KR20230140872A - Composition for preventing or improving uv-induced skin damage comprising pediococcus acidilactici lm1013 - Google Patents
Composition for preventing or improving uv-induced skin damage comprising pediococcus acidilactici lm1013 Download PDFInfo
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- KR20230140872A KR20230140872A KR1020220039668A KR20220039668A KR20230140872A KR 20230140872 A KR20230140872 A KR 20230140872A KR 1020220039668 A KR1020220039668 A KR 1020220039668A KR 20220039668 A KR20220039668 A KR 20220039668A KR 20230140872 A KR20230140872 A KR 20230140872A
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- skin
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- photoaging
- pediococcus acidilactici
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Abstract
Description
본원은, 페디오코쿠스 아시디락티시 LM1013 (KCTC12039BP)을 유효성분으로 포함하는 피부 광노화 개선용 조성물에 관한 것이다. The present application relates to a composition for improving skin photoaging containing Pediococcus acidilactici LM1013 (KCTC12039BP) as an active ingredient.
피부 노화는 일생 동안 다양한 생리적, 환경적 변화로 인해 발생한다. 이러한 피부 노화의 외적 요인들 중 자외선에 의한 광노화는 피부 손상을 유발하는 중요한 원인이다. 지구에 도달하는 자외선은 UVA와 UVB로 구성되어 있으며, 특히 UVB는 피부의 표피층에 흡수되어 세포 내 신호전달 과정을 거쳐 표피의 두께 감소와 극성 손실을 발생시킨다. Skin aging occurs due to various physiological and environmental changes throughout life. Among these external factors for skin aging, photoaging caused by ultraviolet rays is an important cause of skin damage. Ultraviolet rays that reach the Earth are composed of UVA and UVB. In particular, UVB is absorbed by the epidermal layer of the skin and goes through an intracellular signaling process, causing a decrease in the thickness of the epidermis and loss of polarity.
프로바이오틱스는 세계보건기구(WHO)에 의해 "적절한 양으로 투여될 때 숙주에게 건강상의 이점을 주는 살아있는 미생물"로 공식적으로 정의되었다. 이후, 현재는 “숙주에 유익한 작용을 갖는 미생물 또는 미생물의 성분”으로까지 확대되어 사균체 및 그 균체 성분까지 범위를 넓혀 정립되었다. 이러한 프로바이오틱스는 인체에 대하여 항염증성, 항알레르기성, 항당뇨병, 항비만 성 등 다양하고 유익한 효과를 가지고 있다는 연구가 알려졌다. 또한, 프로바이오틱스를 이용한 피부 세포와 인체에서의 피부 광노화 과정의 개선을 위한 연구도 보고되고 있다.Probiotics are officially defined by the World Health Organization (WHO) as “live microorganisms that, when administered in appropriate amounts, confer health benefits to the host.” Afterwards, it has now been expanded to include “microorganisms or components of microorganisms that have beneficial effects on the host” and has been expanded to include dead cells and their components. Studies have shown that these probiotics have various beneficial effects on the human body, including anti-inflammatory, anti-allergic, anti-diabetic, and anti-obesity properties. In addition, research has been reported on using probiotics to improve skin cells and the skin photoaging process in the human body.
본원에서 이용한 젖산균인 페디오코쿠스 아시디락티시는 막걸리에서 분리하였으며, 이전 연구들에서 막걸리 유래성분은 인간 각질세포의 피부장벽 균형 개선에 효과적인 것으로 보고되었지만 인간과 페디오코쿠스 아시디락티시 사이의 건강상의 이점에 대한 연구는 제한적으로 알려져 있다. 최근 연구로는 비만 억제 활성을 갖는 페디오코쿠스 아시디락티시 AO22 (대한민국특허출원 제2017-0168086호)와 당뇨병의 예방 및 치료 효과 페디오코쿠스 아시디락티시 M76 (대한민국특허출원 제2012-0025644호)이 개시된 바 있으나, 페디오코쿠스 아시디락티시가 자외선 UVB 유도 광노화로부터 피부를 보호하는 효과나 기작에 관하여는 보고된 바 없다.Pediococcus acidilactici, the lactic acid bacterium used in our hospital, was isolated from makgeolli, and in previous studies, makgeolli-derived ingredients were reported to be effective in improving the skin barrier balance of human keratinocytes. However, there is a difference between humans and Pediococcus acidilactici. There is limited research on its health benefits. Recent studies include Pediococcus acidiractici AO22 (Korean Patent Application No. 2017-0168086), which has anti-obesity activity, and Pediococcus acidiractici M76 (Korean Patent Application No. 2012-0025644), which has anti-diabetic activity and efficacy in preventing and treating diabetes. ) has been disclosed, but there has been no report on the effect or mechanism of Pediococcus acidilactici protecting the skin from ultraviolet UVB-induced photoaging.
이에, 본 발명자들은 피부 광노화 개선용 균주를 개발하고자 연구하였으며, 그 결과 본원 페디오코쿠스 아시디락티시 유산균이 자외선 UVB 조사에 의한 피부 손상을 회복시키고 피부 보습과 주름 개선에 관련있는 단백질인 콜라겐의 재생성을 유도하는 효과가 있는 것을 확인하고 그 기작에 대해 밝혀, 본 발명을 완성하였다.Accordingly, the present inventors studied to develop a strain for improving skin photoaging, and as a result, our Pediococcus acidilactici lactic acid bacteria repaired skin damage caused by ultraviolet UVB irradiation and regenerated collagen, a protein related to skin moisturization and wrinkle improvement. By confirming that it has an effect of inducing and elucidating the mechanism, the present invention was completed.
