KR20240002618A - Micrococcus flavus strain and skin condition improving uses of thereof - Google Patents
Micrococcus flavus strain and skin condition improving uses of thereof Download PDFInfo
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- KR20240002618A KR20240002618A KR1020220080016A KR20220080016A KR20240002618A KR 20240002618 A KR20240002618 A KR 20240002618A KR 1020220080016 A KR1020220080016 A KR 1020220080016A KR 20220080016 A KR20220080016 A KR 20220080016A KR 20240002618 A KR20240002618 A KR 20240002618A
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- strain
- skin
- culture medium
- micrococcus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
신규한 미생물, 그의 파쇄물, 배양액, 배양액의 추출물, 및 이들의 피부상태 개선 용도에 관한 것이다. It relates to novel microorganisms, their lysates, culture media, extracts of culture media, and their use for improving skin condition.
Description
신규한 미생물, 그의 파쇄물, 배양액, 배양액의 추출물, 및 이들의 피부상태 개선 용도에 관한 것이다.It relates to novel microorganisms, their lysates, culture media, extracts of culture media, and their 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, 피부상재균과 피부의 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 produce effective effects through various beneficial effects on the human body, but research on the relationship between skin flora and skin is insufficient.
이에, 피부 관련된 상태에 유용하게 사용될 수 있는 피부상재균의 개발이 필요한 실정이다.Accordingly, there is a need to develop skin flora that can be useful for skin-related conditions.
일 양상은 마이크로코커스 속(Micrococcus sp.)에 속하는 마이크로코커스 플라버스(Micrococcus flavus) 균주를 제공하는 것이다. One aspect is to provide a strain of Micrococcus flavus belonging to the genus Micrococcus sp.
다른 양상은 상기 균주의 파쇄물, 배양액, 또는 배양액의 추출물을 제공하는 것이다. Another aspect is to provide lysate, culture medium, or extract of the culture medium of the strain.
또 다른 양상은 상기 균주, 그의 파쇄물, 배양액, 배양액의 추출물을 포함하는 조성물을 제공하는 것이다.Another aspect is to provide a composition comprising the strain, its lysate, culture medium, and extract of the culture medium.
일 양상은 마이크로코커스 플라버스(Micrococcus flavus) 균주를 제공한다. One aspect provides a strain of Micrococcus flavus.
상기 마이크로코커스 플라버스(Micrococcus flavus) 균주는 기탁번호 KCCM12997P로 기탁된 균주일 수 있다. The Micrococcus flavus strain may be a strain deposited under deposit number KCCM12997P.
상기 마이크로코커스 플라버스(Micrococcus flavus) 균주는 서열번호 1의 16s rRNA를 포함하는 균주일 수 있다. 일 구체예에 있어서, 상기 균주는 서열번호 1과 약 95% 이상, 약 96% 이상, 약 97% 이상, 약 98% 이상, 약 99% 이상, 약 99.5% 이상, 또는 약 99.9% 이상의 서열 동일성을 갖는 16S rRNA를 포함하는 것일 수 있다.The Micrococcus flavus strain may be a strain containing 16s rRNA of SEQ ID NO: 1. In one embodiment, the strain has at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity with SEQ ID NO. It may contain 16S rRNA having .
일 실시예에서는 피부로부터 분리된 마이크로코커스 플라버스 균주가 COL1A1의 발현을 증가시키고, MMP-1의 발현을 감소시키며, 멜라닌 생성을 억제하는 것을 확인하였다. 또한, 상기 균주가 세포 재생을 촉진하는 것을 확인하였다. In one example, it was confirmed that the Micrococcus flavanus strain isolated from the skin increased the expression of COL1A1, decreased the expression of MMP-1, and inhibited melanin production. Additionally, it was confirmed that the strain promoted cell regeneration.
따라서, 일 구체예에 따른 균주는 피부 상태 개선 효과, 예를 들면, 피부 노화 억제, 주름 개선, 보습 개선, 피부 장벽 강화, 탄력 개선 또는 피부 미백 효과를 갖는 것일 수 있다. 또한, 상기 균주는 피부 재생 또는 피부 상처의 예방, 개선 또는 치료 효과를 갖는 것이 수 있다. Therefore, the strain according to one embodiment may have a skin condition improvement effect, for example, skin aging inhibition, wrinkle improvement, moisturizing improvement, skin barrier strengthening, elasticity improvement, or skin whitening effect. Additionally, the strain may have effects on skin regeneration or prevention, improvement, or treatment of skin wounds.
다른 양상은 상기 균주의 파쇄물, 배양액, 또는 배양액의 추출물을 제공한다. Another aspect provides lysate, culture fluid, or extract of the culture fluid of the strain.
상기 균주에 대해서는 상기한 바와 같다. The strain is as described above.
본 명세서에서 용어"배양액"은 "배양 상층액", "조건 배양액" 또는 "조정 배지"와 호환적으로 사용될 수 있고, 마이크로코커스 플라버스 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 can provide nutrients for Micrococcus flavibus strains to grow and survive in vitro. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain in the medium for a certain period of time. 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 마이크로코커스 플라버스 균주를 배지(예를 들면, R2A 배지 또는 TSA 배지) 에서 10℃ 초과 또는 40℃ 미만 중 어느 온도에서 일정 시간, 예를 들면, 4 내지 50시간 동안 배양하여 수득된 것일 수 있다. The culture medium is obtained by culturing the Micrococcus flavius strain in a medium (e.g., R2A medium or TSA medium) at any temperature above 10°C or below 40°C for a certain period of time, for example, 4 to 50 hours. You can.
