicity was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay. The MTT (Methyl thiazol tetrazolium) assay is used to measure cell viability. MTT converts to a formazan stain that produces a purple color. After 72 hours of incubation, the volume of medium was reduced to 1 ml and 100 μl of 1 mg/ml MTT was added to each wall. Afterwards, human dermal fibroblasts (NHDFs) were cultured in the presence of 5% CO 2 and 95% O 2 at 37°C for 2 hours.
인간피부섬유아세포에 상기 실시예 1에서 제조한 단일 또는 복합추출물을 농도별(50, 100, 250㎍/㎖)로 처리하였으며, 이후 24시간 동안 배양하였다. 24시간 후, 1mg/mL의 MTT를 처리하고 3시간 후, 세포에 MTT 처리로 인해 생성된 포마잔(formazan)을 DMSO 용매로 녹여 570nm에서 흡광도를 측정하였다.Human skin fibroblasts were treated with the single or complex extract prepared in Example 1 at different concentrations (50, 100, 250 μg/ml), and then cultured for 24 hours. 24 hours later, 1 mg/mL of MTT was treated, and 3 hours later, formazan produced by MTT treatment in cells was dissolved in DMSO solvent and absorbance was measured at 570 nm.
흡광도 측정 결과를 정리한 [도 1]을 참조하면, 인간피부섬유아세포(human dermal fibroblasts, NHDFs)는 락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 추출물에 독성을 나타내지 않는 것을 확인할 수 있었으며, 이들 2종 복합추출물을 처리한 시험군(제조예 3)에서는 UVB 조사군(150mJ/cm2)을 기준으로 하여 각각 107%(50㎍/mL), 115%(100㎍/mL) 및 123%(250㎍/mL) 개선된 세포 증식률을 나타내었다. 이러한 실험 결과를 바탕으로 본 발명의 락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 단일추출물 또는 이들의 복합추출물은 세포 독성이 없으며, 피부 세포의 생장을 촉진하는 등 세포 보호 효과가 매우 우수한 것을 확인할 수 있었다.Referring to [Figure 1], which summarizes the absorbance measurement results, human dermal fibroblasts (NHDFs) are Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC It was confirmed that the extract of 15001BP) was not toxic, and in the test group (Preparation Example 3 ) treated with these two composite extracts, the mL), 115% (100 μg/mL), and 123% (250 μg/mL) showed improved cell proliferation rates. Based on these experimental results, the single extract or complex extract of Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP) of the present invention is not cytotoxic and has no cytotoxic effect on the skin. It was confirmed that the cell protection effect was excellent, including promoting cell growth.
실시예 3. 피부 보습 활성Example 3. Skin moisturizing activity
락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물과 이들의 복합추출물이 갖는 피부 보습 효과를 검증하기 위해 시료 처리 유무에 따른 히알루론산(Hyaluronan) 합성량 변화를 분석하였다.To verify the skin moisturizing effect of single extracts of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain and their complex extracts, the change in the amount of hyaluronic acid (Hyaluronan) synthesis according to the presence or absence of sample treatment was analyzed.
히알루론산(HA, Hyaluronan, Hyaluronic acid)은 주로 표피 각질형성세포와 진피 섬유아세포에 의해 합성되며, 수분과 결합하여 세포가 정상적으로 활동할 수 있는 환경을 조성하는 중요한 역할을 하는 물질이다. 피부에서 히알루론산의 감소는 피부 탄력 저하 및 수분 함유량 감소의 직접적인 원인 중 하나이다. 따라서 본 실험에서는 인간각질형성세포 내에서 히알루론산의 생성량을 확인하였다. Hyaluronic acid (HA) is mainly synthesized by epidermal keratinocytes and dermal fibroblasts, and is a substance that plays an important role in creating an environment in which cells can function normally by combining with moisture. A decrease in hyaluronic acid in the skin is one of the direct causes of decreased skin elasticity and reduced moisture content. Therefore, in this experiment, the amount of hyaluronic acid produced in human keratinocytes was confirmed.