본원은, 페디오코쿠스 아시디락티시 LM1013(KCTC12039BP)을 유효성분으로 포함하는 피부 광노화 개선용 조성물에 관한 것이다. The present application relates to a composition for improving skin photoaging containing Pediococcus acidilactici LM1013 (KCTC12039BP) as an active ingredient.
그러나, 본원이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the problem to be solved by the present application is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
본원의 제1측면은, 페디오코쿠스 아시디락티시 LM1013(KCTC12039BP) 사균체를 제공한다.The first aspect of the present application provides dead cells of Pediococcus acidilactici LM1013 (KCTC12039BP).
본원의 제2측면은, 페디오코쿠스 아시디락티시 LM1013(KCTC12039BP)을 유효성분으로 포함하는 피부 광노화 개선용 식품 조성물을 제공한다.The second aspect of the present application provides a food composition for improving skin photoaging containing Pediococcus acidilactici LM1013 (KCTC12039BP) as an active ingredient.
본원의 제3측면은, 페디오코쿠스 아시디락티시 LM1013(KCTC12039BP)을 유효성분으로 포함하는 피부 광노화 관련 질환 예방 또는 치료용 약학 조성물을 제공한다.The third aspect of the present application provides a pharmaceutical composition for preventing or treating diseases related to skin photoaging, containing Pediococcus acidilactici LM1013 (KCTC12039BP) as an active ingredient.
본원의 제4측면은, 페디오코쿠스 아시디락티시 LM1013(KCTC12039BP)을 유효성분으로 포함하는 피부 광노화 개선용 화장료 조성물을 제공한다.The fourth aspect of the present application provides a cosmetic composition for improving skin photoaging containing Pediococcus acidilactici LM1013 (KCTC12039BP) as an active ingredient.
본원의 제5측면은, 페디오코쿠스 아시디락티시 LM1013(KCTC12039BP)을 유효성분으로 포함하는 반려동물 피부 광노화 개선용 사료 조성물을 제공한다.The fifth aspect of the present application provides a feed composition for improving photoaging of pet skin containing Pediococcus acidilactici LM1013 (KCTC12039BP) as an active ingredient.
본원의 일 구현예에 따른 페디오코쿠스 아시디락티시 LM1013을 유효성분으로 포함하는 피부 광노화 개선용 조성물은 자외선 UVB 조사에 의해 유발된 피부 손상을 회복하여 피부 보습과 주름 개선에 효과가 있다. 본원의 조성물을 활용하면 성상이 균질한 제품 생산이 가능하며, 부작용이 적다는 장점이 있다.A composition for improving skin photoaging containing Pediococcus acidilactici LM1013 as an active ingredient according to an embodiment of the present application is effective in moisturizing the skin and improving wrinkles by recovering skin damage caused by ultraviolet UVB irradiation. Using our composition, it is possible to produce products with homogeneous properties and has the advantage of having fewer side effects.
도 1은, HaCaT 세포주에 페디오코쿠스 아시디락티시 LM1013을 처리 후 자외선 UVB를 조사하고 현미경을 이용한 세포형태의 변화(A, B) 및 MTT assay(C, D)로 세포생존율을 비교한 것을 나타낸 도면이다. ###은 무처리 대조군에 비해 유의성이 있으며 (P<0.0001); **/***은 자외선 조사대조군에 비해 유의성이 있다 (P<0.005/P<0.001). Control은 대조군, UV only는 자외선 조사군, V-LM1013 (105 cells/ml)는 페디오코쿠스 아시디락티시 LM1013 생균 단독 처리군, V-LM1013 + UV (105 cells/ml)는 해당 농도구간의 생균 처리 후 자외선 조사군, LM1013-L (107 cells/ml), LM1013-H (108 cells/ml)는 해당 농도구간의 열처리 페디오코쿠스 아시디락티시 LM1013 사균체 처리 후 자외선 조사군을 의미한다.
도 2는, HaCaT 세포주에 페디오코쿠스 아시디락티시 LM1013 처리 후 자외선 UVB를 조사하고 이때 생성된 ROS의 변화량을 형광현미경을 이용한 사진(A, B) 및 DCF-DA assay(C, D)로 확인한 결과를 나타낸 도면이다. ##은 무처리 대조군에 비해 유의성이 있고 (P<0.005) **은 자외선 조사대조군에 비해 유의성이 있다 (P<0.005). ###은 무처리 대조군에 비해 유의성이 있고 (P<0.0001) ***은 자외선 조사대조군에 비해 유의성이 있다 (P<0.0001). Control은 대조군, UV only는 자외선 조사군, V-LM1013는 페디오코쿠스 아시디락티시 LM1013 생균 단독 처리군(105 cells/ml), V-LM1013 + UV는 생균(105 cells/ml) 처리 후 자외선 조사군, LM1013 + UV (108 cells/ml)는 열처리 유산균 사균체의 처리 후 자외선 조사군을 의미한다(실험군 설정 이하 동일).
도 3은, HaCaT 세포주에 페디오코쿠스 아시디락티시 LM1013 사균체 처리 후 자외선 UVB를 조사하고 이때 변화한 사이토카인의 mRNA 양을 qRT-PCR로 확인한 결과를 나타낸 도면이다. ###은 무처리 대조군에 비해 유의성이 있고 (P<0.0001) ***은 자외선 조사대조군에 비해 유의성이 있다 (P<0.0001).