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.
다른 구체예에서, 농축물은 상기 균주 배양액 자체, 또는 상기 배양액을 원심분리나 필터를 이용하여 여과한 후 수득된 상층액을 농축하는 단계에 의해 수득될 수 있다. In another embodiment, the concentrate may be obtained by concentrating the strain culture medium itself, or the supernatant obtained after filtering the culture medium using centrifugation or a filter.
상기 마이크로코커스 플라버스 균주를 배양하기 위한 배양용 배지 및 배양 조건은 통상의 지식을 가진 자가 적절하게 선택하거나 변형하여 이용할 수 있다.Culture media and culture conditions for cultivating the Micrococcus flavius strain can be appropriately selected or modified by those skilled in the art.
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 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.
또 다른 양상은 상기 균주, 상기 균주의 파쇄액, 배양액, 또는 배양액의 추출물의 용도를 제공한다. 상세하게는, 마이크로코커스 플라버스 균주, 그의 파쇄액, 배양액 또는 그의 배양액의 추출물을 포함하는 조성물을 제공한다. Another aspect provides the use of the strain, a lysate of the strain, a culture medium, or an extract of the culture medium. In detail, a composition comprising a Micrococcus flavanus strain, its lysate, culture medium, or extract of the culture medium is provided.
상기 균주의 용도는 피부 상태 개선, 피부 미용 개선, 피부 질환의 예방, 개선 또는 치료, 피부 염증성 질환의 예방, 개선, 또는 치료 등을 포함할 수 있다. Uses of the strain may include improving skin condition, improving skin beauty, preventing, improving or treating skin diseases, preventing, improving or treating skin inflammatory diseases, etc.
상기 피부 상태 개선 또는 피부 미용 개선은 피부 노화 억제 또는 개선, 항 주름, 피부 미백, 피부 장벽 강화, 피부 보습, 상처 개선 또는 치유, 또는 피부 재생일 수 있다. The skin condition improvement or skin beauty improvement may be suppressing or improving skin aging, anti-wrinkle, skin whitening, strengthening the skin barrier, skin moisturizing, wound improvement or healing, or skin regeneration.
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 균주 또는 이의 배양액은 외인성 요인 또는 내인성 요인에 의해 유발되는 피부 노화 개선용일 수 있다. 상기 외인성 요인은 여러 가지 외부 요인, 예컨대 자외선(광)을 말하고 내인성 요인은 연대기적 요인이라고도 지칭되며 주로 시간의 흐름에 의해 발생하는 요인을 말한다. 즉, 상기 피부 노화는 구체적으로는 자외선, 공해, 담배연기, 화학물질 등에 의한 외부 자극에 의해 유도되는 조기 노화 증상 뿐만 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 strain or its culture medium may be used to improve skin aging caused by exogenous factors or endogenous factors. The exogenous factors refer to various external factors, such as ultraviolet rays (light), and the endogenous factors are also referred to as chronological factors and mainly refer to factors that occur due to the passage of time. In other words, the skin aging is not only a premature aging phenomenon induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemicals, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells 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 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 the 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.
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 nutrient loss.
상기 "피부 보습"은 피부 수분을 유지하거나 수분 손실을 방지하는 모든 작용을 의미할 수 있다.The term “skin moisturizing” may refer to any action that maintains skin moisture or prevents moisture loss.
상기 "상처"는 "창상"이라고도 하며, 생체의 손상을 의미한다. 상기 손상은 물리적 손상, 방사선, 화학물질에 의한 자극, 미생물의 증식 등 외부 요인, 스트레스 등의 내부 요인에 의한 것일 수 있으나, 이에 한정되지 않는다. 상기 상처는 절창, 자창, 할창, 좌창, 열창, 사창, 교창 등 모든 종류의 상처를 포함할 수 있다.The “wound” is also called “wound” and refers to damage to a living body. The damage may be caused by external factors such as physical damage, radiation, stimulation by chemicals, proliferation of microorganisms, or internal factors such as stress, but is not limited to these. The wounds may include all types of wounds, such as incisional wounds, puncture wounds, abdominal wounds, acne wounds, fissure wounds, oblique wounds, and vaginal wounds.
상기 "피부 재생"은 피부의 일부분이 소실되었거나 손상되었을 경우 그 부분을 보충하거나 피부 세포의 증식 및 이동을 증진시키는 모든 작용을 의미할 수 있다The "skin regeneration" may refer to any action that replenishes a part of the skin when it is lost or damaged or enhances the proliferation and movement of skin cells.
용어, "항염증"이란 "염증 억제 또는 개선"과 혼용될 수 있으며, 면역 반응이 완화되어 NO 생성이 억제되는 모든 작용을 의미할 수 있다.The term “anti-inflammatory” may be used interchangeably with “inhibiting or improving inflammation,” and may refer to any action that alleviates the immune response and suppresses NO production.
상기 "피부 질환"은 피부 장벽 기능 손상에 의한 질환, 피부 노화, 피부 상처, 피부 흉터, 또는 피부 염증일 수 있다. 용어, "예방"은 질병의 발생을 억제하는 것을 포함한다. 용어, "치료"는 질병의 발전의 억제, 경감, 또는 제거를 포함한다.The “skin disease” may be a disease caused by damage to the skin barrier function, skin aging, skin wound, skin scar, or skin inflammation. The term “prevention” includes suppressing the occurrence of a disease. The term “treatment” includes inhibiting, alleviating, or eliminating the development of a disease.
