KR102582773B1 - Novel Lactobacillus sp. strain and skin condition improving uses of thereof - Google Patents
Novel Lactobacillus sp. strain and skin condition improving uses of thereof Download PDFInfo
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- KR102582773B1 KR102582773B1 KR1020220094201A KR20220094201A KR102582773B1 KR 102582773 B1 KR102582773 B1 KR 102582773B1 KR 1020220094201 A KR1020220094201 A KR 1020220094201A KR 20220094201 A KR20220094201 A KR 20220094201A KR 102582773 B1 KR102582773 B1 KR 102582773B1
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- skin
- lactobacillus
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
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Abstract
Description
신규한 락토바실러스 속 균주 및 그의 피부 상태 개선 용도에 관한 것이다.It relates to a novel Lactobacillus genus strain and its use for improving skin condition.
피부의 생태계는 미생물에게 다양한 형태의 서식처를 제공하며, 광범위한 미생물들이 살고 있다. 숙주인 인간은 이들과 공생관계를 이루고 있으며, 숙주에 많은 긍정적인 영향을 미친다고 알려져 있다. 피부는 함입부, 특화되어있는 틈새 등 다양한 형태의 서식처를 구성하고 있으며, 넓은 분포의 미생물이 자랄 수 있도록 돕는다. 기본적으로 피부는 물리적인 막을 형성하며, 외부로부터 잠재적인 위험요소 및 독성물질들로부터 방어를 하도록 도와준다. 피부는 외부환경과의 접속지점이 되며, 다양한 미생물들(진균, 세균, 바이러스 및 작은 유충)의 집합소이기도 하다. 물리적, 화학적 기능의 선택에 맞게 미생물들은 특화된 틈새에 적응하여 서식처를 마련한다. 일반적으로 피부는 차갑고, 산성 성질을 나타내며, 건조된 상태로 유지된다. 구조적으로 표피(epidermis)는 피부장벽을 이루고 있으며, 미생물과 독소가 침투하는 것을 차단하고, 수분을 유지하는 중요한 역할을 한다. 표피의 최상위층은 각질층(stratum corneum)으로 구성되어있다. 표피는 일명 '벽돌과 몰탈 구조'라고 불리는 형태를 띠고 있는데, 피부 조직은 계속적인 자가 회복 과정을 거치고, 분화과정의 마지막을 거친 비늘(squames)은 끊임없이 피부조직에서 탈락되는 과정을 반복하게 된다.The skin ecosystem provides various types of habitats for microorganisms, and a wide range of microorganisms live there. Humans, the host, have a symbiotic relationship with them and are known to have many positive effects on the host. The skin constitutes various types of habitats, including indentations and specialized crevices, and helps a wide range of microorganisms to grow. Basically, the skin forms a physical membrane and helps defend against potential hazards and toxic substances from the outside. The skin serves as a point of connection with the external environment and is also a collection site for various microorganisms (fungi, bacteria, viruses, and small larvae). Depending on the selection of physical and chemical functions, microorganisms adapt to specialized niches and establish habitats. Typically, the skin remains cool, acidic, and dry. Structurally, the epidermis forms the skin barrier and plays an important role in blocking the penetration of microorganisms and toxins and maintaining moisture. The uppermost layer of the epidermis is composed of the stratum corneum. The epidermis has a form called a 'brick and mortar structure'. The skin tissue goes through a continuous self-recovery process, and the scales (squames) that have completed the differentiation process constantly repeat the process of being shed from the skin tissue.
프로바이오틱스(Probiotics)는 인체에 유익한 작용을 하는 미생물을 총칭하는 말로 우리 몸에 유익(benefit)을 주는 미생물을 말한다. 현재까지 알려진 대부분의 프로바이오틱스는 유산균으로 알려져 있다. 프로바이오틱스는 인체에 여러 가지 유익작용을 통해 효과적인 효능이 발생되는 것으로 보고되었지만, 피부와의 상호관계에 대한 연구는 미비한 실정이다.Probiotics are a general term for microorganisms that have beneficial effects on the human body. They refer to microorganisms that benefit our bodies. Most probiotics known to date are known as lactic acid bacteria. Probiotics have been reported to have effective effects through various beneficial effects on the human body, but research on their relationship with the skin is insufficient.
일 양상은 기탁번호 KCTC 14877BP로 기탁된 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 람노서스 KMH201(Lactobacillus rhamnosus KMH201) 균주, 이의 파쇄액, 배양액 및 배양액의 추출물로 이루어진 군으로부터 선택된 어느 하나; 기탁번호 KCTC 14878BP로 기탁된 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 루테리 KMH202(Lactobacillus reuteri KMH202) 균주, 이의 파쇄액, 배양액, 및 배양액의 추출물로 이루어진 군으로부터 선택된 어느 하나; 및 기탁번호 KCTC 14879BP로 기탁된 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 플란타룸 KMH203(Lactobacillus plantarum KMH203) 균주, 이의 파쇄액, 배양액 및 배양액의 추출물로 이루어진 군으로부터 선택된 어느 하나로 이루어진 군으로부터 선택된 적어도 하나, 둘, 또는 셋 이상을 포함하는 조성물을 제공한다. One aspect is Lactobacillus rhamnosus KMH201 ( Lactobacillus rhamnosus KMH201) strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with the deposit number KCTC 14877BP, any one selected from the group consisting of its lysate, culture medium and extract of the culture medium; Lactobacillus reuteri KMH202 ( Lactobacillus reuteri KMH202) strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with accession number KCTC 14878BP, any one selected from the group consisting of its lysate, culture medium, and extract of the culture medium; and Lactobacillus plantarum KMH203 ( Lactobacillus plantarum KMH203) strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with accession number KCTC 14879BP, its lysate, culture medium, and extracts of the culture medium. From the group consisting of any one selected from the group consisting of A composition comprising at least one, two, or three selected substances is provided.
다른 양상은 상기 조성물을 유효성분으로 포함하는 화장료 조성물을 제공한다.Another aspect provides a cosmetic composition containing the composition as an active ingredient.
또 다른 양상은 상기 조성물을 유효성분으로 포함하는 건강기능식품을 제공한다.Another aspect provides a health functional food containing the composition as an active ingredient.
일 양상은 기탁번호 KCTC 14877BP로 기탁된 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 람노서스 KMH201(Lactobacillus rhamnosus KMH201) 균주, 이의 파쇄액, 배양액 및 배양액의 추출물로 이루어진 군으로부터 선택된 어느 하나; 기탁번호 KCTC 14878BP로 기탁된 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 루테리 KMH202(Lactobacillus reuteri KMH202) 균주, 이의 파쇄액, 배양액, 및 배양액의 추출물로 이루어진 군으로부터 선택된 어느 하나; 및 기탁번호 KCTC 14879BP로 기탁된 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 플란타룸 KMH203(Lactobacillus plantarum KMH203) 균주, 이의 파쇄액, 배양액 및 배양액의 추출물로 이루어진 군으로부터 선택된 어느 하나로 이루어진 군으로부터 선택된 적어도 하나, 둘, 또는 셋 이상을 포함하는 조성물을 제공하는 것이다. One aspect is Lactobacillus rhamnosus KMH201 ( Lactobacillus rhamnosus KMH201) strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with the deposit number KCTC 14877BP, any one selected from the group consisting of its lysate, culture medium and extract of the culture medium; Lactobacillus reuteri KMH202 ( Lactobacillus reuteri KMH202) strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with accession number KCTC 14878BP, any one selected from the group consisting of its lysate, culture medium, and extract of the culture medium; and Lactobacillus plantarum KMH203 ( Lactobacillus plantarum KMH203) strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with accession number KCTC 14879BP, its lysate, culture medium, and extracts of the culture medium. From the group consisting of any one selected from the group consisting of To provide a composition comprising at least one, two, or three or more selected substances.