도 4는, HaCaT 세포주에 페디오코쿠스 아시디락티시 LM1013 사균체 처리 후 자외선 UVB를 조사하고 이때 MAPKs (ERK, JNK)의 단백질 발현량의 변화를 웨스턴 블럿(A)으로 확인하고 활성화 정도를 수치화(B, C)한 것을 나타낸 도면이다. ###은 무처리 대조군에 비해 유의성이 있고 (P<0.0001) ***은 자외선 조사대조군에 비해 유의성이 있다 (P<0.0001).
도 5는, HaCaT 세포주에 페디오코쿠스 아시디락티시 LM1013 사균체 처리 후 자외선 UVB를 조사하고 이때 ECMs (Involucrin, Loricrin, Filaggrin) 단백질 및 MMPs (MMP-1,2)의 단백질 발현량의 변화를 웨스턴 블럿으로 확인한 결과를 나타낸 도면이다.
도 6은, HaCaT 세포주에 페디오코쿠스 아시디락티시 LM1013 사균체 처리 후 자외선 UVB를 조사하고 이때 프로콜라겐 단백질의 발현 변화량을 ELISA (A)로 확인 및 세포수 대비 프로콜라겐의 증감 정도를 수치화 (B) 하여 나타낸 도면이다. ###은 무처리 대조군에 비해 유의성이 있고 (P<0.0001) **은 자외선 조사대조군에 비해 유의성이 있다 (P<0.005).Figure 1 shows the comparison of cell viability by irradiating ultraviolet UVB after treating HaCaT cell line with Pediococcus acidilactici LM1013 and changing cell shape using a microscope (A, B) and MTT assay (C, D). This is the drawing shown. ### is significant compared to the untreated control group (P<0.0001); **/*** is significant compared to the ultraviolet irradiation control group (P<0.005/P<0.001). Control is the control group, UV only is the UV irradiation group, V-LM1013 (10 5 cells/ml) is the Pediococcus acidilactici LM1013 live cell treatment group alone, and V-LM1013 + UV (10 5 cells/ml) is the corresponding concentration. The UV irradiated group, LM1013-L (10 7 cells/ml) and LM1013-H (10 8 cells/ml), were treated with heat-treated Pediococcus acidilactici LM1013 dead cells in the corresponding concentration range and then irradiated with UV rays. It means military.
Figure 2 shows the HaCaT cell line treated with Pediococcus acidilactici LM1013 and then irradiated with ultraviolet UVB, and the change in ROS generated at this time was measured using fluorescence microscopy (A, B) and DCF-DA assay (C, D). This is a drawing showing the confirmed results. ## is significant compared to the untreated control group (P<0.005) and ** is significant compared to the ultraviolet irradiated control group (P<0.005). ### is significant compared to the untreated control group (P<0.0001) and *** is significant compared to the ultraviolet irradiated control group (P<0.0001). Control is the control group, UV only is the UV irradiation group, V-LM1013 is the group treated with Pediococcus acidilactici LM1013 live cells only (10 5 cells/ml), and V-LM1013 + UV is the group treated with live cells (10 5 cells/ml). Post-UV irradiation group, LM1013 + UV (10 8 cells/ml) refers to the UV irradiation group after treatment of heat-treated dead lactic acid bacteria cells (same as experimental group settings below).
Figure 3 is a diagram showing the results of treating the HaCaT cell line with Pediococcus acidilactici LM1013 dead cells, irradiating ultraviolet UVB, and confirming the changed amount of cytokine mRNA by qRT-PCR. ### is significant compared to the untreated control group (P<0.0001) and *** is significant compared to the ultraviolet irradiated control group (P<0.0001).
Figure 4 shows that the HaCaT cell line was treated with Pediococcus acidilactici LM1013 dead cells and then irradiated with ultraviolet UVB. At this time, changes in protein expression levels of MAPKs (ERK, JNK) were confirmed by Western blot (A), and the degree of activation was quantified. (B, C) This is a drawing showing what was done. ### is significant compared to the untreated control group (P<0.0001) and *** is significant compared to the ultraviolet irradiated control group (P<0.0001).
Figure 5 shows changes in the protein expression levels of ECMs (Involucrin, Loricrin, Filaggrin) proteins and MMPs (MMP-1, 2) when HaCaT cell line was treated with dead cells of Pediococcus acidilactici LM1013 and then irradiated with ultraviolet UVB. This is a diagram showing the results confirmed by Western blot.
Figure 6 shows the HaCaT cell line treated with Pediococcus acidilactici LM1013 dead cells and then irradiated with ultraviolet UVB. At this time, the change in expression of procollagen protein was confirmed by ELISA (A) and the degree of increase or decrease in procollagen compared to the number of cells was quantified ( B) This is a drawing shown. ### is significant compared to the untreated control group (P<0.0001) and ** is significant compared to the ultraviolet irradiated control group (P<0.005).
아래에서는 첨부한 도면을 참조하여 본원이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본원의 실시예를 상세히 설명한다. 그러나 본원은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다. 그리고 도면에서 본원을 명확하게 설명하기 위해서 설명과 관계없는 부분은 생략하였으며, 명세서 전체를 통하여 유사한 부분에 대해서는 유사한 도면 부호를 붙였다.Below, with reference to the attached drawings, embodiments of the present application will be described in detail so that those skilled in the art can easily implement them. However, the present application may be implemented in various different forms and is not limited to the embodiments described herein. In order to clearly explain the present application in the drawings, parts that are not related to the description are omitted, and similar reference numerals are assigned to similar parts throughout the specification.
본원 명세서 전체에서, 어떤 부재가 다른 부재 “상에” 위치하고 있다고 할 때, 이는 어떤 부재가 다른 부재에 접해 있는 경우뿐 아니라 두 부재 사이에 또 다른 부재가 존재하는 경우도 포함한다.Throughout the specification of the present application, when a member is said to be located “on” another member, this includes not only the case where the member is in contact with the other member, but also the case where another member exists between the two members.