상기 피부 장벽 기능 손상은 피부 장벽의 기능이 저하되거나 손상되어 피부에 나타나는 모든 변화를 의미할 수 있다. 예를 들어, 피부 주름 증가, 건조, 피부염, 아토피 피부염, 알레르기성 피부염, 여드름 등을 포함할 수 있다. The damage to the skin barrier function may mean any change that appears in the skin due to decreased or damaged skin barrier function. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, etc.
상기 피부 염증성 질환은 피부 상처, 피부염, 아토피 피부염, 소양증, 습진성 피부질환, 건성 습진, 홍반, 두드러기, 건선, 약발진, 및 여드름으로 이루어진 군으로부터 선택된 어느 하나인 것일 수 있다. The skin inflammatory disease may be any one selected from the group consisting of skin wounds, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, drug rash, and acne.
또 다른 구체예에 있어서, 상기 균주는 피부 개선 효과를 갖는 다른 균주 마이크로코커스 속에 속하는 균주와 함께 사용되어 시너지 효과를 나타낸 것일 수 있다. 상기 마이크로코커스 속에 속하는 균주는 예를 들어, 마이크로코커스 안타르크티쿠스(Micrococcus Antarcticacus), 마이크로코커스 리래(Micrococcus rirae), 마이크로코커스 루테우스(Micrococcus luteus), 마이크로코커스 테리우스(Micrococcus terreus) 등인 것일 수 있다. In another embodiment, the strain may be used together with a strain belonging to the genus Micrococcus, another strain that has a skin-improving effect, and may exhibit a synergistic effect. Strains belonging to the Micrococcus genus may be, for example, Micrococcus Antarcticacus , Micrococcus rirae, Micrococcus luteus , Micrococcus terreus , etc. .
상기 조성물은 조성물 총 중량에 대하여 0.001 중량% 내지 80 중량%, 예를 들면, 0.01 중량% 내지 60 중량%, 0.01 중량% 내지 40 중량%, 0.01 중량% 내지 30 중량%, 0.01 중량% 내지 20 중량%, 0.01 중량% 내지 10 중량%, 0.01 중량% 내지 5 중량%, 0.05 중량% 내지 60 중량%, 0.05 중량% 내지 40 중량%, 0.05 중량% 내지 30 중량%, 0.05 중량% 내지 20 중량%, 0.05 중량% 내지 10 중량%, 0.05 중량% 내지 5 중량%, 0.1 중량% 내지 60 중량%, 0.1 중량% 내지 40 중량%, 0.1 중량% 내지 30 중량%, 0.1 중량% 내지 20 중량%, 0.1 중량% 내지 10 중량%, 또는 0.1 중량% 내지 5 중량%의 균주, 이의 파쇄액, 배양액, 또는 이의 배양액의 추출물을 포함할 수 있다.The composition is 0.001% to 80% by weight, for example, 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 0.01% to 20% by weight, based on the total weight of the composition. %, 0.01% to 10% by weight, 0.01% to 5% by weight, 0.05% to 60% by weight, 0.05% to 40% by weight, 0.05% to 30% by weight, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight It may include % to 10% by weight, or 0.1% to 5% by weight of the strain, its lysate, culture medium, or extract of the culture medium.
용어, "유효성분으로 포함"은 상기에서 언급한 효과를 나타낼 수 있는 정도로 본 명세서의 균주, 이의 파쇄액, 배양액, 또는 이의 배양액의 추출물이 첨가되는 것을 의미하고, 약물전달 및 안정화 등을 위하여 다양한 성분을 부성분으로 첨가하여 다양한 형태로 포뮬레이션 (formulation)되는 것을 포함하는 의미이다.The term "included as an active ingredient" means that the strain of the present specification, its lysate, culture medium, or extract of the culture medium is added to the extent that it can exhibit the effects mentioned above, and is used for various purposes such as drug delivery and stabilization. It means adding ingredients as sub-ingredients and formulating them into various forms.
상기 조성물은 화장료 조성물일 수 있다. The composition may be a cosmetic composition.
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 조성물에 포함되는 성분은 유효성분으로서 상기 조성물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있으며, 예를 들면, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제 및 담체를 포함할 수 있다.Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances. may include.
또한, 상기 조성물은 피부외용제용 조성물일 수 있다. Additionally, the composition may be a composition for external skin application.
본 명세서에서, 상기 피부외용제는 크림, 겔, 연고, 피부 유화제, 피부 현탁액, 경피전달성 패치, 약물 함유 붕대, 로션, 또는 그 조합일 수 있다. 상기 피부외용제는 통상 화장품이나 의약품 등의 피부외용제에 사용되는 성분, 예를 들면 수성성분, 유성성분, 분말성분, 알코올류, 보습제, 증점제, 자외선흡수제, 미백제, 방부제, 산화방지제, 계면활성제, 향료, 색제, 각종 피부 영양제, 또는 이들의 조합과 필요에 따라서 적절하게 배합될 수 있다. 상기 피부외용제는, 에데트산이나트륨, 에데트산삼나트륨, 시트르산나트륨, 폴리인산나트륨, 메타인산나트륨, 글루콘산 등의 금속봉쇄제, 카페인, 탄닌, 벨라파밀, 감초추출물, 글라블리딘, 칼린의 과실의 열수추출물, 각종생약, 아세트산토코페롤, 글리틸리틴산, 트라넥삼산 및 그 유도체 또는 그 염등의 약제, 비타민 C, 아스코르브산인산마그네슘, 아스코르브산글루코시드, 알부틴, 코지산, 글루코스, 프룩토스, 트레할로스 등의 당류등도 적절하게 배합할 수 있다.In this specification, the external skin agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The skin external preparations include ingredients commonly used in external skin preparations such as cosmetics and medicines, such as aqueous ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or a combination thereof may be appropriately mixed according to need. The skin external preparations include metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidin, and calin. Hot water extract of fruit, various herbal medicines, drugs such as tocopherol acetate, glytylitinic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, Sugars such as trehalose can also be appropriately mixed.