인간각질형성세포(HaCaT)를 24 well 세포배양 접시에 1㎖의 배양액을 넣고 약 1×105 농도로 세포를 접종한 후, 37℃, 5% CO2 환경에서 24시간 동안 배양하였다. 그 후, 상기 실시예 1에 따라 준비된 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물과 이들의 복합추출물(제조예 1 내지 3)을 각각 50, 100, 및 250㎍/㎖ 농도로 포함한 배지로 교체하고 24시간 배양하였다. 이후 배양액을 회수(harvest)하여 4℃, 7,500rpm 환경에서 5분간 원심분리(centrifuge)한 뒤, ELISA 방법을 이용하여 Hyaluronan(Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA)의 발현량 변화를 확인하였다. Human keratinocytes (HaCaT) were placed in 1 ml of culture medium in a 24-well cell culture dish, inoculated with cells at a concentration of approximately 1×10 5 , and cultured for 24 hours at 37°C in a 5% CO 2 environment. Thereafter, the single extract of the Lactococcus lactis Nabysol07 strain and the Streptococcus thermophilus Nabysol07 strain prepared according to Example 1 and their complex extracts (Preparation Examples 1 to 3) were added at 50, 100, and 250 μg/ml, respectively. It was replaced with medium containing the correct concentration and cultured for 24 hours. Afterwards, the culture medium was harvested and centrifuged for 5 minutes at 4°C at 7,500 rpm, and the expression level of Hyaluronan (Hyalunan kit, R&D Systems, Inc., Minneapolis, MN, USA) was determined using the ELISA method. Changes were confirmed.
실험 결과를 정리한 [도 2]를 참조하면, 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물과 이들의 복합추출물을 처리한 실험군에서는 히알루론산(Hyaluronan)의 생성량이 농도 의존적으로 증가하는 경향을 확인할 수 있었는데, 단일추출물들에 비해 이들의 복합추출물의 처리에 따른 히알루론산 생성량 증가 현상이 보다 뚜렷하게 관찰되었다. Referring to [Figure 2], which summarizes the experimental results, in the experimental group treated with single extracts of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain and their complex extracts, the production of hyaluronic acid (Hyalunan) was concentration dependent. A tendency to increase was confirmed, and the increase in hyaluronic acid production due to the treatment of these complex extracts was observed more clearly compared to the single extracts.
구체적으로, 락토코커스 락티스 Nabysol07 균주의 추출물을 처리한 실험군에서는 무처리군에 비해 50, 100 및 250㎍/ml의 농도에서 각각 106%, 108% 및 115% 증가한 히알루론산의 생성량을 확인할 수 있었고, 스트렙토코커스 써모필러스 Nabysol07 균주의 추출물을 처리한 실험군에서는 50, 100 및 250㎍/ml의 농도에서 각각 102%, 109% 및 116% 증가한 히알루론산의 생성량을 확인할 수 있었으나, 이들 2종 균주 복합추출물을 처리한 실험군에서는 무처리군에 비해 50, 100 및 250㎍/ml의 농도에서 각각 118%, 136% 및 147% 증가한 히알루론산의 생성량을 확인할 수 있었고, 각각의 단일 추출물에 비해 피부 보습에 우수한 시너지 효과가 있음을 확인할 수 있었다.Specifically, in the experimental group treated with extract of Lactococcus lactis Nabysol07 strain, the amount of hyaluronic acid produced increased by 106%, 108%, and 115% at concentrations of 50, 100, and 250 μg/ml, respectively, compared to the untreated group. In the experimental group treated with the extract of the Streptococcus thermophilus Nabysol07 strain, the amount of hyaluronic acid produced increased by 102%, 109%, and 116% at concentrations of 50, 100, and 250㎍/ml, respectively, but the combination of these two strains In the experimental group treated with the extract, the amount of hyaluronic acid produced was confirmed to be increased by 118%, 136%, and 147%, respectively, at concentrations of 50, 100, and 250㎍/ml compared to the untreated group, and it was found to increase skin moisturization compared to each single extract. It was confirmed that there was an excellent synergy effect.
상기와 같은 결과를 통해, 본 발명의 2종 복합추출물은 피부 세포에서 히알루론산 생성을 효과적으로 증가시켜 피부 보습과 관련된 용도로 매우 효과적으로 사용될 수 있음을 확인할 수 있었다. Through the above results, it was confirmed that the two complex extracts of the present invention can be used very effectively for purposes related to skin moisturization by effectively increasing hyaluronic acid production in skin cells.
실시예 4. 피부 장벽 강화 활성Example 4. Skin barrier strengthening activity
락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물 또는 이들의 복합추출물 처리에 따른 필라그린(Filaggrin, FLG, 각질층 형성 인자) 유전자 발현 변화를 분석하였다.Changes in filaggrin (FLG, stratum corneum formation factor) gene expression according to treatment with single extracts or complex extracts of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain were analyzed.