본원 명세서 전체에서, 어떤 부분이 어떤 구성 요소를 “포함” 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성 요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다. 본원 명세서 전체에서 사용되는 정도의 용어 “약”, “실질적으로” 등은 언급된 의미에 고유한 제조 및 물질 허용오차가 제시될 때 그 수치에서 또는 그 수치에 근접한 의미로 사용되고, 본원의 이해를 돕기 위해 정확하거나 절대적인 수치가 언급된 개시 내용을 비양심적인 침해자가 부당하게 이용하는 것을 방지하기 위해 사용된다. 본원 명세서 전체에서 사용되는 정도의 용어 “~(하는) 단계” 또는 “~의 단계”는 “~ 를 위한 단계”를 의미하지 않는다.Throughout the specification of the present application, when a part “includes” a certain component, this means that it may further include other components rather than excluding other components unless specifically stated to the contrary. As used throughout the specification, the terms “about,” “substantially,” and the like are used to mean at or close to a numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to convey the understanding of the present application. Precise or absolute figures are used to assist in preventing unscrupulous infringers from taking unfair advantage of stated disclosures. The term “step of” or “step of” as used throughout the specification does not mean “step for.”
본원 명세서 전체에서, 마쿠시 형식의 표현에 포함된 “이들의 조합(들)”의 용어는 마쿠시 형식의 표현에 기재된 구성 요소들로 이루어진 군에서 선택되는 하나 이상의 혼합 또는 조합을 의미하는 것으로서, 상기 구성 요소들로 이루어진 군에서 선택되는 하나 이상을 포함하는 것을 의미한다.Throughout this specification, the term “combination(s) thereof” included in the Markushi format expression refers to a mixture or combination of one or more selected from the group consisting of the components described in the Markushi format expression, It means containing one or more selected from the group consisting of the above components.
본원 명세서 전체에서, “A 및/또는 B”의 기재는 “A 또는 B, 또는 A 및 B”를 의미한다.Throughout this specification, references to “A and/or B” mean “A or B, or A and B.”
이하, 첨부된 도면을 참조하여 본원의 구현예 및 실시예를 상세히 설명한다. 그러나, 본원이 이러한 구현예 및 실시예와 도면에 제한되지 않을 수 있다.Hereinafter, implementation examples and examples of the present application will be described in detail with reference to the attached drawings. However, the present application may not be limited to these implementations, examples, and drawings.
본원의 제 1 측면은, 페디오코쿠스 아시디락티시 LM1013 사균체를 제공한다.The first aspect of the present application provides dead cells of Pediococcus acidilactici LM1013.
상기 유산균은 페디오코쿠스 (Pediococcus) 속이며 막걸리에서 분리 동정 후 이용하였다. 상기 유산균 배양 과정은 함수포도당, 효모 추출물, 소이펩톤 및 이온성분, 그 외 생육인자를 첨가하는 것이 바람직하다. 상기 유산균의 열처리는 상기 유산균의 열처리는 80~120℃에서 5~120분 동안 가열공정으로 사균화한 사균체를 의미한다. 또한 상기 열처리 유산균 사균체는 동결건조한 분말 형태인 것이 바람직하지만 이에 한정하지 않는다.The lactic acid bacteria belong to the genus Pediococcus and were used after isolation and identification from makgeolli. In the lactic acid bacteria culture process, it is preferable to add hydrated glucose, yeast extract, soy peptone, ionic components, and other growth factors. The heat treatment of the lactic acid bacteria refers to dead cells killed by a heating process at 80 to 120°C for 5 to 120 minutes. In addition, the heat-treated dead lactic acid bacteria cells are preferably in the form of freeze-dried powder, but are not limited to this.
본 발명의 페디오코쿠스 아시디락티시 LM1013유산균은 그대로 섭취하거나 다른 식품 또는 식품 성분과 함께 제조될 수 있고, 통상적인 방법에 따라 적절하게 제조될 수 있다. 상기 열처리 유산균을 첨가할 수 있는 통상적인 의미에서의 모든 식품에서 가능하며, 식품의 종류에는 특별한 제한은 없다.The Pediococcus acidilactici LM1013 lactic acid bacterium of the present invention can be consumed as is or manufactured with other foods or food ingredients, and can be appropriately manufactured according to conventional methods. The heat-treated lactic acid bacteria can be added to any food in the conventional sense, and there is no particular limitation on the type of food.
본원의 제 2측면은, 페디오코쿠스 아시디락티시 LM1013을 유효성분으로 포함하는 피부 광노화 개선용 식품 조성물을 제공한다. 제1측면과 중복되는 내용은 제2측면의 식품 조성물에도 공히 적용된다.The second aspect of the present application provides a food composition for improving skin photoaging containing Pediococcus acidilactici LM1013 as an active ingredient. Content that overlaps with the first aspect also applies to the food composition of the second aspect.
본원의 페디오코쿠스 아시디락티시 LM1013유산균은 그대로 섭취하거나 다른 식품 또는 식품 성분과 함께 제조될 수 있고, 통상적인 방법에 따라 적절하게 제조될 수 있다. 상기 유산균을 첨가할 수 있는 통상적인 의미에서의 모든 식품에서 가능하며, 식품의 종류에는 특별한 제한은 없다.The Pediococcus acidilactici LM1013 lactic acid bacterium of the present application can be consumed as is or manufactured with other foods or food ingredients, and can be appropriately manufactured according to conventional methods. It is possible to add the above-mentioned lactic acid bacteria to all foods in the conventional sense, and there is no particular limitation on the type of food.