다른 구체예에 있어서, 상기 조성물은 약학적 조성물일 수 있다. In another embodiment, the composition may be a pharmaceutical composition.
상기 약학적 조성물은 약제학적으로 허용가능한 희석제 또는 담체를 추가적으로 포함할 수 있다. 상기 희석제는 유당, 옥수수 전분, 대두유, 미정질 셀룰로오스, 또는 만니톨, 활택제로는 스테아린산 마그네슘, 탈크, 또는 그 조합일 수 있다. 상기 담체는 부형제, 붕해제, 결합제, 활택제, 또는 그 조합일 수 있다. 상기 부형제는 미결정 셀룰로오즈, 유당, 저치환도 히드록시셀룰로오즈, 또는 그 조합일 수 있다. 상기 붕해제는 카르복시메틸셀룰로오스 칼슘, 전분글리콜산 나트륨, 무수인산일수소 칼슘, 또는 그 조합일 수 있다. 상기 결합제는 폴리비닐피롤리돈, 저치환도 히드록시프로필셀룰로오즈, 히드록시프로필셀룰로오즈, 또는 그 조합일 수 있다. 상기 활택제는 스테아린산 마그네슘, 이산화규소, 탈크, 또는 그 조합일 수 있다.The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, disintegrant, binder, lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate anhydride, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.
상기 약학적 조성물은 경구 또는 비경구 투여 제형으로 제형화될 수 있다. 경구 투여 제형은 과립제, 산제, 액제, 정제, 캅셀제, 건조시럽제, 또는 그 조합일 수 있다. 비경구 투여 제형은 주사제일 수 있다.The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrup, or combinations thereof. Parenteral dosage forms may be injectable.
상기 조성물은 건강기능식품 조성물을 일 수 있다. The composition may be a health functional food composition.
상기 건강기능식품 조성물은 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 composition 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.
또한, 다른 양상은 유효한 양의 상기한 조성물을 그를 필요로 하는 개체에 처리 또는 투여하는 단계를 포함하는 개체의 상태를 예방, 개선, 또는 치료하는 방법을 제공한다. Additionally, another aspect provides a method of preventing, ameliorating, or treating a condition of a subject comprising treating or administering an effective amount of the composition described above to the subject in need thereof.
상기 개체의 상태는 피부와 관련된 상태, 또는 염증과 관련된 상태일 수 있다. The condition of the subject may be a skin-related condition or an inflammation-related condition.
용어, "투여하는", "도입하는", 및 "이식하는"은 상호교환적으로 사용되고 일 구체예에 따른 조성물의 원하는 부위로의 적어도 부분적 국소화를 초래하는 방법 또는 경로에 의한 개체 내로의 일 구체예에 따른 조성물의 배치를 의미할 수 있다. The terms “administering,” “introducing,” and “implanting” are used interchangeably and refer to a method or route that results in at least partial localization of the composition according to one embodiment to the desired site. It may refer to the arrangement of the composition according to the example.
투여는 당업계에 알려진 방법에 의하여 투여될 수 있다. 투여는 예를 들면, 정맥내, 근육내, 경구, 경피 (transdermal), 점막, 코안 (intranasal), 기관내 (intratracheal) 또는 피하 투여와 같은 경로로, 임의의 수단에 의하여 개체로 직접적으로 투여될 수 있다. 상기 투여는 전신적으로 또는 국부적으로 투여될 수 있다.Administration can be done by methods known in the art. Administration may be administered directly to the subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. You can. The administration may be administered systemically or locally.
상기 개체는 포유동물, 예를 들면, 사람, 소, 말, 돼지, 개, 양, 염소, 또는 고양이일 수 있다. 상기 개체는 피부미용 개선, 예를 들어 피부 보습, 피부 장벽 강화, 피부 염증 억제, 피부 주름 개선 효과를 필요로 하는 개체일 수 있다.The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of skin beauty improvement, for example, skin moisturizing, skin barrier strengthening, skin inflammation inhibition, and skin wrinkle improvement effects.
상기 투여는 일 구체예에 따른 조성물을 개체당 일당 0.1 mg 내지 1,000 mg, 예를 들면, 0.1 mg 내지 500 mg, 0.1 mg 내지 100 mg, 0.1 mg 내지 50 mg, 0.1 mg 내지 25 mg, 1 mg 내지 1,000 mg, 1 mg 내지 500 mg, 1 mg 내지 100 mg, 1 mg 내지 50 mg, 1 mg 내지 25 mg, 5mg 내지 1,000 mg, 5 mg 내지 500 mg, 5 mg 내지 100 mg, 5 mg 내지 50 mg, 5 mg 내지 25 mg, 10mg 내지 1,000 mg, 10 mg 내지 500 mg, 10 mg 내지 100 mg, 10 mg 내지 50 mg, 또는 10 mg 내지 25 mg을 투여하는 것일 수 있다. 다만, 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있고, 당업자라면 이러한 요인들을 고려하여 투여량을 적절히 조절할 수 있다. 투여 횟수는 1일 1회 또는 임상적으로 용인가능한 부작용의 범위 내에서 2회 이상이 가능하고, 투여 부위에 대해서도 1개소 또는 2개소 이상에 투여할 수 있으며, 매일 또는 2 내지 5일 간격으로 총 투여 일수는 한번 치료 시 1일에서 30일까지 투여될 수 있다. 필요한 경우, 적정 시기 이후에 동일한 치료를 반복할 수 있다. 인간 이외의 동물에 대해서도, kg당 인간과 동일한 투여량으로 하거나, 또는 예를 들면 목적의 동물과 인간과의 기관(심장 등)의 용적비(예를 들면, 평균값) 등으로 상기의 투여량을 환산한 양을 투여할 수 있다.The administration of the composition according to one embodiment is 0.1 mg to 1,000 mg per day, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1 mg. 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The frequency of administration can be once a day or two or more times within the range of clinically acceptable side effects, and can be administered at one or two or more locations, daily or at intervals of 2 to 5 days. The number of days of administration can be from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the dosage per kg is the same as for humans, or the above dosage is converted into, for example, the volume ratio (e.g., average value) of the organs (heart, etc.) between the target animal and human. One dose can be administered.