먼저, 인간각질형성세포(HaCaT)를 세포주로 사용하였고, 100mm 세포 배양 접시에 10% FBS를 첨가한 DMEM 배지를 사용하여 세포를 1×106 농도로 접종하여 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. 배양 후, 상기 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주 배양액의 단일추출물과 이들의 복합추출물을 각각 50, 100, 및 250㎍/㎖ 농도가 되도록 DMEM 배지에 희석하여 제조한 희석용액을 첨가하여, 24시간 동안 동일 조건에서 추가 배양하였다. 이후 Trizol reagent(invitrogen, USA)를 이용하여 세포를 회수하고, RNA를 분리하였다. RT-PCR은 PCR 기기(Step One Plus, Applied Biosystems, 미국)를 이용하였고, 사이버그린(SYBR Green supermix, Applied Biosystems, 미국) 시약을 프로필라그린(profilaggrin) 및 GAPDH, 그리고 cDNA와 함께 첨가하여 94℃에서 5분 동안 중합효소 활성화, 95℃ 30초, 54℃ 1분, 72℃ 1분간 40 사이클로 중합 반응을 수행하였다. First, human keratinocytes (HaCaT) were used as a cell line, and the cells were inoculated at a concentration of 1 × 10 6 using DMEM medium supplemented with 10% FBS in a 100 mm cell culture dish and incubated at 37°C in a 5% CO 2 incubator. Cultured for 24 hours. After culturing, a dilution solution prepared by diluting the single extract of the Lactococcus lactis Nabysol07 strain and the Streptococcus thermophilus Nabysol07 strain culture medium and their composite extract in DMEM medium to concentrations of 50, 100, and 250 ㎍/㎖, respectively. was added and further cultured under the same conditions for 24 hours. Afterwards, cells were recovered using Trizol reagent (invitrogen, USA), and RNA was isolated. RT-PCR was performed using a PCR device (Step One Plus, Applied Biosystems, USA), and Cybergreen (SYBR Green supermix, Applied Biosystems, USA) reagent was added along with profilaggrin, GAPDH, and cDNA to obtain 94 The polymerization reaction was performed with 40 cycles of polymerase activation at °C for 5 minutes, 95°C for 30 seconds, 54°C for 1 minute, and 72°C for 1 minute.
사용한 프라이머는 다음과 같다:The primers used were:
- Propilaggrin : 정방향 5′-AAG CTT CAT GGT GAT GCG AC-3′, 역방향 5′-TCA AGC AGA AGA GGA AGG CA-3′- Propilaggrin: forward 5′-AAG CTT CAT GGT GAT GCG AC-3′, reverse 5′-TCA AGC AGA AGA GGA AGG CA-3′
- GAPDH: 정방향 5'-ACC ACA GTC CAT GCC ATC AC-3', 역방향 5'-CCA CCA CCC TGT TGC TGT AG-3'.- GAPDH: forward 5'-ACC ACA GTC CAT GCC ATC AC-3', reverse 5'-CCA CCA CCC TGT TGC TGT AG-3'.
상기와 같은 프라이머를 이용하여 RT-PCR 수행하고, 실험 결과를 [도 5]에 정리하였다.RT-PCR was performed using the above primers, and the experimental results are summarized in [Figure 5].
필라그린은 표피 과립층에서 케라토히알린과립(keratohyalin granule)을 형성하는 단백질인 프로필라그린(profilaggrin)의 형태로 존재하다가, 각질형성세포의 최종 분화과정에서 필라그린으로 분해된 후에 각질세포 막을 형성할 때 케라틴 필라멘트를 응집하여 각질세포의 단단하고 편평한 구조를 형성함으로써 피부장벽에서 벽돌(brick) 역할을 수행하게 된다.Filaggrin exists in the form of profilaggrin, a protein that forms keratohyalin granules in the granular layer of the epidermis, and is decomposed into filaggrin during the final differentiation of keratinocytes to form a keratinocyte membrane. When keratin filaments are aggregated to form a hard, flat structure of keratinocytes, they play the role of a brick in the skin barrier.