본원의 페디오코쿠스 아시디락티시 LM1013유산균 외에 피부 건강 관련 성분을 추가로 포함할 수 있고 이는 본 발명의 균주를 이용한 피부 광노화 개선 효과는 더욱 증진될 수 있다. 상기 추가적인 피부 광노화 개선 성분은 식품의약품안정청의 건강기능식품으로 고시된 고시원료 및 개별인정형 원료를 이용할 수 있다.In addition to the Pediococcus acidilactici LM1013 lactic acid bacteria of the present application, skin health-related ingredients may be additionally included, which can further enhance the effect of improving skin photoaging using the strain of the present invention. The additional skin photoaging improvement ingredients can be used as notified raw materials and individually approved raw materials notified as health functional foods by the Food and Drug Safety Administration.
본원의 제 3측면은, 페디오코쿠스 아시디락티시 LM1013을 유효성분으로 포함하는 피부 광노화 관련 질환 예방 또는 치료용 약학 조성물을 제공한다. 제1측면 및 제2측면과 중복되는 내용은 제3측면의 약학 조성물에도 공히 적용된다.The third aspect of the present application provides a pharmaceutical composition for preventing or treating diseases related to skin photoaging, containing Pediococcus acidilactici LM1013 as an active ingredient. Contents that overlap with the first and second aspects also apply to the pharmaceutical composition of the third aspect.
본원의 일 구현예에 있어서, 상기 피부 광노화 관련 질환은 기미, 주근깨, 색소침착, 흑자, 만성 광선피부염, 다형광발진, 피부 노화, 주름, 안면홍조, 피부암, 광선각화증 및 지루각화증으로 이루어진 군에서 선택된 하나 이상의 것이나, 이에 제한되는 것은 아니다.In one embodiment of the present application, the skin photoaging-related diseases are from the group consisting of freckles, freckles, pigmentation, lentigo, chronic actinic dermatitis, polymorphic light rash, skin aging, wrinkles, facial flushing, skin cancer, actinic keratosis, and seborrheic keratosis. One or more selected ones, but are not limited thereto.
본원의 일 구현예에 있어서, 상기 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제제화하여 사용될 수 있으나, 이에 제한되지 않을 수 있다. In one embodiment of the present application, the pharmaceutical composition is administered in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. It may be formulated and used, but may not be limited thereto.
본원의 일 구현예에 있어서, 상기 약학 조성물을 제제화할 경우, 일반적으로 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 또는 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있으나, 이에 제한되지 않을 수 있다.In one embodiment of the present application, when formulating the pharmaceutical composition, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, or surfactants, but is not limited thereto. It may not be possible.
본원의 일 구현예에 있어서, 경구투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 또는 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 균주의 사균체에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스 (sucrose), 락토오스 (lactose), 또는 젤라틴 등을 섞어 조제될 수 있다. 또한, 예를 들어, 단순한 부형제 이외에도 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있으나, 이에 제한되지 않을 수 있다. In one embodiment of the present application, solid preparations for oral administration include tablets, pills, powders, granules, or capsules, and such solid preparations include dead cells of the above-mentioned strain with at least one excipient, for example, It can be prepared by mixing starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, for example, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used, but may not be limited thereto.
본원의 일 구현예에 있어서, 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있으나, 이에 제한되지 않을 수 있다. In one embodiment of the present application, liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, Sweeteners, fragrances, preservatives, etc. may be included, but may not be limited thereto.
본원의 일 구현예에 있어서, 비경구 투여를 위한 제제로는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함될 수 있으나, 이에 제한되지 않을 수 있다. 예를 들어, 상기 비수성용제 또는 현탁제로는, 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있으나, 이에 제한되지 않을 수 있다. 예를 들어, 상기 좌제로는, 위텝솔(witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있으나, 이에 제한되지 않을 수 있다.In one embodiment of the present application, preparations for parenteral administration may include, but are not limited to, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. For example, the non-aqueous solvent or suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc., but may not be limited thereto. For example, the suppository may include witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc., but may not be limited thereto.
본원의 일 구현예에 따른 약학 조성물은 의약품 조성물 또는 의약외품 조성물일 수 있다.The pharmaceutical composition according to one embodiment of the present application may be a pharmaceutical composition or a quasi-drug composition.
본원 명세서 전체에서 사용되는 용어 "의약외품"은 사람이나 동물의 질병을 진단, 치료, 개선, 경감, 처치 또는 예방할 목적으로 사용되는 물품들 중 의약품보다 작용이 경미한 물품들을 의미하는 것으로, 예를 들어 약사법에 따르면 의약외품이란 의약품의 용도로 사용되는 물품을 제외한 것으로, 사람ㆍ동물의 질병 치료나 예방에 쓰이는 제품, 인체에 대한 작용이 경미하거나 직접 작용하지 않는 제품 등이 포함된다.The term "quasi-drugs" used throughout the specification herein refers to products with a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals. For example, the Pharmaceutical Affairs Act According to the Act, quasi-drugs exclude products used for medicinal purposes and include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.
본원의 상기 의약외품 조성물은 바디 클렌저, 소독 청결제, 세정제, 주방용 세정제, 청소용 세정제, 치약, 가글제, 물티슈, 세제, 비누, 핸드 워시, 헤어세정제, 헤어 유연제, 가습기 충진제, 마스크, 연고제 및 필터 충진제로 이루어진 군에서 선택되는 제형으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The quasi-drug composition of the present application consists of body cleanser, disinfectant cleaner, detergent, kitchen cleaner, cleaning cleaner, toothpaste, mouthwash, wet tissue, detergent, soap, hand wash, hair cleaner, hair softener, humidifier filler, mask, ointment, and filter filler. It can be manufactured in a formulation selected from the group, but is not limited thereto.