일 구체예에 있어서, 상기 락토바실러스 람노서스 KHM201 균주는 서열번호 1의 16s rRNA를 포함하는 균주일 수 있다.In one embodiment, the Lactobacillus rhamnosus KHM201 strain may be a strain containing 16s rRNA of SEQ ID NO: 1.
일 구체예에 있어서, 상기 락토바실러스 루테리 KHM202 균주는 서열번호 2의 16s rRNA를 포함하는 균주일 수 있다.In one embodiment, the Lactobacillus reuteri KHM202 strain may be a strain containing 16s rRNA of SEQ ID NO: 2.
일 구체예에 있어서, 상기 락토바실러스 플란타룸 KHM203 균주는 서열번호 3의 16s rRNA를 포함하는 균주일 수 있다.In one embodiment, the Lactobacillus plantarum KHM203 strain may be a strain containing 16s rRNA of SEQ ID NO: 3.
일 구체예에 있어서, 상기 락토바실러스 람노서스 KHM201 균주, 락토바실러스 루테리 KMH202 균주, 및 락토바실러스 플란타룸 KMH203 균주는 생균(살아있는 균) 또는 사균(죽어있는 균)일 수 있다. 상기 사균은 열처리에 의한 사균일 수 있다.In one embodiment, the Lactobacillus rhamnosus KHM201 strain, Lactobacillus reuteri KMH202 strain, and Lactobacillus plantarum KMH203 strain may be viable (living bacteria) or dead bacteria (dead bacteria). The dead cells may be dead cells caused by heat treatment.
본 명세서에서 용어 "소포체(vesicle)"는 세포에서 분비되어 세포외 공간으로 방출된 입자를 의미하는 것으로서, 엑소좀(exosome), 엑토좀(ectosome), 마이크로소낭(microvesicle), 마이크로입자(microparticle), 엑소좀 유사 소포체 (exosome like vesicle) 등의 다수의 상이한 종을 포함할 수 있다. 세포 밖 소포체는 분비하는 기원 세포(공여 세포)의 상태를 반영할 수 있으며, 어떤 세포에서 분비되었는가에 따라 다양한 생물학적 활성을 나타내고, 세포들 사이에 유전 물질과 단백질을 옮기면서 세포 간 상호작용에 중요한 역할을 할 수 있다. 또한, 상기 소포체를 포함하는 세포 유래 물질들은 질병을 일으키거나 또는 면역세포를 자극하여 질병에 대항하게 하며, 미생물의 대사과정을 통해 사람이 소화시키지 못하는 물질들을 분해하여 흡수할 수 있도록 도와주는 효과가 있다. 상기 소포체는 막 구조 소포체로 내부와 외부가 구분되며, 세포의 세포막 지질(plasma membrane lipid)과 세포막 단백질(plasma membrane protein), 핵산(nucleic acid), 및 세포질 성분 등을 가지고 있고, 원래 세포보다 크기가 작은 것일 수 있다.As used herein, the term "vesicle" refers to particles secreted from cells and released into the extracellular space, including exosomes, ectosomes, microvesicles, and microparticles. , exosome-like vesicles, etc. Extracellular endoplasmic reticulum can reflect the state of the secreting cell of origin (donor cell), exhibits various biological activities depending on which cell it is secreted from, and plays an important role in intercellular interactions by transferring genetic material and proteins between cells. can do. In addition, cell-derived substances containing the endoplasmic reticulum cause disease or stimulate immune cells to fight against disease, and have the effect of helping humans break down and absorb substances that cannot be digested through the metabolic process of microorganisms. there is. The endoplasmic reticulum is a membrane-structured endoplasmic reticulum that is divided into an inside and an outside, and contains cell membrane lipids, plasma membrane proteins, nucleic acids, and cytoplasmic components, and is larger than the original cell. may be small.
상기 소포체는 상기 락토바실러스 람노서스 KHM201 균주의 배양액의 세포 파쇄물, 락토바실러스 루테리 KMH202 균주의 배양액의 세포 파쇄물, 또는 락토바실러스 플란타룸 KMH203 균주의 배양액의 세포 파쇄물로부터 분리된 것일 수 있다.The endoplasmic reticulum may be separated from cell lysate of the culture medium of the Lactobacillus rhamnosus KHM201 strain, cell lysate of the culture medium of the Lactobacillus reuteri KMH202 strain, or cell lysate of the culture medium of the Lactobacillus plantarum KMH203 strain.
일 구체예에 있어서, 상기 세포 외 소포체는 10 nm 내지 400 nm의 직경을 갖는 것일 수 있다. 예를 들어, 10 nm 내지 400 nm, 10 nm 내지 350 nm, 10 nm 내지 300 nm, 10 nm 내지 250 nm 일 수 있다. In one embodiment, the extracellular vesicles may have a diameter of 10 nm to 400 nm. For example, it may be 10 nm to 400 nm, 10 nm to 350 nm, 10 nm to 300 nm, or 10 nm to 250 nm.
본 명세서에서 용어 "파쇄액"은 균주 자체를 화학적 또는 물리적 힘에 의하여 균주의 세포벽을 파쇄하여 얻은 산물을 의미할 수 있다.As used herein, the term “lysate” may refer to a product obtained by breaking the cell wall of the strain itself using chemical or physical force.
본 명세서에서 용어"배양액"은 "배양 상층액", "조건 배양액" 또는 "조정 배지"와 호환적으로 사용될 수 있고, 락토바실러스 람노서스, 락토바실러스 루테리, 락토바실러스 플란타룸 또는 이들의 복합 균주가 시험관 내에서 성장 및 생존할 수 있도록 영양분을 공급할 수 있는 배지에 상기 균주를 일정기간 배양하여 얻는 상기 균주, 이의 대사물, 여분의 영양분 등을 포함하는 전체 배지를 의미할 수 있다. 또한, 상기 배양액은 균주를 배양하여 얻은 균체 배양액에서 균체를 제거한 배양액을 의미할 수 있다. 한편, 상기 배양액 중 균체를 제거한 액체를 "상등액"이라고도 하며, 배양액을 일정시간 가만히 두어 하층에 가라앉은 부분을 제외한 상층의 액체만을 취하거나, 여과를 통해 균체를 제거하거나, 배양액을 원심분리하여 하부의 침전을 제거하고 상부의 액체만을 취하여 획득할 수 있다. 상기 "균체"는 본 발명의 균주 자체를 의미하는 것으로 피부 샘플 등으로부터 분리하여 선별한 균주 자체 또는 상기 균주를 배양하여 배양액으로부터 분리한 균주를 포함한다. 상기 균체는 배양액을 원심분리하여 하층에 가라앉은 부분을 취하여 획득할 수 있고, 또는 중력에 의해 배양액의 하층으로 가라앉으므로 일정 시간동안 가만히 두었다가 상부의 액체를 제거함으로써 획득할 수 있다.As used herein, the term “culture medium” may be used interchangeably with “culture supernatant,” “conditioned culture medium,” or “conditioned medium,” and may be used interchangeably with Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, or a complex strain thereof. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time in a medium that can supply nutrients for growth and survival in a test tube. Additionally, the culture medium may refer to a culture medium obtained by removing the bacterial cells from the bacterial culture medium obtained by culturing the strain. On the other hand, the liquid from which the bacteria have been removed from the culture medium is also called "supernatant", and the culture medium is left alone for a certain period of time and only the liquid in the upper layer excluding the part that has settled in the lower layer is taken, the bacteria are removed through filtration, or the culture medium is centrifuged and the lower layer is removed. It can be obtained by removing the precipitation and taking only the upper liquid. The "bacteria" refers to the strain itself of the present invention, and includes the strain itself isolated and selected from a skin sample, etc., or a strain isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and taking the part that has sunk to the lower layer. Alternatively, since they sink to the lower layer of the culture medium by gravity, they can be obtained by leaving them still for a certain period of time and then removing the upper liquid.