실험 결과를 정리한 [도 3]을 참조하면, 시료 무처리군에 비해 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물 또는 이들의 복합추출물 처리군에서는 프로필라그린(profilaggrin)의 mRNA 발현량이 증가한 것으로 나타났는데, 단일추출물 처리군에 비해 복합추출물 처리군에서 발현량 증가가 더욱 뚜렷하게 관찰되었다. Referring to [Figure 3], which summarizes the experimental results, compared to the untreated group, the group treated with single extracts or complex extracts of Lactococcus lactis Nabysol07 strain and Streptococcus thermophilus Nabysol07 strain had profilaggrin. It was found that the mRNA expression level increased, and the increase in expression level was more clearly observed in the complex extract treatment group compared to the single extract treatment group.
구체적으로 시료 무처리군에 비해 락토코커스 락티스 Nabysol07 균주의 단일추출물 처리군에서는 프로필라그린의 mRNA 발현량이 각각 104%(100μg/mL) 및 108%(250μg/mL) 증가하였고, 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물 처리군에서는 프로필라그린의 mRNA 발현량이 각각 106%(50μg/mL), 109%(100μg/mL) 및 114%(250μg/mL) 증가한 것으로 나타났다. Specifically, compared to the untreated sample group, in the group treated with a single extract of Lactococcus lactis Nabysol07 strain, the mRNA expression levels of profilaggrin increased by 104% (100 μg/mL) and 108% (250 μg/mL), respectively, and Streptococcus thermophiler In the group treated with a single extract of the Nabysol07 strain, the mRNA expression level of profilaggrin increased by 106% (50 μg/mL), 109% (100 μg/mL), and 114% (250 μg/mL), respectively.
이러한 단일추출물의 효과에 비해, 본 발명의 2종 균주의 복합추출물 처리군에서는 시료 무처리군에 비해 프로필라그린의 mRNA 발현량이 각각 121%(50μg/mL), 139%(100μg/mL) 및 154%(250μg/mL) 증가한 것으로 관찰되어 프로필라그린(profilaggrin)의 mRNA 발현량이 현저하게 증가된 것을 확인할 수 있었고, 2종 균주 조합에 따른 시너지 효과가 관찰되었다. Compared to the effect of this single extract, in the group treated with the complex extract of the two strains of the present invention, the mRNA expression level of profilaggrin was 121% (50 μg/mL), 139% (100 μg/mL), and 139% (100 μg/mL), respectively, compared to the untreated group. An increase of 154% (250 μg/mL) was observed, confirming that the mRNA expression level of profilaggrin was significantly increased, and a synergistic effect was observed due to the combination of the two strains.
상기와 같은 결과를 통해, 본 발명의 락토코커스 락티스 Nabysol07 균주 및 스트렙토코커스 써모필러스 Nabysol07 균주의 2종 복합추출물은 프로필라그린(profilaggrin) 유전자의 발현을 증가시켜 피부 장벽 형성에 기여함으로써 피부 상태 개선에 우수한 효과를 나타냄을 확인할 수 있었다. Through the above results, the two complex extracts of the Lactococcus lactis Nabysol07 strain and the Streptococcus thermophilus Nabysol07 strain of the present invention contribute to the formation of the skin barrier by increasing the expression of the profilaggrin gene, thereby improving skin condition. It was confirmed that it had an excellent effect on improvement.
실시예 5. 피부 주름 개선 활성Example 5. Skin wrinkle improvement activity
5-1. MMP-1 유전자 발현 억제5-1. Inhibition of MMP-1 gene expression
락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 단일추출물과 이들의 복합추출물이 MMP-1 유전자 발현에 미치는 영향을 분석하였다.The effects of single extracts of Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP) and their complex extracts on MMP-1 gene expression were analyzed.