본원의 일 구현예에 있어서, 상기 약학 조성물은 약제학적으로 유효한 양으로 투여될 수 있는데, 본원의 용어 "약제학적으로 유효한 양"이란 의학적 치료 또는 예방에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료 또는 예방하기에 충분한 양을 의미하며, 유효 용량 수준은 질환의 중증도, 약물의 활성, 환자의 연령, 체중, 건강, 성별, 환자의 약물에 대한 민감도, 사용된 본 발명 조성물의 투여 시간, 투여 경로 및 배출 비율 치료기간, 사용된 본 발명의 조성물과 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본원의 약학 조성물은 단독으로 투여하거나 공지된 장 질환에 대한 치료 효과를 나타내는 것으로 알려진 성분과 병용하여 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하다.In one embodiment of the present application, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" herein refers to the treatment of diseases with a reasonable benefit/risk ratio applicable to medical treatment or prevention. Or it means an amount sufficient for prevention, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, and the administration route. and excretion rate can be determined based on factors including treatment duration, drugs used in combination or concurrently with the compositions of the invention used, and other factors well known in the medical field. The pharmaceutical composition of the present application can be administered alone or in combination with ingredients known to exhibit therapeutic effects on known intestinal diseases. It is important to consider all of the above factors and administer the amount that will achieve the maximum effect with the minimum amount without side effects.
본원의 일 구현예에 있어서, 상기 약학 조성물의 투여량은 사용목적, 질환의 중독도, 환자의 연령, 체중, 성별, 기왕력, 또는 유효성분으로서 사용되는 물질의 종류 등을 고려하여 당업자가 결정할 수 있다. 예를 들어, 본 발명의 약학 조성물은 성인 1인당 약 0.1ng 내지 약 1,000 mg/kg, 바람직하게는 1 ng 내지 약 100 mg/kg로 투여할 수 있고, 본원의 조성물의 투여빈도는 특별히 이에 제한되지 않으나, 1일 1회 투여하거나 또는 용량을 분할하여 수회 투여할 수 있다. 상기 투여량 또는 투여횟수는 어떠한 면으로든 본원의 범위를 한정하는 것은 아니다.In one embodiment of the present application, the dosage of the pharmaceutical composition can be determined by a person skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, gender, antecedent history, or the type of substance used as an active ingredient. there is. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg per adult, and the frequency of administration of the composition of the present invention is specifically limited thereto. However, it can be administered once a day or the dose can be divided and administered several times. The above dosage or frequency of administration does not limit the scope of the present application in any way.
본원의 약학 조성물은 특별히 이에 제한되지 않으나, 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경피패치투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여 등의 경로를 통해 투여될 수 있다. 다만, 경구 투여 시에는 제형화되지 않은 형태로도 투여할 수 있고, 위산에 의하여 상기 락토바실러스 파라카제이 LM1014 균주가 변성 또는 분해될 수 있기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화된 형태 또는 경구용 패치형태로 구강내에 투여할 수도 있다. 또한, 상기 조성물은 활성 물질이 표적세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The pharmaceutical composition herein is not particularly limited, but depending on the purpose, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. It can be administered through the route. However, when administered orally, it can also be administered in an unformulated form, and since the Lactobacillus paracasei LM1014 strain may be denatured or decomposed by stomach acid, the oral composition may be coated with the active agent or protected from decomposition in the stomach. It can also be administered orally in a protective form or in the form of an oral patch. Additionally, the composition can be administered by any device that allows the active substance to move to target cells.
본원의 제 4측면은, 페디오코쿠스 아시디락티시 LM1013을 유효성분으로 포함하는 피부 광노화 개선용 화장료 조성물을 제공한다. 제1측면 내지 제3측면과 중복되는 내용은 제4측면의 화장료 조성물에도 공히 적용된다.The fourth aspect of the present application provides a cosmetic composition for improving skin photoaging containing Pediococcus acidilactici LM1013 as an active ingredient. Contents that overlap with the first to third aspects also apply to the cosmetic composition of the fourth aspect.
상기 화장료 조성물은 유연화장수, 영양화장수, 수렴화장수, 스킨, 로션, 에센스, 세럼, 크림, 마사지 크림, 팩, 메이크업 베이스, 비비크림, 파운데이션, 파우더, 클렌징 폼, 클렌징 크림 및 클렌징 워터로 이루어진 군에서 선택된 하나의 제형일 수 있다. The cosmetic composition is from the group consisting of softening lotion, nourishing lotion, astringent lotion, skin, lotion, essence, serum, cream, massage cream, pack, makeup base, BB cream, foundation, powder, cleansing foam, cleansing cream and cleansing water. There may be only one formulation selected.
본원의 제 5측면은, 페디오코쿠스 아시디락티시 LM1013을 유효성분으로 포함하는 반려동물 피부 광노화 개선용 사료 조성물을 제공한다. 제1측면 내지 제4측면과 중복되는 내용은 제5측면의 사료 조성물에도 공히 적용된다.The fifth aspect of the present application provides a feed composition for improving photoaging of pet skin containing Pediococcus acidilactici LM1013 as an active ingredient. Contents that overlap with the first to fourth aspects also apply to the feed composition of the fifth aspect.