상기 배양액은 균주를 배양하여 수득된 배양액 자체, 그의 농축물, 또는 동결건조물 또는 배양액로부터 균주를 제거하여 수득된 배양 상층액, 그의 농축물 또는 동결건조물을 포함할 수 있다. The culture medium may include the culture medium itself, its concentrate, or freeze-dried product obtained by cultivating the strain, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or freeze-dried product.
상기 배양액은 상기 락토바실러스 람노서스, 락토바실러스 루테리, 락토바실러스 플란타룸 또는 이들의 복합 균주가 적절한 배지(예를 들면, R2A 배지, TSA 배지, 또는 MRS 배지)에서 10 ℃ 초과 또는 40 ℃ 미만 중 어느 온도에서 일정 시간, 예를 들면, 4 내지 50 시간 동안 배양하여 수득된 것일 수 있다. MRS 배지는 펩톤, 소고기 추출물, 효모 추출물, 포도당, 아세트산나트륨, 폴리소르베이트 80, 디포타슘 하이드로젠 포스페이트, 암모늄 시트레이트, 황산마그네슘, 황산망간, 아가 및 증류수로 이루어진 군으로부터 선택된 어느 하나, 이들의 2 이상의 조합, 또는 이들 모두를 포함하는 것일 수 있다. 상기 언급된 배지 내 성분들의 농도는 적절히 변경가능하며, MRS 배지는 상업적으로 구매 가능한 MRS라 불리는 모든 배지를 포함하는 개념일 수 있다.The culture medium is the Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, or a composite strain thereof in an appropriate medium (e.g., R2A medium, TSA medium, or MRS medium) at a temperature of more than 10°C or less than 40°C. It may be obtained by culturing at a certain temperature for a certain period of time, for example, 4 to 50 hours. MRS medium is any one selected from the group consisting of peptone, beef extract, yeast extract, glucose, sodium acetate, polysorbate 80, dipotassium hydrogen phosphate, ammonium citrate, magnesium sulfate, manganese sulfate, agar, and distilled water. It may include a combination of two or more, or all of them. The concentrations of the components in the above-mentioned medium can be appropriately changed, and the MRS medium may be a concept that includes all commercially available media called MRS.
일 구체예에서, 균주의 배양 상층액은 균주 배양액을 원심분리나 여과시켜 균주를 제거하는 단계에 의해 수득될 수 있다.In one embodiment, the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture medium to remove the strain.
본 명세서에서 용어 "배양액 추출물"은 상기 배양액 또는 그의 농축액로부터 추출한 것을 의미하며, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들 조정제물 또는 정제물, 이를 분획한 분획물을 포함할 수 있다. As used herein, the term "culture extract" refers to an extract from the culture medium or its concentrate, and may include extracts, diluted or concentrated extracts, dried products obtained by drying the extracts, crude or purified products thereof, and fractions thereof. You can.
상기 락토바실러스 람노서스 KHM201 균주, 이의 파쇄액, 배양액 및 배양액의 추출물은 피부 상태 개선 효과가 있는 것일 수 있다.The Lactobacillus rhamnosus KHM201 strain, its lysate, culture medium, and extract of the culture medium may have an effect of improving skin condition.
상기 락토바실러스 루테리 KMH202 균주, 이의 파쇄액, 배양액 및 배양액의 추출물은 피부 상태 개선 효과가 있는 것일 수 있다.The Lactobacillus reuteri KMH202 strain, its lysate, culture medium, and extract of the culture medium may have an effect of improving skin condition.
상기 락토바실러스 플란타룸 KMH203 균주, 이의 파쇄액, 배양액 및 배양액의 추출물은 피부 상태 개선 효과가 있는 것일 수 있다.The Lactobacillus plantarum KMH203 strain, its lysate, culture medium, and extract of the culture medium may have an effect of improving skin condition.
일 구체예에 따르면, 상기 피부 상태는 피부 노화, 피부 주름, 피부 탄력, 피부 장벽, 피부 재생, 또는 피부 미백일 수 있다.According to one embodiment, the skin condition may be skin aging, skin wrinkles, skin elasticity, skin barrier, skin regeneration, or skin whitening.
본 명세서에서 용어 "피부 노화"란 나이가 들어가면서 피부에 나타나게 되는 유형과 무형상의 변화를 통틀어 말하는 것으로, 예컨대 표피 두께가 얇아지는 현상, 진피의 세포 수나 혈관 수, DNA 손상복구 능력, 세포교체주기, 상처 회복, 피부장벽기능, 표피의 수분 유지, 땀분비, 피지분비, 비타민D 생산, 물리적 손상방어, 화학물질 제거능력, 면역반응, 감각 기능, 체온조절의 감소를 말한다.As used herein, the term "skin aging" refers to both tangible and intangible changes that occur in the skin as one ages, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to a decrease in wound recovery, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical removal ability, immune response, sensory function, and temperature regulation.
상기 피부 노화는 구체적으로는 자외선, 공해, 담배연기, 화학물질 등에 의한 외부 자극에 의해 유도되는 조기 노화 증상뿐 아니라, 나이가 들어감에 의해 피부세포의 증식이 감소함에 따라 발생하는 자연노화 현상을 포함하며 주름, 탄력 감소, 피부 쳐짐 및 건조 현상 등을 모두 포함하는 개념이다. 또한 주름은 내ㆍ외부 요인의 변화에 의한 자극이 피부조직을 구성하고 있는 성분을 변화시켜 주름을 유발하는 것을 포함한다.The skin aging specifically includes premature aging symptoms induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemicals, etc., as well as natural aging phenomenon that occurs as skin cell proliferation decreases with age. It is a concept that includes wrinkles, loss of elasticity, skin sagging, and dryness. In addition, wrinkles include stimulation caused by changes in internal and external factors that change the components that make up skin tissue, causing wrinkles.
상기 노화는 광노화일 수 있다. 용어 "광노화(Photoaging)"는 외부 환경적인 요인에 의해 유발되는 현상으로, 가장 대표적인 인자로는 자외선이 있다. 자외선은 단백질 분해효소의 활성화와 기질단백질의 사슬절단 및 비정상적인 교차결합 등의 생체 구성 성분들의 손상을 가져오고, 이러한 메커니즘의 반복은 외관상으로도 확연한 피부노화를 초래하게 된다.The aging may be photoaging. The term “photoaging” is a phenomenon caused by external environmental factors, the most representative factor being ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cutting of matrix proteins, and abnormal cross-linking, and repetition of this mechanism causes skin aging that is evident in appearance.
본 명세서에서 용어 "주름"은 피부의 탄력성이 상실되어 느슨해진 상태를 의미하며, 예를 들면 피부가 접히는 것일 수 있다. 색소 침착은 체내에 있는 색소의 양이 비정상적인 경우 또는 색소가 나타나는 장소에 이상이 있는 상태를 의미하며, 예를 들면 기미 및 주근깨 등인 것일 수 있다. 상기 "피부 주름 예방 또는 개선"이란 주름과 관련된 인자의 발현을 억제하여 주름을 방지 또는 개선하거나, 콜라겐 총량을 증가시키는 모든 작용을 의미할 수 있다.As used herein, the term “wrinkle” refers to a state in which the skin loses its elasticity and becomes loose, for example, the skin may be folded. Pigmentation refers to a condition in which the amount of pigment in the body is abnormal or there is an abnormality in the area where the pigment appears, for example, blemishes and freckles. The term “prevention or improvement of skin wrinkles” may refer to any action that prevents or improves wrinkles by suppressing the expression of factors related to wrinkles, or increases the total amount of collagen.
상기 "피부 장벽 강화"는 피부 가장 바깥쪽에 위치하여 수분과 영양 손실을 막아주는 피부 장벽의 기능이 증진되는 모든 작용을 의미할 수 있다.The “skin barrier strengthening” may refer to any action that improves the function of the skin barrier, which is located on the outermost layer of the skin and prevents moisture and nutrition loss.