실험에 사용되는 세포는 40mm 세포 배양 접시에 2mL의 DMEM 배양액을 넣고, 인간피부섬유아세포(human dermal fibroblasts, NHDFs)를 약 1.2x105 농도로 접종한 후, 37℃, 5% CO2 환경에서 24시간 동안 배양하여 사용하였다. 그 후, 실시예 1에서 제조된 제조예 1 내지 3의 배양액 추출물(각각 50㎍/㎖, 100㎍/㎖ 및 250㎍/㎖)을 포함하는 배지로 교체하여 3일간 배양하였다. 이후 배양액을 회수(harvest)하여 4℃, 7,500rpm 환경에서 5분간 원심분리(centrifuge)하여 ELISA 방법을 이용하여 MMP-1(Human Total MMP-1 kit, R&D Systems, Inc., Minneapolis, MN, USA) 단백질의 발현량 변화를 확인하였다. 모든 실험은 3회 반복하여 측정되었다.The cells used in the experiment were placed in 2 mL of DMEM culture medium in a 40 mm cell culture dish, inoculated with human dermal fibroblasts (NHDFs) at a concentration of approximately 1.2x10 5 , and incubated at 37°C in a 5% CO 2 environment for 24 hours. It was cultured for some time before use. Thereafter, the medium was replaced with a medium containing the culture extracts of Preparation Examples 1 to 3 prepared in Example 1 (50 μg/ml, 100 μg/ml, and 250 μg/ml, respectively) and cultured for 3 days. Afterwards, the culture medium was harvested and centrifuged for 5 minutes at 4°C at 7,500 rpm to detect MMP-1 (Human Total MMP-1 kit, R&D Systems, Inc., Minneapolis, MN, USA) using the ELISA method. ) Changes in protein expression were confirmed. All experiments were measured in triplicate.
MMP-1 단백질 발현량 변화를 정량한 [도 4]를 참조하면, 단일 균주 배양액을 처리한 시험군들 중 락토코커스 락티스 Nabysol07 균주 배양액 추출물이 스트렙토코커스 써모필러스 Nabysol07 균주의 배양액 추출물에 비해 MMP-1 단백질 발현 억제 효과가 다소 높은 것으로 나타났으나, 단일 추출물의 전반적인 효과는 미미한 것으로 나타났다. 한편, 본 발명의 2종 균주의 복합추출물을 처리한 시험군에서는 MMP-1 단백질 생성량이 단일 균주 추출물 처리군에 비해 농도 의존적으로 더욱 크게 감소하는 양상을 확인할 수 있었다. Referring to [Figure 4], which quantifies the change in MMP-1 protein expression, among the test groups treated with the single strain culture medium, the Lactococcus lactis Nabysol07 strain culture extract had MMP higher than the culture extract of the Streptococcus thermophilus Nabysol07 strain. -1 The effect of suppressing protein expression was found to be somewhat high, but the overall effect of a single extract was found to be minimal. Meanwhile, in the test group treated with the complex extract of the two strains of the present invention, it was confirmed that the amount of MMP-1 protein production decreased more significantly in a concentration-dependent manner compared to the group treated with the single strain extract.
구체적으로, UVB 조사(150mJ/cm2)로 증가한 MMP-1 단백질 함량(Con.)을 기준으로 하여 락토코커스 락티스 Nabysol07 균주의 단일추출물 처리군은 MMP-1 단백질 발현이 각각 6%(100㎍/㎖) 및 9%(250㎍/㎖)로 감소하였고, 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물 처리군은 MMP-1 단백질 발현이 각각 5%(100㎍/㎖) 및 3%(250㎍/㎖)로 감소하여, 단일 균주의 처리로 인한 MMP-1 단백질 발현 감소 폭은 상대적으로 효과적이지 않은 것으로 분석되었다. Specifically, based on the MMP-1 protein content (Con.) increased by UVB irradiation (150 mJ/cm 2 ), the MMP-1 protein expression in the group treated with a single extract of Lactococcus lactis Nabysol07 strain was reduced by 6% (100 μg each). /㎖) and 9% (250㎍/㎖), and in the group treated with a single extract of Streptococcus thermophilus Nabysol07 strain, MMP-1 protein expression decreased by 5% (100㎍/㎖) and 3% (250㎍), respectively. /ml), it was analyzed that the decrease in MMP-1 protein expression due to treatment of a single strain was relatively ineffective.
이에 반해, 본 발명의 2종 균주의 복합추출물 처리군은 UVB 조사(150mJ/cm2)로 증가한 MMP-1 단백질 함량(Con.)을 기준으로 MMP-1 단백질 발현이 각각 9%(50㎍/㎖), 12%(100㎍/㎖) 및 24%(250㎍/㎖)로 감소하여 2종 균주의 혼합으로 MMP-1 단백질 발현이 현저하게 감소하는 피부 주름 개선에 대한 시너지 효과가 있음을 확인할 수 있었다. On the other hand , in the group treated with the complex extract of the two strains of the present invention, MMP-1 protein expression was 9% (50 ㎍/ ㎖), 12% (100㎍/㎖) and 24% (250㎍/㎖), confirming that there is a synergistic effect on improving skin wrinkles by significantly reducing MMP-1 protein expression by mixing the two strains. I was able to.