상기 사료 조성물은 유효성분으로 페디오코쿠스 아시디락티시 LM1013 이외에, 식품의 기준 및 규격('식품공전')에 기재된 식품으로 사용가능한 식품 원료, 식품첨가물 공전에 기재된 식품첨가물을 사용할 수 있고, 식품으로 사용가능한 식품 원료 또는 식품첨가물이 아니더라도 '사료 등의 기준 및 규격' 별표 1의 단미사료의 범위에 해당하는 원료, 별표 2의 보조사료의 범위에 해당하는 원료를 사용할 수 있다.In addition to Pediococcus acidilactici LM1013 as an active ingredient, the feed composition can use food raw materials and food additives listed in the Food Additives Code as described in the Food Standards and Specifications ('Food Code'), and food Even if they are not food raw materials or food additives that can be used as food ingredients, raw materials that fall within the scope of single feed in Annex 1 of ‘Standards and Specifications for Feed, etc.’ and raw materials that fall within the scope of supplementary feed in Annex Table 2 can be used.
상기 사료 조성물은 '사료 등의 기준 및 규격'에 따른 보조사료 중 추출제일 수 있고, 상기 보조사료를 포함하는 배합사료일 수 있다.The feed composition may be an extractant among supplementary feeds according to 'Standards and specifications for feed, etc.', or may be a compound feed containing the supplementary feed.
이하, 본원의 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 하나, 하기의 실시예는 본원의 이해를 돕기 위하여 예시하는 것 일뿐, 본원의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples of the present application. However, the following examples are merely illustrative to aid understanding of the present application, and the content of the present application is not limited to the following examples.
[실시예][Example]
실시예 1. 세포 회복능 측정 (MTT assay)과 광학현미경 관찰Example 1. Cell recovery ability measurement (MTT assay) and light microscope observation
인간 유래 표피세포주인 HaCaT의 배양을 위해 10% 소태아 혈청 (Gibco-BRL, USA)과 1& 페니실린-스트렙토마이신 (5,000 Units/mL 페니실린, 5,000 μg/μL 스트렙토마이신, Gibco-BRL, USA)을 넣은 DMEM 배양액을 사용하였다. HaCaT 세포주를 1×105 cells/mL로 6-웰 플레이트 (SPL, Korea)에 첨가한 후 18 시간 동안 37℃, 5% CO2 인큐베이터에서 배양한 다음, 페디오코쿠스 아시디락티시 LM1013의 생균 105 cells/mL 또는 사균체를 각각 107 과 108 cells/mL의 농도로 제조하여 HaCaT 세포주 실험군에 처리하고 추가로 24 시간 동안 배양하였다. 다음, 양성대조군과 실험군에 UV crosslinker (CL-508, UVITEC, GBR)를 이용하여 18 mJ/cm2으로 자외선 UVB를 조사하고 다시 24 시간 동안 배양 후 광학현미경 (Observer D.1, ZIEZZ, DEU)을 이용하여 200배율로 확대 관찰 후 사진을 촬영하였다. 이후, 6-웰에 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL 용액을 10% 용량으로 첨가하여 1 시간 동안 배양시켰다. 포르마잔 (Formazan) 형성을 확인한 후, 배지를 완전히 제거하고, 웰의 바닥에 형성된 포르마잔을 녹이기 위해 1mL의 DMSO를 첨가한 후, 마이크로플레이트 리더기를 이용하여 570 nm에서 흡광도를 측정하였다. 그 결과, 페디오코쿠스 아시디락티시 LM1013의 생균 105 cells/mL 또는 사균체 107 내지 108 cells/mL 농도의 첨가가 HaCaT 세포주의 자외선 UVB에 대한 손상을 회복시켜주는 것을 확인하였다.For the culture of HaCaT, a human-derived epidermal cell line, 10% fetal bovine serum (Gibco-BRL, USA) and 1&penicillin-streptomycin (5,000 Units/mL penicillin, 5,000 μg/μL streptomycin, Gibco-BRL, USA) were added. DMEM culture medium was used. The HaCaT cell line was added at 1×10 5 cells/mL to a 6-well plate (SPL, Korea) and cultured in an incubator at 37°C, 5% CO 2 for 18 hours, and then incubated with viable cells of Pediococcus acidilactici LM1013. 10 5 cells/mL or dead cells were prepared at a concentration of 10 7 and 10 8 cells/mL, respectively, treated with the HaCaT cell line experimental group, and cultured for an additional 24 hours. Next, the positive control group and the experimental group were irradiated with ultraviolet UVB at 18 mJ/cm 2 using a UV crosslinker (CL-508, UVITEC, GBR), incubated for another 24 hours, and then examined with an optical microscope (Observer D.1, ZIEZZ, DEU). The photograph was taken after magnifying the observation at 200x magnification. Afterwards, 10% volume of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL solution) was added to 6-wells and incubated for 1 hour. Formazan ( After confirming the formation of formazan, the medium was completely removed, 1 mL of DMSO was added to dissolve the formazan formed at the bottom of the well, and the absorbance was measured at 570 nm using a microplate reader. As a result, Pedioco It was confirmed that adding 10 5 cells/mL of live cells or 10 7 to 10 8 cells/mL of dead cells of Cus acidilactici LM1013 restored damage caused by ultraviolet UVB in the HaCaT cell line.