상기 "피부 미백"은 멜라닌 색소의 합성을 저해함으로써 피부 톤을 밝게 할 뿐만 아니라, 자외선, 호르몬 또는 유전에 기인한 기미나 주근깨 등의 피부 과색소 침착을 개선하는 것을 의미할 수 있다.The “skin whitening” may mean not only brightening skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as spots or freckles caused by ultraviolet rays, hormones, or genetics.
따라서, 상기 조성물은 COL1A1(collagen, type I, alpha 1), 또는 COL3A1(collagen, type III, alpha 1)의 발현을 촉진시켜 콜라겐 합성을 촉진시키고, MMP1(Matrix metalloproteinase-1)의 발현을 억제시켜 콜라겐 분해를 억제시킴으로써 피부 주름 또는 피부 탄력을 개선하는 효과가 있을 수 있다. Therefore, the composition promotes collagen synthesis by promoting the expression of COL1A1 (collagen, type I, alpha 1) or COL3A1 (collagen, type III, alpha 1) and inhibits the expression of MMP1 (Matrix metalloproteinase-1). By inhibiting collagen breakdown, it may have the effect of improving skin wrinkles or skin elasticity.
상기 조성물은 필라그린(Filaggrin)의 발현을 촉진시켜 피부 장벽을 강화시키는 효과가 있을 수 있다.The composition may have the effect of strengthening the skin barrier by promoting the expression of filaggrin.
상기 조성물은 상처 수복률을 향상시켜 피부 재생 효과를 향상시키는 것일 수 있다.The composition may improve the skin regeneration effect by improving the wound healing rate.
상기 조성물은 멜라닌(melanin) 생성을 억제함으로써, 피부 톤 개선 효과, 예를 들면 미백 효과가 있을 수 있다.The composition may have a skin tone improvement effect, for example, a whitening effect, by suppressing melanin production.
다른 양상은 상기 조성물을 유효성분으로 포함하는 화장료 조성물을 제공한다. Another aspect provides a cosmetic composition containing the composition as an active ingredient.
상기 조성물은 상기한 바와 같다.The composition is as described above.
본 명세서에서 용어, "유효성분으로 포함"은 상기에서 언급한 효과를 나타낼 수 있는 정도로 본 명세서의 균주, 상기 균주의 파쇄액, 배양액, 또는 이의 배양액의 추출물이 첨가되는 것을 의미하고, 안정화 등을 위하여 다양한 성분을 부성분으로 첨가하여 다양한 형태로 포뮬레이션 (formulation)되는 것을 포함하는 의미이다.As used herein, the term "included as an active ingredient" means that the strain of the present specification, the lysate of the strain, the culture medium, or the extract of the culture medium are added to an extent that can exhibit the effects mentioned above, stabilization, etc. This means that it is formulated into various forms by adding various ingredients as sub-ingredients.
일 구체예에 따르면, 상기 화장료 조성물은 피부 상태 개선용일 수 있다.According to one embodiment, the cosmetic composition may be used to improve skin condition.
상기 피부 상태는 상기한 바와 같다.The skin condition is as described above.
상기 화장료 조성물은 예를 들면, 유연화장수, 영양화장수, 마사지크림, 영양크림, 에센스, 팩, 젤, 앰플 또는 피부 점착 타입의 화장료 제형을 갖는 것일 수 있다.The cosmetic composition may have, for example, an softening lotion, nourishing lotion, massage cream, nourishing cream, essence, pack, gel, ampoule, or skin-adhesive type cosmetic formulation.
상기 화장료 조성물은 화장품에 통상 사용되는 추가 성분을 더 포함할 수 있다. 당업자는 본 명세서에 따른 조성물의 유리한 특성이 예상된 첨가에 의해 악영향을 받지 않거나 실질적으로 받지 않도록, 임의의 추가 성분 및/또는 이의 양을 선택할 수 있다.The cosmetic composition may further include additional ingredients commonly used in cosmetics. A person skilled in the art will be able to select any additional ingredients and/or their amounts such that the advantageous properties of the compositions according to the present disclosure are not or substantially not adversely affected by the envisaged additions.
또한, 상기 화장료 조성물은 단독 또는 중복으로 도포하여 사용하거나, 본 발명 이외의 다른 화장료 조성물과 중복 도포하여 사용할 수 있다. 또한, 본 발명에 따른 화장료 조성물은 통상적인 사용방법에 따라 사용될 수 있으며, 사용자의 피부 상태 또는 취향에 따라 그 사용횟수를 달리할 수 있다.Additionally, the cosmetic composition may be used alone or in combination, or may be used in combination with other cosmetic compositions other than those of the present invention. In addition, the cosmetic composition according to the present invention can be used according to conventional usage methods, and the number of times of use can be varied depending on the user's skin condition or preference.
또 다른 양상은 상기 조성물을 유효성분으로 포함하는 건강기능식품을 제공한다.Another aspect provides a health functional food containing the composition as an active ingredient.
상기 조성물은 상기한 바와 같다.The composition is as described above.
일 구체예에 따르면, 상기 건강기능식품은 피부 상태 개선용일 수 있다.According to one embodiment, the health functional food may be used to improve skin condition.
상기 피부 상태는 상기한 바와 같다.The skin condition is as described above.
상기 건강기능식품은 상기 균주 또는 이의 배양액 단독 또는 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 명세서의 조성물은 원료에 대하여 15 중량부 이하의 양으로 첨가될 수 있다. 상기 건강기능식품의 종류에는 특별한 제한은 없다. 건강기능식품의 종류 중 음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 건강식품 조성물은 또한 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제, 또는 그 조합을 함유할 수 있다. 상기 건강기능식품 조성물은 또한, 천연 과일쥬스, 과일쥬스 음료, 야채 음료의 제조를 위한 과육, 또는 그 조합을 함유할 수 있다.The health functional food can be used alone or with other foods or food ingredients, such as the strain or its culture medium, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw materials. There are no particular restrictions on the types of health functional foods. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The health food composition also contains nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. It may contain the carbonating agent used, or a combination thereof. The health functional food composition may also contain pulp for the production of natural fruit juice, fruit juice beverage, vegetable beverage, or a combination thereof.
일 양상에 따른 신규한 락토바실러스 람노서스, 락토바실러스 루테리, 락토바실러스 플란타룸 균주 및 이의 복합물에 의하면, 피부 상태 개선 효과가 있다. 예를 들면, 피부 주름 개선, 피부 탄력 개선, 피부 노화 억제, 피부 재생, 피부 장벽 강화, 및 미백 효과가 있다. According to one aspect, the novel Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus plantarum strains and their complexes have an effect of improving skin condition. For example, it has the effect of improving skin wrinkles, improving skin elasticity, inhibiting skin aging, skin regeneration, strengthening the skin barrier, and whitening.
도 1은 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203 각각의 균주, 및 이의 복합 균주의 처리에 따른 COL1A1 및 COL3A1의 mRNA 발현 정도를 나타낸 그래프이다.
도 2는 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 처리에 따른 COL1A1 및 MMP1의 mRNA 발현 정도를 나타낸 그래프이다.
도 3은 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 발효 일 수에 따른 필라그린 mRNA의 발현 정도를 나타낸 그래프이다.
도 4는 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 농도에 따른 필라그린 mRNA의 발현 정도를 나타낸 그래프이다.
도 5는 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 처리에 따른 상처 수복률을 나태낸 그래프 및 이미지이다.
도 6은 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 처리에 따른 멜라닌 생성 억제 효과를 나타낸 그래프이다.Figure 1 is a graph showing the mRNA expression levels of COL1A1 and COL3A1 according to treatment of each strain of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203, and a composite strain thereof.
Figure 2 is a graph showing the mRNA expression levels of COL1A1 and MMP1 according to treatment of complex strains of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
Figure 3 is a graph showing the expression level of filaggrin mRNA according to the number of days of fermentation of the complex strain of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
Figure 4 is a graph showing the expression level of filaggrin mRNA according to the concentration of complex strains of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
Figure 5 is a graph and image showing the wound recovery rate according to treatment with complex strains of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
Figure 6 is a graph showing the effect of suppressing melanin production according to the treatment of complex strains of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
이하 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. This will be described in more detail through examples below. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.