5-2. 타입 1 프로콜라겐 유전자 발현 증가5-2. Increased type 1 procollagen gene expression
실시예 5-1과 동일한 조건으로 동일 시료를 처리하여 배양하였다. 이후 배양액을 회수(harvest)하여 4℃, 7,500rpm 환경에서 5분간 원심분리(centrifuge)하였고, ELISA 방법으로 타입 1 프로콜라겐(Procollagen Type I C Peptide EIA Kit, Takara, Shiga, Japan)의 단백질 발현량 변화를 확인하였다. 모든 실험은 3회 반복하여 측정되었다.The same sample was treated and cultured under the same conditions as in Example 5-1. Afterwards, the culture medium was harvested and centrifuged for 5 minutes at 4°C and 7,500 rpm, and the protein expression level of type 1 procollagen (Procollagen Type I C Peptide EIA Kit, Takara, Shiga, Japan) was measured using ELISA. was confirmed. All experiments were measured in triplicate.
타입 1 프로콜라겐 단백질 발현량 변화를 정량한 [도 5]를 참조하면, 본 발명의 2종 균주의 복합추출물을 처리한 시험군에서 타입 1 프로콜라겐 단백질 발현이 단일추출물 처리군에 비해 농도 의존적으로 크게 증가하는 양상을 확인할 수 있었다. Referring to [Figure 5], which quantifies the change in type 1 procollagen protein expression, in the test group treated with the complex extract of the two strains of the present invention, type 1 procollagen protein expression was concentration-dependent compared to the single extract treatment group. A significant increase was observed.
구체적으로, UVB 조사(150mJ/cm2)로 인해 2/3 수준으로 감소한 타입 1 프로콜라겐 단백질 발현(Con.)을 기준으로, 락토코커스 락티스 Nabysol07 균주의 단일추출물 처리군은 타입 1 프로콜라겐 단백질 발현이 각각 103%(50㎍/㎖), 105%(100㎍/㎖) 및 108%(250㎍/㎖)로 증가하였고, 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물 처리군은 타입 1 프로콜라겐 단백질 발현이 각각 105%(250㎍/㎖)로 증가하여, 단일 균주의 처리로 인한 타입 1 프로콜라겐 단백질 발현량 증가 폭은 전반적으로 미미한 것으로 분석되었다. Specifically, based on the type 1 procollagen protein expression (Con.), which was reduced by 2/3 due to UVB irradiation (150mJ/cm 2 ), the group treated with a single extract of Lactococcus lactis Nabysol07 strain had type 1 procollagen protein. Expression increased to 103% (50㎍/㎖), 105% (100㎍/㎖), and 108% (250㎍/㎖), respectively, and the group treated with a single extract of Streptococcus thermophilus Nabysol07 strain had type 1 procollagen. Protein expression increased by 105% (250 μg/ml), respectively, and the overall increase in type 1 procollagen protein expression due to treatment of a single strain was analyzed to be insignificant.
이에 반해, 본 발명의 2종 균주의 복합추출물 처리군은 타입 1 프로콜라겐 단백질 발현은 UVB 조사(150mJ/cm2) 무처리군을 기준으로 각각 109%(50㎍/㎖), 118%(100㎍/㎖) 및 129%(250㎍/㎖)로 증가하여 2종 균주의 혼합으로 타입 1 프로콜라겐 단백질 발현이 현저하게 증가하는 피부 주름 개선에 대한 시너지 효과가 있음을 확인할 수 있었다. On the other hand, in the group treated with the complex extract of the two strains of the present invention, the type 1 procollagen protein expression was 109% (50㎍/㎖) and 118% (100㎍), respectively, based on the UVB irradiation (150mJ/cm 2 ) untreated group. ㎍/㎖) and 129% (250㎍/㎖), confirming that there is a synergistic effect on improving skin wrinkles by significantly increasing type 1 procollagen protein expression by mixing the two strains.