일 양상에 신규 마이크로코커스 플라버스 균주에 의하면, 피부 관련 상태 또는 염증 관련 상태의 예방, 개선, 또는 치료에 유용하게 사용될 수 있는 효과가 있다.In one aspect, the novel Micrococcus flavanus strain has effects that may be useful for preventing, improving, or treating skin-related conditions or inflammation-related conditions.
도 1은 일 구체예에 따른 균주가 COL1A1의 발현에 미치는 영향을 나타낸 결과이다.
도 2는 일 구체예에 따른 균주가 MMP-1의 발현에 미치는 영향을 나타낸 결과이다.
도 3은 일 구체예에 따른 균주가 멜라닌의 생성에 미치는 영향을 나타낸 결과이다.
도 4는 일 구체예에 따른 균주가 피부 재생 및 상처 회복에 미치는 영향을 나타낸 결과이다. Figure 1 shows the results showing the effect of a strain on the expression of COL1A1 according to one embodiment.
Figure 2 is a result showing the effect of a strain on the expression of MMP-1 according to one embodiment.
Figure 3 is a result showing the effect of a strain according to one embodiment on the production of melanin.
Figure 4 is a result showing the effect of the strain according to one embodiment on skin regeneration and wound recovery.
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][Example]
실시예 1. 균주의 분리 및 동정Example 1. Isolation and identification of strains
건강한 여성의 피부를 멸균 증류수로 세척하여 확보된 샘플(human epidermal keratinocyte)을 R2A 배지(Becton Dickinson, Cockeysville, MD)에 접종하였다. 접종 후 48시간 동안 28℃ 인큐베이터에서 배양한 뒤 형성된 집락 100개를 순수 분리 배양하여, 48시간 동안 28℃ 인큐베이터에서 재배양 하였다. 배양이 완료된 집락은 16s rRNA 유전자 서열 동정을 실시하였다. 이때 사용된 프라이머는 박테리아에만 반응하여 증폭하도록 고안(서열번호 2 및 3)되었다. PCR 증폭은 95℃ 1분, 55℃ 1분, 75℃ 1분30초씩 30 사이클로 실시 하였으며, 마지막으로 72℃에서 8분간 처리한 후 4℃에서 보관하였다. PCR 반응이 끝난 뒤 분리 배양된 종들의 DNA 서열은 ABI-3730XL(ABI, USA)를 이용하여 결정하였다. 분리 배양된 미생물 집락 중 결정된 16S rRNA부위의 염기서열을 미국 국립생물정보센터(NCBI, National Center for Biotechnology Information) 홈페이지에서 제공되는 BLAST 프로그램으로 등록된 다른 균주들과 비교 분석 하였을 때 상동성 99% 이하의 신규 미생물 마이크로코커스 플라버스(Micrococcus flavus) 균주(이하 "MC-4"이라 함)을 선별하였다. 선별된 MC-4 균주를 2021년 5월 28일자로 한국 미생물 보존센터에 기탁하여 기탁번호 KCCM12997P를 부여 받았고, MC-4 균주는 서열번호 1(complementary DNA)의 16s rRNA 서열을 갖는다. A sample (human epidermal keratinocyte) obtained by washing the skin of a healthy woman with sterile distilled water was inoculated into R2A medium (Becton Dickinson, Cockeysville, MD). After inoculation, 100 colonies formed after culturing in an incubator at 28°C for 48 hours were isolated and cultured in pure form, and re-cultured in an incubator at 28°C for 48 hours. Colonies whose culture was completed were identified by 16s rRNA gene sequence. The primers used at this time were designed to amplify by reacting only with bacteria (SEQ ID NOs. 2 and 3). PCR amplification was performed in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, and 75°C for 1 minute and 30 seconds, and was finally treated at 72°C for 8 minutes and stored at 4°C. After the PCR reaction was completed, the DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA). When the nucleotide sequence of the 16S rRNA region determined among the isolated and cultured microbial colonies was compared and analyzed with other strains registered with the BLAST program provided on the website of the U.S. National Center for Biotechnology Information (NCBI), the homology was less than 99%. A new microbial Micrococcus flavus strain (hereinafter referred to as “MC-4”) was selected. The selected MC-4 strain was deposited at the Korea Microbial Conservation Center on May 28, 2021 and given the deposit number KCCM12997P, and the MC-4 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).
[실험예] [Experimental example]
항노화 및 항주름 활성 분석Anti-aging and anti-wrinkle activity analysis
일 구체예에 따른 균주가 자외선(UV) 조사에 의한 피부 노화 및 주름 생성과 관련된 인자에 미치는 영향을 분석하였다. The effects of strains according to one embodiment on factors related to skin aging and wrinkle formation caused by ultraviolet (UV) irradiation were analyzed.