실시예 1. 균주의 분리 및 동정Example 1. Isolation and identification of strains
1.1 락토바실러스 람노서스 KMH201의 분리 및 동정1.1 Isolation and identification of Lactobacillus rhamnosus KMH201
수제요거트로부터 균주를 분리 및 동정하기 위해 다음과 같이 수행하였다.To isolate and identify strains from homemade yogurt, the following was performed.
먼저, 수제요거트 1 g과 MRS media 20 ml을 혼합한 후, vortexing하여 시료를 현탁하였다. 그 후, 현탁된 시료를 멸균증류수를 이용하여 연속희석(Serial dilution)하였고, MRS agar plate에 10-3 내지 10-5으로 pour plating하였다. 이 후, 37 ℃에서 1 일 이상 배양하여 균을 선별하였다.First, 1 g of homemade yogurt and 20 ml of MRS media were mixed and then vortexed to suspend the sample. Afterwards, the suspended sample was serially diluted using sterilized distilled water and poured onto an MRS agar plate at a concentration of 10 -3 to 10 -5 . Afterwards, the bacteria were selected by culturing at 37°C for more than 1 day.
선별한 균체 집락 중 결정된 16S rRNA부위의 염기서열을 미국 국립생물정보센터(NCBI, National Center for Biotechnology Information) 홈페이지에서 제공되는 BLAST 프로그램으로 등록된 다른 균주들과 비교 분석하였다. 비교 분석 결과 상동성 99%의 락토바실러스 람노서스 KMH201을 분리하였다. 선별된 락토바실러스 람노서스 KMH201 균주를 2022년 02월 25일자로 한국생물자원센터에 기탁하여 기탁번호 KCTC 14877BP를 부여받았고, 락토바실러스 람노서스 KMH201 균주는 서열번호 1(complementary DNA)의 16s rRNA 서열을 갖는다.The base sequence of the 16S rRNA region determined among the selected bacterial colonies was compared and analyzed with other strains registered with the BLAST program provided on the US National Center for Biotechnology Information (NCBI) website. As a result of comparative analysis, Lactobacillus rhamnosus KMH201 with 99% homology was isolated. The selected Lactobacillus rhamnosus KMH201 strain was deposited at the Korea Center for Biological Resources on February 25, 2022 and given the deposit number KCTC 14877BP. The Lactobacillus rhamnosus KMH201 strain has the 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA). have
1.2 락토바실러스 루테리 KMH202의 분리 및 동정1.2 Isolation and identification of Lactobacillus reuteri KMH202
석류로부터 균주를 분리 및 동정하기 위해 다음과 같이 수행하였다.To isolate and identify strains from pomegranate, the following procedures were performed.
먼저, 껍질 채 파쇄한 석류 1 g과 MRS media 20 ml을 혼합 후 vortexing하여 시료를 현탁하였다. 그 후, 현탁된 시료를 멸균증류수를 이용하여 연속희석 하였고, MRS agar plate에 10-4 내지 10-6으로 pour plating하였다. 이 후, 37 ℃에서 1 일 이상 배양하여 균을 선별하였다.First, 1 g of shredded pomegranate and 20 ml of MRS media were mixed and vortexed to suspend the sample. Afterwards, the suspended sample was serially diluted using sterilized distilled water, and poured and plated at 10 -4 to 10 -6 on an MRS agar plate. Afterwards, the bacteria were selected by culturing at 37°C for more than 1 day.
선별한 균체 집락 중 결정된 16S rRNA부위의 염기서열을 미국 국립생물정보센터(NCBI, National Center for Biotechnology Information) 홈페이지에서 제공되는 BLAST 프로그램으로 등록된 다른 균주들과 비교 분석하였다. 비교 분석 결과 상동성 99%의 락토바실러스 루테리 KMH202를 분리하였다. 선별된 락토바실러스 루테리 KMH202 균주를 2022년 02월 25일자로 한국생물자원센터에 기탁하여 기탁번호 KCTC 14878BP를 부여받았고, 락토바실러스 루테리 KMH202 균주는 서열번호 2(complementary DNA)의 16s rRNA 서열을 갖는다.The base sequence of the 16S rRNA region determined among the selected bacterial colonies was compared and analyzed with other strains registered with the BLAST program provided on the US National Center for Biotechnology Information (NCBI) website. As a result of comparative analysis, Lactobacillus reuteri KMH202 with 99% homology was isolated. The selected Lactobacillus reuteri KMH202 strain was deposited at the Korea Center for Biological Resources on February 25, 2022 and given the deposit number KCTC 14878BP, and the Lactobacillus reuteri KMH202 strain has a 16s rRNA sequence of SEQ ID NO: 2 (complementary DNA).
1.3 락토바실러스 플란타룸 KMH203의 분리 및 동정1.3 Isolation and identification of Lactobacillus plantarum KMH203
샤인머스켓으로부터 균주를 분리 및 동정하기 위해 다음과 같이 수행하였다.To isolate and identify strains from Shine Musket, the following was performed.
먼저, 껍질 채 파쇄한 샤인머스켓 1 g과 MRS media 20 ml을 혼합 후 vortexing하여 시료를 현탁하였다. 그 후, 현탁된 시료를 멸균증류수를 이용하여 연속희석하였고, MRS agar plate에 10-4 내지 10-6으로 pour plating하였다. 이 후, 37 ℃에서 1 일 이상 배양하여 균을 선별하였다.First, 1 g of shredded Shine Musket was mixed with 20 ml of MRS media and then vortexed to suspend the sample. Afterwards, the suspended sample was serially diluted using sterilized distilled water, and poured and plated at 10 -4 to 10 -6 on an MRS agar plate. Afterwards, the bacteria were selected by culturing at 37°C for more than 1 day.
선별한 균체 집락 중 결정된 16S rRNA부위의 염기서열을 미국 국립생물정보센터(NCBI, National Center for Biotechnology Information) 홈페이지에서 제공되는 BLAST 프로그램으로 등록된 다른 균주들과 비교 분석하였다. 비교 분석 결과 상동성 99%의 락토바실러스 플란타룸 KMH203을 분리하였다. 선별된 락토바실러스 플란타룸 KMH203 균주를 2022년 02월 25일자로 한국생물자원센터에 기탁하여 기탁번호 KCTC 14879BP를 부여받았고, 락토바실러스 플란타룸 KMH203 균주는 서열번호 3(complementary DNA)의 16s rRNA 서열을 갖는다.The base sequence of the 16S rRNA region determined among the selected bacterial colonies was compared and analyzed with other strains registered with the BLAST program provided on the US National Center for Biotechnology Information (NCBI) website. As a result of comparative analysis, Lactobacillus Plantarum KMH203 with 99% homology was isolated. The selected Lactobacillus Plantarum KMH203 strain was deposited at the Korea Center for Biological Resources on February 25, 2022 and given the deposit number KCTC 14879BP, and the Lactobacillus Plantarum KMH203 strain has 16s rRNA of SEQ ID NO: 3 (complementary DNA). It has a hierarchy.
실시예 2. 분리된 균주 및 이의 복합 균주의 발효 및 배양Example 2. Fermentation and cultivation of isolated strains and complex strains thereof
2.1 락토바실러스 람노서스 KMH201의 수득2.1 Obtainment of Lactobacillus rhamnosus KMH201
상기 실시예 1에서 분리된 락토바실러스 람노서스 KMH201을 MRS 배지에서 배양하였다.Lactobacillus rhamnosus KMH201 isolated in Example 1 was cultured in MRS medium.