상기와 같은 결과를 통해, 본 발명의 락토코커스 락티스 Nabysol07 및 스트렙토코커스 써모필러스 Nabysol07의 2종 균주의 복합추출물은 피부 세포에서 MMP-1 단백질의 발현을 효과적으로 억제하고, 타입 1 프로콜라겐 단백질 발현을 증가시켜 피부 주름 개선 용도로 효과적으로 사용될 수 있음을 확인할 수 있었다. Through the above results, the complex extract of the two strains of Lactococcus lactis Nabysol07 and Streptococcus thermophilus Nabysol07 of the present invention effectively inhibits the expression of MMP-1 protein in skin cells and type 1 procollagen protein expression. It was confirmed that it can be effectively used to improve skin wrinkles by increasing .
실시예 6. TSLP 발현 억제를 통한 피부 가려움 개선Example 6. Improvement of skin itching through inhibition of TSLP expression
사이토카인인 TSLP(thymic stromal lymphopoietin)는 IL-17, IL-6, CD11c+ 골수 수지상세포(myeloid dendritic cell)를 유도하여 Th2 염증반응을 일으키는 것으로 알려져 있으며, 아토피 피부염도 Th2 매개성 면역질환으로 알려져 있다.The cytokine TSLP (thymic stromal lymphopoietin) is known to induce a Th2 inflammatory response by inducing IL-17, IL-6, and CD11c+ bone marrow dendritic cells, and atopic dermatitis is also known to be a Th2-mediated immune disease. .
또한, 최근에는 TSLP(Thymic stromal lymphopoietin)가 피부염의 중증도와 연관되어 있고, 가려움 증세의 원인이라고 알려져 피부 가려움증의 마커로 사용되고 있다(Wilson SR et al., The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell. 2013;155(2):285-95).Additionally, TSLP (Thymic stromal lymphopoietin) has recently been associated with the severity of dermatitis and is known to be the cause of itching symptoms, so it is being used as a marker for skin itching (Wilson SR et al., The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce it. Cell 2013;155(2):285-95).
락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 단일추출물과 이들의 복합추출물이 TSLP 단백질의 발현에 미치는 영향을 검증하기 위해 인간각질형성세포(HaCaT)를 대상으로 실험을 수행하였다.To verify the effect of single extracts of Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP) and their complex extracts on the expression of TSLP protein, human keratinocytes were used. An experiment was performed targeting (HaCaT).
인간각질형성세포(HaCaT, 1×106/well)를 FBS DMEM 배지에서 24시간 배양한 후, FBS 없는 DMEM 배지에 실시예 1에서 제조된 제조예 1 내지 3의 배양액 추출물을 각각 50㎍/㎖, 100㎍/㎖ 및 250㎍/㎖ 농도로 첨가하고, TNF-α 및 IFN-γ(10 ng/mL)을 희석하여 처리한 후, 추가 배양하였다. 24시간 경과 후, 상층액을 7,500rpm, 5min, 4℃ 조건에서 원심분리하여 400-800μL 정도의 부피로 회수하여 새로운 e-tube에 옮겨준 후, 각 well의 상층액을 human TSLP ELISA kit(R&D Systems, Inc., Minneapolis, MN, USA)를 사용하여 제조사 프로토콜에 따라 정량하였다.After culturing human keratinocytes (HaCaT, 1×10 6 /well) in FBS DMEM medium for 24 hours, 50 μg/ml of the culture extracts of Preparation Examples 1 to 3 prepared in Example 1 were added to DMEM medium without FBS. , were added at concentrations of 100 ㎍/㎖ and 250 ㎍/㎖, diluted with TNF-α and IFN-γ (10 ng/mL), and then further cultured. After 24 hours, the supernatant was centrifuged at 7,500 rpm, 5 min, and 4°C to recover a volume of about 400-800 μL and transferred to a new e-tube. Then, the supernatant from each well was analyzed with a human TSLP ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) was used for quantification according to the manufacturer's protocol.
실험 결과를 정리한 [도 6]을 참조하면, TNF-α 및 IFN-γ(10 ng/mL) 처리에 의해 과발현된 TSLP 단백질의 생성량이 본 발명에 따른 락토코커스 락티스 Nabysol07 균주(기탁번호 KCTC 15002BP) 및 스트렙토코커스 써모필러스 Nabysol07 균주(기탁번호 KCTC 15001BP)의 단일 또는 복합 추출물의 처리로 유의적으로 억제됨을 확인할 수 있었다.Referring to [FIG. 6], which summarizes the experimental results, the amount of TSLP protein overexpressed by treatment with TNF-α and IFN-γ (10 ng/mL) was determined by the Lactococcus lactis Nabysol07 strain according to the present invention (accession number KCTC). 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP) were confirmed to be significantly inhibited by treatment with single or combined extracts.