구체적으로, 인간 섬유아세포 세포주(Human dermal fibroblast, Hs68)를 6 웰 플레이트에 3.5x105로 분주한 후, 37℃, 5% CO2 조건의 배양기에서 24시간 동안 배양하였다. 이후 배지를 제거하고 DPBS를 넣은 후 12 mJ/cm2의 UVB를 조사하거나 조사하지 않았다. UVB 조사 직후 DPBS를 제거하고 FBS가 없는 배지로 갈아 준 후, 상기 MC-4 균주의 배양액(1%(w/w))을 처리하고 24시간 동안 추가 배양하였다. 음성 대조군으로 자외선 처리 및 균주 배양액을 포함하지 않는 R2A 배지를 사용하였다. 그 후 각 시료의 세포에서 트리졸(RNA iso, DAKARA, 일본)을 이용하여 RNA를 분리한 뒤 nanodrop으로 260㎚에서 RNA를 정량한 후, 각각 2㎍의 RNA를 사용하여 증폭기에서 cDNA를 합성하였다(C1000 Thermal Cycler, Bio-Rad, 미국). 합성된 cDNA를 주형으로 타겟 유전자인 COL1A1 및 MMP-1(Matrix metalloproteinase-1)에 대하여 사이버그린 (SYBR Green supermix, Applied Biosystems, USA)을 프라이머 및 cDNA와 함께 첨가하여 실시간(real-time) PCR 기계 (Step One Plus, Applied Biosystems, 미국)에서 실시간 중합효소 연쇄반응을 수행하였다. 유전자의 발현량은 β-actin 유전자에 대한 보정을 통해 최종적으로 분석하였고, 그 결과는 도 1 및 도 2에 나타내었다. Specifically, the human fibroblast cell line (Human dermal fibroblast, Hs68) was distributed at 3.5x10 5 in a 6-well plate and then cultured in an incubator at 37°C and 5% CO 2 conditions for 24 hours. Afterwards, the medium was removed, DPBS was added, and UVB of 12 mJ/cm 2 was irradiated or not. Immediately after UVB irradiation, DPBS was removed and the medium was replaced with FBS-free medium, and the culture medium (1% (w/w)) of the MC-4 strain was treated and further cultured for 24 hours. As a negative control, R2A medium without UV treatment and without strain culture was used. Afterwards, RNA was isolated from the cells of each sample using Trizol (RNA iso, DAKARA, Japan), RNA was quantified at 260 nm with nanodrop, and cDNA was synthesized in an amplifier using 2 μg of RNA each. (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, Cyber Green (SYBR Green supermix, Applied Biosystems, USA) was added together with primers and cDNA for the target genes COL1A1 and MMP-1 (Matrix metalloproteinase-1), and a real-time PCR machine was used. Real-time polymerase chain reaction was performed (Step One Plus, Applied Biosystems, USA). The expression level of the gene was finally analyzed through correction for the β-actin gene, and the results are shown in Figures 1 and 2.
도 1은 일 구체예에 따른 균주가 COL1A1의 발현에 미치는 영향을 나타낸 결과이다. Figure 1 shows the results showing the effect of a strain on the expression of COL1A1 according to one embodiment.
도 2는 일 구체예에 따른 균주가 MMP-1의 발현에 미치는 영향을 나타낸 결과이다.Figure 2 is a result showing the effect of a strain on the expression of MMP-1 according to one embodiment.
그 결과, 도 1에 나타낸 바와 같이, 섬유아세포에 자외선을 처리함으로써 COL1A1 발현이 감소하였고, 실시예 1의 균주 배양액 처리에 의하여 COL1A1의 발현이 증가하는 것을 확인할 수 있었다. As a result, as shown in Figure 1, it was confirmed that treatment of fibroblasts with ultraviolet rays decreased the expression of COL1A1, and that treatment of the strain culture medium of Example 1 increased the expression of COL1A1.
또한, 도 2에 나타낸 바와 같이, 섬유아세포에 자외선을 처리함으로써 MMP-1의 발현이 증가하였고, 실시예 1의 균주 배양액 처리에 의하여 MMP-1의 발현이 감소하는 것을 확인할 수 있었다.In addition, as shown in Figure 2, the expression of MMP-1 was increased by treating fibroblasts with ultraviolet rays, and it was confirmed that the expression of MMP-1 was decreased by treatment with the strain culture medium of Example 1.
즉, 일 구체예에 따른 마이크로코커스 플라버스 MC-4 균주는 COL1A1의 발현을 증가시키고, MMP-1의 발현을 감소시킴으로써 노화(예를 들면, 광노화)의 예방 또는 개선에 이용될 수 있다.That is, the Micrococcus flavanus MC-4 strain according to one embodiment can be used to prevent or improve aging (eg, photoaging) by increasing the expression of COL1A1 and decreasing the expression of MMP-1.
피부 미백 활성 분석Skin whitening activity analysis
일 구체예에 따른 균주가 피부 미백에 영향을 미치는지 여부를 분석하였다. It was analyzed whether the strain according to one embodiment affects skin whitening.