구체적으로, 플라스크에 1 리터를 기준으로 각 55 g의 MRS 배지를 물과 혼합하여 121 ℃의 조건에서 15 분 동안 멸균하고, 상온에서 상기 플라스크를 보관하였다. 그리고, 동결 보관된 락토바실러스 람노서스 KMH201 400 μl를 상기 플라스크의 MRS 배지에 접종하였다. 상기 MRS 배지를 37 ℃의 조건의 혐기 배양기에서 약 1 일 동안 배양 및 발효하여 배양액을 수득하고, 상기 배양액을 원심분리(4000 rpm, 10 분)하여 균체를 수득하였다. Specifically, 55 g of MRS medium per 1 liter of flask was mixed with water and sterilized at 121°C for 15 minutes, and the flask was stored at room temperature. Then, 400 μl of frozen Lactobacillus rhamnosus KMH201 was inoculated into the MRS medium of the flask. The MRS medium was cultured and fermented in an anaerobic incubator at 37°C for about 1 day to obtain a culture solution, and the culture solution was centrifuged (4000 rpm, 10 minutes) to obtain bacterial cells.
2.2 락토바실러스 루테리 KMH202의 수득2.2 Obtainment of Lactobacillus reuteri KMH202
상기 실시예 2.1과 동일한 방법으로 실시예 1에서 분리된 락토바실러스 루테리 KMH202를 MRS 배지에서 배양하고, 균체를 수득하였다.Lactobacillus reuteri KMH202 isolated in Example 1 was cultured in MRS medium in the same manner as Example 2.1, and bacterial cells were obtained.
2.3 락토바실러스 플란타룸 KMH203의 수득2.3 Obtainment of Lactobacillus plantarum KMH203
상기 실시예 2.1과 동일한 방법으로 실시예 1에서 분리된 락토바실러스 플란타룸 KMH203을 MRS 배지에서 배양하고, 균체를 수득하였다.Lactobacillus Plantarum KMH203 isolated in Example 1 was cultured in MRS medium in the same manner as Example 2.1, and bacterial cells were obtained.
2.4 단독 균주의 발효 및 배양2.4 Fermentation and cultivation of single strains
상기 실시예 2.1 내지 2.3에서 수득한 균주를 각각 37 ℃의 조건의 혐기 배양기에서 약 1 일 동안 배양 및 발효하여 배양액을 회수하였다. 상기 회수한 배양액을 원심분리(4000 rpm, 10 분)하여 배양상등액(상층액)을 수득하였다. 수득한 배양상등액에 대해 0.22 μM 필터를 통해 필터멸균을 수행함으로써, 각 균주에 대한 무색 무취의 멸균된 배양 상등액을 수득하였다.The strains obtained in Examples 2.1 to 2.3 were each cultured and fermented in an anaerobic incubator at 37°C for about 1 day to recover the culture solution. The recovered culture medium was centrifuged (4000 rpm, 10 minutes) to obtain a culture supernatant (supernatant). By filter sterilizing the obtained culture supernatant through a 0.22 μM filter, colorless and odorless sterilized culture supernatant for each strain was obtained.
2.5 3 종의 복합 균주의 발효 및 배양2.5 Fermentation and cultivation of three complex strains
상기 실시예 2.1 내지 2.3에서 수득한 균주를 1 : 1 : 1의 중량비로 혼합하고, 37 ℃의 조건의 혐기 배양기에서 약 1 일 동안 배양 및 발효하여 배양액을 회수하였다. 상기 회수한 배양액을 원심분리(4000 rpm, 10 분)하여 배양상등액(상층액)을 수득하였다. 수득한 배양상등액에 대해 0.22 μM 필터를 통해 필터멸균을 수행함으로써, 무색 무취의 멸균된 배양 상등액을 수득하였다.The strains obtained in Examples 2.1 to 2.3 were mixed at a weight ratio of 1:1:1, and cultured and fermented in an anaerobic incubator at 37°C for about 1 day to recover the culture solution. The recovered culture medium was centrifuged (4000 rpm, 10 minutes) to obtain a culture supernatant (supernatant). The obtained culture supernatant was filter sterilized through a 0.22 μM filter to obtain a colorless, odorless sterilized culture supernatant.
실험예 1. 항주름 효과 확인Experimental Example 1. Confirmation of anti-wrinkle effect
실시예 2에서 분리 및 배양한 균주 및 이의 복합 균주의 항주름 효과를 확인하기 위해, 인간 섬유아세포(human fibroblast)인 Hs68 세포주에서 상기 균주 및 복합 균주의 처리에 따른 COL1A1(collagen type 1, alpha 1), COL3A1(collagen, type 3, alpha 1), MMP1(matrix metalloproteinase-1) 및 β-actin의 mRNA 발현량을 real-time PCR로 확인하였다.In order to confirm the anti-wrinkle effect of the strain isolated and cultured in Example 2 and its complex strain, COL1A1 (collagen type 1, alpha 1) was observed in the Hs68 cell line, which is a human fibroblast, upon treatment of the strain and the complex strain. ), COL3A1 (collagen, type 3, alpha 1), MMP1 (matrix metalloproteinase-1), and β-actin mRNA expression levels were confirmed by real-time PCR.
우선, 상기 Hs68 세포를 6 well 세포배양 플레이트(plate)에 5 x 105 cells/well의 밀도로 접종한 후, 37 ℃, 5 % CO2 조건의 배양기에서 24 시간 동안 배양하였다. 상기 복합 균주를 FBS를 포함하지 않는 배지(FBS free)로 희석한 후, 상기 플레이트에 첨가한 뒤 20 시간 동안 배양하였다. RNA는 RNeasy Mini Kit (QIAGEN)를 사용하여 분리하였으며, 분리한 RNA는 NanoDrop Onec(Thermo Scientific)를 이용해 순도(purity)를 확인한 후 정량하여 SuperScript IV VILO Master Mix(Invitrogen)를 사용하여 cDNA로 합성하였다. RT-PCR 장비로는 Step One Plus(applied biosystems)를 사용하였으며, PowerUp SYBR Green Master Mix(applied biosystems)를 합성한 cDNA 및 프라이머(COL3A1, MMP1, 또는 β-actin)를 함께 첨가하여, 95 ℃에서 2 분간 중합효소를 활성화시킨 후 중합반응을 진행하였다: 총 40 사이클(1 사이클당 95 ℃에서 15 초, 55 ℃에서 15 초, 72 ℃에서 1 분). 대조군으로 D-판테놀(D-panthenol)을 사용하였고, 사용된 프라이머의 서열은 하기 표 1과 같다.First, the Hs68 cells were inoculated into a 6-well cell culture plate at a density of 5 x 10 5 cells/well, and then cultured for 24 hours in an incubator at 37°C and 5% CO 2 conditions. The complex strain was diluted with medium not containing FBS (FBS free), added to the plate, and cultured for 20 hours. RNA was isolated using the RNeasy Mini Kit (QIAGEN), and the purity of the isolated RNA was confirmed using NanoDrop Onec (Thermo Scientific), quantified, and synthesized into cDNA using SuperScript IV VILO Master Mix (Invitrogen). . Step One Plus (applied biosystems) was used as the RT-PCR equipment, and cDNA and primers (COL3A1, MMP1, or β-actin) synthesized with PowerUp SYBR Green Master Mix (applied biosystems) were added together, and incubated at 95°C. After activating the polymerase for 2 minutes, the polymerization reaction was performed: a total of 40 cycles (15 seconds at 95°C, 15 seconds at 55°C, and 1 minute at 72°C per cycle). D-panthenol was used as a control, and the sequences of the primers used are shown in Table 1 below.
락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203, 이의 복합 균주에 대한 소재 번호 및 발효 일수는 하기 표 2에 나타내었다.The material numbers and fermentation days for Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202 and Lactobacillus plantarum KMH203, and their complex strains are shown in Table 2 below.