구체적으로 시료 무처리군에 비해 락토코커스 락티스 Nabysol07 균주의 단일추출물 처리군에서는 TLSP의 mRNA 발현량이 각각 5%(100μg/mL) 및 13%(250μg/mL) 감소하였고, 스트렙토코커스 써모필러스 Nabysol07 균주의 단일추출물 처리군에서는 TSLP의 mRNA 발현량이 각각 5%(50μg/mL), 12%(100μg/mL) 및 14%(250μg/mL) 감소한 것으로 분석되어, 단일 추출물의 TLSP 발현 감소 효과는 다소 미미한 것으로 나타났다. Specifically, compared to the untreated sample group, in the group treated with a single extract of Lactococcus lactis Nabysol07 strain, the mRNA expression levels of TLSP decreased by 5% (100 μg/mL) and 13% (250 μg/mL), respectively, and Streptococcus thermophilus Nabysol07 In the group treated with the single extract of the strain, the mRNA expression level of TSLP was analyzed to be reduced by 5% (50 μg/mL), 12% (100 μg/mL), and 14% (250 μg/mL), respectively, so the effect of the single extract on reducing TLSP expression was somewhat small. It was found to be insignificant.
이러한 단일추출물들에 비해, 본 발명의 2종 균주 복합추출물 처리군에서는 시료 무처리군에 비해 TSLP mRNA 발현량이 각각 18%(50μg/mL), 33%(100μg/mL) 및 51%(250μg/mL) 감소한 것으로 관찰되어 단일추출물에 비해 TLSP 발현이 현저하게 감소된 것을 확인할 수 있었고, 2종 균주 조합에 따른 시너지 효과가 관찰되었다. Compared to these single extracts, in the group treated with the complex extract of the two strains of the present invention, the TSLP mRNA expression level was 18% (50 μg/mL), 33% (100 μg/mL), and 51% (250 μg/mL), respectively, compared to the untreated sample group. mL) was observed to decrease, confirming that TLSP expression was significantly reduced compared to the single extract, and a synergistic effect was observed due to the combination of the two strains.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (6)
상기 조성물은 필라그린 유전자 발현을 증가시켜 피부 장벽 강화 활성을 갖는 것을 특징으로 하는,
화장료 조성물.Contains two complex strains of Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP), their cultures, or their complex extracts as active ingredients,
The composition is characterized in that it has skin barrier strengthening activity by increasing filaggrin gene expression,
Cosmetic composition.
상기 조성물은 히알루론산 합성을 촉진하여 피부 보습 활성을 나타내는 것을 특징으로 하는 화장료 조성물.According to paragraph 1,
A cosmetic composition characterized in that the composition promotes hyaluronic acid synthesis and exhibits skin moisturizing activity.
상기 조성물은 MMP-1(matrix metalloproteinase-1) 유전자의 발현을 억제하고, 타입 1 프로콜라겐 유전자의 발현을 증가시켜 피부 탄력 개선 활성을 나타내는 것을 특징으로 하는 화장료 조성물.According to paragraph 1,
The composition is a cosmetic composition characterized in that it exhibits skin elasticity improvement activity by suppressing the expression of the MMP-1 (matrix metalloproteinase-1) gene and increasing the expression of the type 1 procollagen gene.
상기 조성물은 TSLP(Thymic stromal lymphopoietin) 생성을 억제하여 아토피 피부염 또는 피부 가려움증 개선 활성을 나타내는 것을 특징으로 하는 화장료 조성물.According to paragraph 1,
A cosmetic composition characterized in that the composition exhibits atopic dermatitis or skin itching improvement activity by inhibiting the production of TSLP (Thymic stromal lymphopoietin).
상기 조성물은 필라그린 유전자 발현을 증가시켜 피부 장벽 강화 활성을 갖는 것을 특징으로 하는,
식품 조성물.Contains two complex strains of Lactococcus lactis Nabysol07 strain (accession number KCTC 15002BP) and Streptococcus thermophilus Nabysol07 strain (accession number KCTC 15001BP), their cultures, or their complex extracts as active ingredients,
The composition is characterized in that it has skin barrier strengthening activity by increasing filaggrin gene expression,
Food composition.
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