구체적으로, 색소 세포주(melanoma)인 B16F10를 12-웰 플레이트에 접종하여 24시간 배양하였다. 이후 배지를 교체하고, α-멜라닌세포자극 호르몬(α-melanocyte stimulating hormone: α-MSH) 10 nM과 상기 MC-4 균주의 배양액(1%(w/w) 를 처리하여 48시간 동안 추가로 배양하였다. 음성대조군으로 비히클 및 균주 배양액을 포함하지 않는 R2A 배지를 사용하였다. 이후, 인산완충생리식염수(Phosphate Buffered Saline: PBS)로 세척하고, 트립신(Trypsin)- EDTA를 처리하여 세포를 회수하였다. 회수한 세포를 PBS로 세척한 후, 10% DMSO가 함유된 1N NaOH 100 ㎕를 처리하고 80 ℃에서 1시간 열을 가한 후 이를 식혔다. 이후, ELISA microplate reader를 이용하여 405 ㎚에서 흡광도를 측정하여 멜라닌 생성능을 확인하였다.Specifically, B16F10, a pigmented cell line (melanoma), was inoculated into a 12-well plate and cultured for 24 hours. Afterwards, the medium was changed, treated with 10 nM of α-melanocyte stimulating hormone (α-MSH) and the culture medium (1% (w/w)) of the MC-4 strain, and cultured for an additional 48 hours. As a negative control, R2A medium containing no vehicle or strain culture medium was used. Afterwards, cells were recovered by washing with Phosphate Buffered Saline (PBS) and treatment with Trypsin-EDTA. After washing the recovered cells with PBS, they were treated with 100 ㎕ of 1N NaOH containing 10% DMSO, heated at 80 ° C for 1 hour, and then cooled. Afterwards, absorbance was measured at 405 nm using an ELISA microplate reader. The melanin production ability was confirmed.
도 3은 일 구체예에 따른 균주가 멜라닌의 생성에 미치는 영향을 나타낸 결과이다.Figure 3 is a result showing the effect of a strain according to one embodiment on the production of melanin.
그 결과, 도 3에 나타낸 바와 같이, 실시예 1의 균주 배양액은 음성대조군과 비교하여 멜라닌 함량을 유의하게 감소시키는 것을 확인할 수 있었다. As a result, as shown in Figure 3, it was confirmed that the strain culture medium of Example 1 significantly reduced the melanin content compared to the negative control group.
따라서, 일 구체예에 따른 마이크로코커스 플라버스 MC-4 균주는 멜라닌 생성을 억제함으로써 피부 미백에 유용하게 사용될 수 있다. Therefore, the Micrococcus flavus MC-4 strain according to one embodiment can be usefully used for skin whitening by inhibiting melanin production.
피부 재생 및 상처 회복 활성 분석Skin regeneration and wound recovery activity analysis
일 구체예에 따른 균주가 피부 재생 및 상처 회복에 영향을 미치는지 여부를 분석하였다. It was analyzed whether the strain according to one embodiment affects skin regeneration and wound recovery.
구체적으로, 인간 섬유아세포 세포주 (Human dermal fibroblast, Hs68)를 6-웰 플레이트에 4Х105의 밀도로 접종한 후, 37℃ 5% CO2 조건의 배양기에서 24시간 동안 배양하여 100% 포화된(confluent) 상태를 만들어 주었다. 이후 세포 스크래퍼(cell scrapper)를 이용하여 배양된 세포 중심을 긁어 상처를 낸 후, 상기 MC-4 균주의 배양액(1%(w/w))을 처리하여 24시간 동안 추가로 배양하였다. 음성대조군으로 배양액 비처리군 및 균주 배양액을 포함하지 않는 R2A 배지를 사용하였다. 배양 기간 동안 2시간에 한번씩, 세포에 임의로 낸 상처가 어느 정도 회복되었는지 촬영하여 상처 부위의 세포 밀도를 측정하였다. Specifically, the human fibroblast cell line (Human dermal fibroblast, Hs68) was inoculated into a 6-well plate at a density of 4Х10 5 and then cultured for 24 hours in an incubator under 37°C and 5% CO 2 conditions to reach 100% confluence. ) state was created. Afterwards, the center of the cultured cells was scratched using a cell scraper to create a wound, and then treated with culture medium (1% (w/w)) of the MC-4 strain and cultured for an additional 24 hours. As a negative control group, R2A medium containing no culture media treatment group and strain culture media was used. During the culture period, once every 2 hours, the extent to which wounds randomly applied to cells had recovered was photographed and the cell density of the wound area was measured.
도 4는 일 구체예에 따른 균주가 피부 재생 및 상처 회복에 미치는 영향을 나타낸 결과이다.Figure 4 is a result showing the effect of the strain according to one embodiment on skin regeneration and wound recovery.
그 결과, 도 4에 나타낸 바와 같이, 실시예 1의 균주 배양액은 시간이 증가함에 따라 손상 부위로 섬유아세포가 이동(migration)함으로써 상처 회복에 효과적인 것을 확인할 수 있었다. 구체적으로, 음성대조군과 비교하여 회복된 부위의 세포 밀도가 증가함에 따라 상처 회복 효과가 더욱 효과적인 것을 확인할 수 있었다. As a result, as shown in Figure 4, it was confirmed that the strain culture medium of Example 1 was effective in wound recovery as fibroblasts migrated to the damaged area as time increased. Specifically, it was confirmed that the wound recovery effect was more effective as the cell density in the recovered area increased compared to the negative control group.
즉, 일 구체예에 따른 마이크로코커스 플라버스 MC-4 균주는 피부 재생을 촉진하고 상처의 예방 또는 치료에 유용하게 사용될 수 있다.That is, the Micrococcus flavus MC-4 strain according to one embodiment promotes skin regeneration and can be usefully used to prevent or treat wounds.