도 1은 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203 각각의 균주, 및 이의 복합 균주의 처리에 따른 COL1A1 및 COL3A1의 mRNA 발현 정도를 나타낸 그래프이다. Figure 1 is a graph showing the mRNA expression levels of COL1A1 and COL3A1 according to treatment of each strain of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203, and a composite strain thereof.
도 2는 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 처리에 따른 MMP1의 mRNA 발현 정도를 나타낸 그래프이다.Figure 2 is a graph showing the mRNA expression level of MMP1 according to treatment of complex strains of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
실험예 2. 피부 장벽 강화 효과 확인Experimental Example 2. Confirmation of skin barrier strengthening effect
실시예 2에서 분리 및 배양한 복합 균주의 피부 장벽 강화 효과를 확인하기 위해, 인간 각질형성세포(human keratinocyte)인 HaCaT 세포주에서 필라그린(Filaggrin) 유전자의 mRNA 발현량을 real-time PCR로 확인하였다. In order to confirm the skin barrier strengthening effect of the complex strain isolated and cultured in Example 2, the mRNA expression level of the filaggrin gene in the HaCaT cell line, a human keratinocyte, was confirmed by real-time PCR. .
우선, 상기 HaCaT 세포를 6 well 세포배양 플레이트에 3 x 105 cells/well의 밀도로 접종한 후, 37 ℃, 5 % CO2 조건의 배양기에서 24 시간 동안 배양하였다. 상기 복합 균주를 FBS free 배지로 희석한 후, 상기 플레이트에 첨가하여 20 시간 동안 배양하였다. RNA는 RNeasy Mini Kit (QIAGEN)를 사용하여 분리하였으며, 분리한 RNA는 NanoDrop Onec(Thermo Scientific)를 이용해 순도를 확인한 후 정량하여 SuperScript IV VILO Master Mix(Invitrogen)를 사용하여 cDNA로 합성하였다. RT-PCR 장비로는 Step One Plus(applied biosystems)를 사용하였으며, PowerUp SYBR Green Master Mix(applied biosystems)를 합성한 cDNA 및 프라이머(filaggrin 또는 β-actin)를 함께 첨가하여, 95 ℃에서 2 분간 중합효소를 활성화시킨 후, 중합반응을 진행하였다: 총 40 사이클(1 사이클당 95 ℃에서 15 초, 55 ℃에서 15 초, 72 ℃에서 1 분). 대조군으로 D-판테놀을 사용하였고, 사용된 프라이머의 서열은 하기 표 3과 같다.First, the HaCaT cells were inoculated into a 6-well cell culture plate at a density of 3 x 10 5 cells/well, and then cultured in an incubator at 37°C and 5% CO 2 for 24 hours. The complex strain was diluted with FBS free medium, added to the plate, and cultured for 20 hours. RNA was isolated using the RNeasy Mini Kit (QIAGEN), and the purity of the isolated RNA was confirmed using NanoDrop Onec (Thermo Scientific), quantified, and synthesized into cDNA using SuperScript IV VILO Master Mix (Invitrogen). Step One Plus (applied biosystems) was used as the RT-PCR equipment, cDNA synthesized with PowerUp SYBR Green Master Mix (applied biosystems) and primer (filaggrin or β-actin) were added together and polymerized at 95°C for 2 minutes. After activating the enzyme, the polymerization reaction was performed: a total of 40 cycles (95°C for 15 seconds, 55°C for 15 seconds, and 72°C for 1 minute per cycle). D-panthenol was used as a control, and the sequences of the primers used are shown in Table 3 below.
도 3은 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 발효 일 수에 따른 필라그린 mRNA의 발현 정도를 나타낸 그래프이다.Figure 3 is a graph showing the expression level of filaggrin mRNA according to the number of days of fermentation of the complex strain of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
도 4는 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 농도에 따른 필라그린 mRNA의 발현 정도를 나타낸 그래프이다.Figure 4 is a graph showing the expression level of filaggrin mRNA according to the concentration of complex strains of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
도 3에 나타낸 바와 같이, 필라그린 mRNA의 발현 정도가 상기 복합 균주의 처리에 따라 증가하며, 특히, 상기 복합 균주의 발효 일 수에 비례하여 증가하는 것을 확인하였다.As shown in Figure 3, it was confirmed that the expression level of filaggrin mRNA increased with treatment of the complex strain, and in particular, increased in proportion to the number of days of fermentation of the complex strain.
도 4에 나타낸 바와 같이, 필라그린 mRNA의 발현 정도가 상기 복합 균주의 농도에 비례하여 증가하는 것을 확인하였다. 특히, 1.25 % 농도의 복합 균주의 경우 판테놀 대비 2 배 이상의 필라그린 mRNA가 발현되어, 현저한 피부 장벽 강화 효과가 있음을 확인하였다.As shown in Figure 4, it was confirmed that the expression level of filaggrin mRNA increased in proportion to the concentration of the complex strain. In particular, in the case of the complex strain at a concentration of 1.25%, filaggrin mRNA was expressed more than twice that of panthenol, confirming that it had a significant skin barrier strengthening effect.
실험예 3. 피부 재생 효과 확인Experimental Example 3. Confirmation of skin regeneration effect
실시예 2에서 분리 및 배양한 복합 균주의 피부 재생 효과를 확인하기 위해, 인간 각질형성세포인 HaCaT 세포주를 사용하여 상처 치유 분석(wound healing assay)을 수행하였다. To confirm the skin regenerative effect of the complex strain isolated and cultured in Example 2, a wound healing assay was performed using the HaCaT cell line, a human keratinocyte.
보다 구체적으로, 상기 HaCaT 세포를 6 well 세포배양 플레이트에 3 x 104 cells/well의 밀도로 접종한 후, 37 ℃, 5 % CO2 조건의 배양기에서 24 시간 동안 배양하였다. 그리고 96-well wound maker(sartorius stedim biotech)를 이용하여 상처를 생성한 뒤, PBS로 2 번 세척하였다. 이후 FBS free 배지로 교체한 뒤, 상기 복합 균주를 2.5%(v/v) 농도가 되도록 첨가한 후, 37 ℃, 5% CO2 조건에서 8 시간 동안 배양하였다. 그리고 IncuCyte(Satorius)를 사용하여 배양한 HaCaT 세포의 세포 사진을 촬영하였다. 대조군으로는 균주를 처리하지 않은 세포주를 사용하였다. More specifically, the HaCaT cells were inoculated into a 6-well cell culture plate at a density of 3 x 10 4 cells/well, and then cultured for 24 hours in an incubator at 37°C and 5% CO 2 conditions. Then, a wound was created using a 96-well wound maker (sartorius stedim biotech) and washed twice with PBS. After replacing with FBS free medium, the complex strain was added to a concentration of 2.5% (v/v) and cultured for 8 hours at 37°C and 5% CO 2 conditions. Then, cell photos of cultured HaCaT cells were taken using IncuCyte (Satorius). As a control group, a cell line that was not treated with the strain was used.
상처 수복률(wound closure rate)은 Incucyte Software(Scratch Wound Cell Migration and Invastion Analysis Metrics)를 사용하여 계산하였으며, 계산식은 하기 식 1과 같다: Wound closure rate was calculated using Incucyte Software (Scratch Wound Cell Migration and Invastion Analysis Metrics), and the calculation formula is as follows:
[식 1][Equation 1]
상처 수복률(%) = [(8 시간 세포면적 - 0 시간 세포면적)/(전체면적 - 0 시간 세포면적)] x 100.Wound repair rate (%) = [(cell area at 8 hours - cell area at 0 hours)/(total area - cell area at 0 hours)] x 100.
도 5는 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 처리에 따른 상처 수복률을 나태낸 그래프 및 이미지이다.Figure 5 is a graph and image showing the wound recovery rate according to treatment with complex strains of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
도 5에 나타낸 바와 같이, 복합 균주를 처리함에 따라 상처 수복 효과가 현저히 증가함을 확인하였다. As shown in Figure 5, it was confirmed that the wound repair effect significantly increased as the complex strain was treated.