SEQUENCE LISTING <110> Cosmax, INC. <120> Micrococcus flavus strain and skin condition improving uses of thereof <130> PN144142 <160> 3 <170> PatentIn version 3.2 <210> 1 <211> 1410 <212> DNA <213> Artificial <220> <223> Micrococcus flavus 16s rRNA <400> 1 atcctggctc aggatgaacg ctggcggcgt gcttaacaca tgcaagtcga acgatgaagc 60 ccagcttgct gggtggatta gtggcgaacg ggtgagtaac acgtgagtaa cctgccctta 120 actctgggat aagcctggga aactgggtct aataccggat aggagcgtcc accgcatggt 180 gggtgttgga aagatttatc ggttttggat ggactcgcgg cctatcagct tgttggtgag 240 gtaatggctc accaaggcga cgacgggtag ccggcctgag agggtgaccg gccacactgg 300 gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg cacaatgggc 360 gaaagcctga tgcagcgacg ccgcgtgagg gatgacggcc ttcgggttgt aaacctcttt 420 cagtagggaa gaagcgaaag tgacggtacc tgcagaagaa gcaccggcta actacgtgcc 480 agcagccgcg gtaatacgta gggtgcgagc gttatccgga attattgggc gtaaagagct 540 cgtaggcggt ttgtcgcgtc tgtcgtgaaa gtccggggct taaccccgga tctgcggtgg 600 gtacgggcag actagagtgc agtaggggag actggaattc ctggtgtagc ggtggaatgc 660 gcagatatca ggaggaacac cgatggcgaa ggcaggtctc tgggctgtaa ctgacgctga 720 ggagcgaaag catggggagc gaacaggatt agataccctg gtagtccatg ccgtaaacgt 780 tgggcactag gtgtggggac cattccacgg tttccgcgcc gcagctaacg cattaagtgc 840 cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg ggcccgcaca 900 agcggcggag catgcggatt aattcgatgc aacgcgaaga accttaccaa ggcttgacat 960 gttctcgatc gccgtagaga tacggtttcc cctttggggc gggttcacag gtggtgcatg 1020 gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctcg 1080 ttccatgttg ccagcacgtc gtggtgggga ctcatgggag actgccgggg tcaactcgga 1140 ggaaggtgag gacgacgtca aatcatcatg ccccttatgt cttgggcttc acgcatgcta 1200 caatggccgg tacaatgggt tgcgatactg tgaggtggag ctaatcccaa aaagccggtc 1260 tcagttcgga ttggggtctg caactcgacc ccatgaagtc ggagtcgcta gtaatcgcag 1320 atcagcaacg ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcaagtcacg 1380 aaaagtcggt aacacccgaa agccggtggc 1410 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> Forward primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial <220> <223> Reverse primer <400> 3 ggttaccttg ttacgactt 19 SEQUENCE LISTING <110> Cosmax, INC. <120> Micrococcus flavus strain and skin condition improving uses of of that <130> PN144142 <160> 3 <170> PatentIn version 3.2 <210> 1 <211> 1410 <212> DNA <213> Artificial <220> <223> Micrococcus flavus 16s rRNA <400> 1 atcctggctc aggatgaacg ctggcggcgt gcttaacaca tgcaagtcga acgatgaagc 60 ccagcttgct gggtggatta gtggcgaacg ggtgagtaac acgtgagtaa cctgccctta 120 actctgggat aagcctggga aactgggtct aataccggat aggagcgtcc accgcatggt 180 gggtgttgga aagatttatc ggttttggat ggactcgcgg cctatcagct tgttggtgag 240 gtaatggctc accaaggcga cgacgggtag ccggcctgag agggtgaccg gccacactgg 300 gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg cacaatgggc 360 gaaagcctga tgcagcgacg ccgcgtgagg gatgacggcc ttcgggttgt aaacctcttt 420 cagtagggaa gaagcgaaag tgacggtacc tgcagaagaa gcaccggcta actacgtgcc 480 agcagccgcg gtaatacgta gggtgcgagc gttatccgga attattgggc gtaaagagct 540 cgtaggcggt ttgtcgcgtc tgtcgtgaaa gtccggggct taaccccgga tctgcggtgg 600 gtacgggcag actagagtgc agtaggggag actggaattc ctggtgtagc ggtggaatgc 660 gcagatatca ggaggaacac cgatggcgaa ggcaggtctc tgggctgtaa ctgacgctga 720 ggagcgaaag catggggagc gaacaggatt agataccctg gtagtccatg ccgtaaacgt 780 tgggcactag gtgtggggac cattccacgg tttccgcgcc gcagctaacg cattaagtgc 840 cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg ggcccgcaca 900 agcggcggag catgcggatt aattcgatgc aacgcgaaga accttaccaa ggcttgacat 960 gttctcgatc gccgtagaga tacggtttcc cctttggggc gggttcacag gtggtgcatg 1020 gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctcg 1080 ttccatgttg ccagcacgtc gtggtgggga ctcatgggga actgccgggg tcaactcgga 1140 ggaaggtgag gacgacgtca aatcatcatg ccccttatgt cttgggcttc acgcatgcta 1200 caatggccgg tacaatgggt tgcgatactg tgaggtggag ctaatcccaa aaagccggtc 1260 tcagttcgga ttggggtctg caactcgacc ccatgaagtc ggagtcgcta gtaatcgcag 1320 atcagcaacg ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcaagtcacg 1380 aaaagtcggt aacacccgaa agccggtggc 1410 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> Forward primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial <220> <223> Reverse primer <400> 3 ggttaccttg ttacgactt 19
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