실험예 4. 미백 효과 확인Experimental Example 4. Confirmation of whitening effect
실시예 2에서 분리 및 배양한 복합 균주의 미백 효과 확인을 위해, 멜라닌 세포에서 멜라닌 생합성량을 정량화하였다.To confirm the whitening effect of the complex strain isolated and cultured in Example 2, the amount of melanin biosynthesis in melanocytes was quantified.
보다 구체적으로, B16F1 세포주(5 X 104 cell/ml)을 24 시간 배양하고, B16F1 세포의 배양액을 제거한 뒤 1 mL의 DPBS 로 세척하였다. 그 후, FBS를 포함하는 배지로 희석한 상기 복합 균주를 각 2 mL/well씩 B16F1 세포에 첨가하였다. 그 이후, 상기 B16F1 세포를 72 시간 동안 37 ℃, 5 % CO2의 조건으로 배양기(incubator)에서 배양하였다. 72 시간 동안 배양한 후, 상기 B16F1 세포에서 배양액을 제거한 후, 상기 B16F1 세포에 0.2 mL의 trypsin-EDTA를 처리하고 37 ℃, 5 % CO2의 조건으로 배양기에서 5 분 동안 반응시켰다. 그 후, 0.5 mL의 배양액을 첨가하고 세포를 회수한 뒤, 25 ℃, 10,000 rpm에서 10 분간 원심 분리하여 상층액을 제거하였다. 상층액을 제거한 후, DPBS 1 mL를 첨가하여 세척하고 25 ℃, 10,000 rpm에서 10 분간 다시 원심 분리하고, 상층액을 제거하여 펠릿(pellet)을 수득하였다. 상기 수득한 펠릿을 70 ℃의 건조 오븐(drying oven)에서 1시간 건조시켰다. 완전히 건조된 펠릿에 수산화 나트륨(NaOH) 용액 0.4 mL를 첨가한 후, 상기 펠릿을 70 ℃의 건조 오븐에서 10 분간 녹였다. 그 후, 펠릿을 교반기에서 1,800 rpm으로 5 분간 교반한 후, 96 well plate에 펠릿 100 uL을 분주하고 microplate reader 490 nm에서 흡광도를 측정하였다. 대조군으로는 알부틴(arbutin)을 사용하였으며, 그 결과를 도 6에 나타내었다.More specifically, the B16F1 cell line ( 5 Afterwards, 2 mL/well of the above complex strains diluted with medium containing FBS was added to B16F1 cells. Afterwards, the B16F1 cells were cultured in an incubator at 37°C and 5% CO 2 for 72 hours. After culturing for 72 hours, the culture medium was removed from the B16F1 cells, and the B16F1 cells were treated with 0.2 mL of trypsin-EDTA and incubated for 5 minutes in an incubator at 37°C and 5% CO 2 . Afterwards, 0.5 mL of culture medium was added, the cells were recovered, and the supernatant was removed by centrifugation at 25°C and 10,000 rpm for 10 minutes. After removing the supernatant, it was washed by adding 1 mL of DPBS, centrifuged again at 25°C and 10,000 rpm for 10 minutes, and the supernatant was removed to obtain a pellet. The obtained pellets were dried in a drying oven at 70°C for 1 hour. After adding 0.4 mL of sodium hydroxide (NaOH) solution to the completely dried pellet, the pellet was melted in a drying oven at 70°C for 10 minutes. Afterwards, the pellet was stirred in a stirrer at 1,800 rpm for 5 minutes, and then 100 uL of the pellet was dispensed into a 96 well plate and the absorbance was measured at 490 nm with a microplate reader. Arbutin was used as a control, and the results are shown in Figure 6.
도 6은 락토바실러스 람노서스 KMH201, 락토바실러스 루테리 KMH202 및 락토바실러스 플란타룸 KMH203의 복합 균주의 처리에 따른 멜라닌 생성 억제 효과를 나타낸 그래프이다.Figure 6 is a graph showing the effect of suppressing melanin production according to the treatment of complex strains of Lactobacillus rhamnosus KMH201, Lactobacillus reuteri KMH202, and Lactobacillus plantarum KMH203.
도 6에 나타낸 바와 같이, 멜라닌 생성 억제 효과가 상기 복합 균주의 처리에 따라 증가하며, 특히, 상기 복합 균주의 농도에 비례하여 증가하는 것을 확인하였다.As shown in Figure 6, it was confirmed that the melanin production inhibition effect increases with treatment of the complex strain, and in particular, increases in proportion to the concentration of the complex strain.
Claims (13)
기탁번호 KCTC14878BP로 기탁된 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 루테리 KMH202(Lactobacillus reuteri KMH202) 균주, 이의 파쇄액, 배양액, 및 배양액의 추출물로 이루어진 군으로부터 선택된 적어도 어느 하나; 및
기탁번호 KCTC14879BP로 기탁된 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 플란타룸 KMH203(Lactobacillus plantarum KMH203) 균주, 이의 파쇄액, 배양액 및 배양액의 추출물로 이루어진 군으로부터 선택된 적어도 어느 하나를 포함하는 조성물.At least one selected from the group consisting of the Lactobacillus rhamnosus KMH201 strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with accession number KCTC14877BP, its lysate, culture medium, and extract of the culture medium;
Lactobacillus reuteri KMH202 ( Lactobacillus reuteri KMH202) strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with accession number KCTC14878BP, at least one selected from the group consisting of its lysate, culture medium, and extract of the culture medium; and
A composition containing at least one selected from the group consisting of Lactobacillus plantarum KMH203 ( Lactobacillus plantarum KMH203) strain belonging to the Lactobacillus genus ( Lactobacillus sp .) deposited with accession number KCTC14879BP, its lysate, culture medium, and extract of the culture medium. .
상기 락토바실러스 람노서스 KMH201는 서열번호 1의 16s rRNA를 포함하는 것인, 조성물.In claim 1,
A composition wherein the Lactobacillus rhamnosus KMH201 includes 16s rRNA of SEQ ID NO: 1.
상기 락토바실러스 루테리 KMH202는 서열번호 2의 16s rRNA를 포함하는 것인, 조성물.In claim 1,
A composition wherein the Lactobacillus reuteri KMH202 includes 16s rRNA of SEQ ID NO: 2.
상기 락토바실러스 플란타룸 KMH203는 서열번호 3의 16s rRNA를 포함하는 것인, 조성물. In claim 1,
The composition of which the Lactobacillus Plantarum KMH203 includes 16s rRNA of SEQ ID NO: 3.
상기 균주는 생균 또는 사균인 것인, 조성물.In claim 1,
A composition wherein the strain is live or dead.
상기 조성물은 피부 상태 개선용인 것인, 조성물.In claim 1,
The composition is for improving skin condition.
상기 피부 상태는 피부 주름, 피부 탄력, 피부 장벽, 피부 재생, 또는 피부 미백인 것인, 조성물.In claim 6,
The composition, wherein the skin condition is skin wrinkles, skin elasticity, skin barrier, skin regeneration, or skin whitening.
상기 화장료 조성물은 피부 상태 개선용인 것인, 화장료 조성물.In claim 8,
The cosmetic composition is for improving skin condition.
상기 피부 상태는 피부 주름, 피부 탄력, 피부 장벽, 피부 재생, 또는 피부 미백인 것인, 화장료 조성물. In claim 9,
A cosmetic composition wherein the skin condition is skin wrinkles, skin elasticity, skin barrier, skin regeneration, or skin whitening.
상기 건강기능식품은 피부 상태 개선용인 것인, 건강기능식품. In claim 11,
The health functional food is a health functional food for improving skin condition.
상기 피부 상태는 피부 주름, 피부 탄력, 피부 장벽, 피부 재생, 또는 피부 미백인 것인, 건강기능식품.In claim 12,
A health functional food wherein the skin condition is skin wrinkles, skin elasticity, skin barrier, skin regeneration, or skin whitening.
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