실시예 2. ROS 생성능 확인 (DCF-DA assay)Example 2. Confirmation of ROS generation ability (DCF-DA assay)
HaCaT 세포주를 1×105 cells/mL로 6-웰 플레이트에 첨가한 후 18 시간 동안 37℃, 5% CO2 인큐베이터에서 배양한 다음, 페디오코쿠스 아시디락티시 LM1013의 생균 105 cells/mL 농도 또는 사균체를 108 cells/mL의 농도로 제조하여 HaCaT 세포주 실험군에 처리하고 추가로 24 시간 동안 배양하였다. 그 다음, 양성대조군과 실험군에 UV crosslinker를 이용하여 18 mJ/cm2으로 자외선 UVB를 조사하고 다시 24 시간 동안 배양하였다. The HaCaT cell line was added to a 6-well plate at 1×10 5 cells/mL, cultured in a 5% CO2 incubator at 37°C for 18 hours, and then incubated with viable cells of Pediococcus acidilactici LM1013 at a concentration of 10 5 cells/mL. Alternatively, dead cells were prepared at a concentration of 10 8 cells/mL, treated with the HaCaT cell line experimental group, and cultured for an additional 24 hours. Next, the positive control group and the experimental group were irradiated with ultraviolet UVB at 18 mJ/cm 2 using a UV crosslinker and incubated for another 24 hours.
HBSS (Hanks balanced salts solution)에 1mM DCF-DA (2’, 7’ -dichlorofluorescin diacetate) 용액을 넣어 최종 농도가 40μM이 되도록 희석하고 배양액을 제거한 6-웰 플레이트에 40μM DCF-DA 용액을 1mL씩 넣고 빛을 차단하여 37℃, 30분 동안 배양하였다. 그 다음, 인산완충식염수로 2회 씻어주고 1M NaOH를 1mL을 넣어 세포를 용해시키고 원심분리기를 이용하여 상온에서 8000rpm, 5분간 가동하여 상등액만 분리하였다. 상등액 중에서 200μL을 덜어서 마이크로플레이트 리더기를 이용하여 들뜬상태 파장 485nm 와 방출상태 파장 535nm를 설정하여 형광을 측정하였다. Add 1mM DCF-DA (2', 7'-dichlorofluorescin diacetate) solution to HBSS (Hanks balanced salts solution), dilute to a final concentration of 40μM, and add 1mL of 40μM DCF-DA solution to each 6-well plate from which the culture medium was removed. Light was blocked and incubated at 37°C for 30 minutes. Next, the cells were washed twice with phosphate-buffered saline, 1 mL of 1 M NaOH was added to lyse the cells, and the supernatant was separated by centrifuging at 8000 rpm for 5 minutes at room temperature. 200 μL of the supernatant was taken out and fluorescence was measured by setting the excited state wavelength to 485 nm and the emission state wavelength to 535 nm using a microplate reader.
본 실험 시 DCF-DA가 ROS와 반응하여 형광을 나타내는 원리를 이용하였으며, HaCaT 세포주에 페디오코쿠스 아시디락티시 LM1013의 생균을 105 cells/mL의 농도로 처리하거나, 사균체를 108 cells/mL의 농도로 처리 후 자외선 UVB를 조사하면 염증반응을 유도하는 ROS (Reactive oxygen species)의 발현량이 억제되는 것을 형광현미경 사진과 마이크로 플레이트 리더기로 측정한 형광값 그래프로 확인하였다(도 2 참조).In this experiment, the principle of DCF-DA reacting with ROS and producing fluorescence was used, and the HaCaT cell line was treated with live cells of Pediococcus acidilactici LM1013 at a concentration of 10 5 cells/mL, or dead cells were treated with 10 8 cells. It was confirmed by fluorescence micrographs and a graph of fluorescence values measured with a microplate reader that the expression of ROS (Reactive oxygen species), which induces inflammatory reactions, was suppressed when irradiated with ultraviolet UVB after treatment at a concentration of /mL (see Figure 2). .
실시예 3. 사이토카인 측정 (qRT-PCR)Example 3. Cytokine measurement (qRT-PCR)
실시예 3-1. 인간 유래 표피세포주인 HaCaT에서의 RNA 분리Example 3-1. Isolation of RNA from HaCaT, a human-derived epidermal cell line
HaCaT 세포주를 1×105 cells/mL로 6-웰 플레이트에 첨가한 후 18 시간 동안 37℃, 5% CO2 인큐베이터에서 배양한 다음, 페디오코쿠스 아시디락티시 LM1013의 사균체를 각각 108 cells/mL의 농도로 제조하여 HaCaT 세포주 실험군에 처리하고 추가로 24 시간 동안 배양하였다. 그 다음, 양성대조군과 실험군에 UV 가교제(UV crosslinker)를 이용하여 18 mJ/cm2으로 자외선 UVB를 조사하고 다시 24 시간 동안 배양하였다. 인산완충식염수로 씻어낸 후 RNA 추출용 키트 (MiniBEST universal RNA eztraction kit, Takara, JPN)를 이용하여 해당 제품의 메뉴얼에 따라서 RNA를 추출하였다.HaCaT cell line was added to a 6-well plate at 1×10 5 cells/mL, cultured in a 37°C, 5% CO2 incubator for 18 hours, and then dead cells of Pediococcus acidilactici LM1013 were grown at 10 8 cells each. It was prepared at a concentration of /mL, treated with the HaCaT cell line experimental group, and cultured for an additional 24 hours. Next, the positive control group and the experimental group were irradiated with ultraviolet UVB at 18 mJ/cm 2 using a UV crosslinker and incubated for another 24 hours. After washing with phosphate-buffered saline, RNA was extracted using an RNA extraction kit (MiniBEST universal RNA eztraction kit, Takara, JPN) according to the product's manual.
실시예 3-2. 인간 유래 표피세포주인 HaCaT에서의 cDNA 합성Example 3-2. cDNA synthesis in HaCaT, a human-derived epidermal cell line
상기 실시예 3-1에서 회수한 RNA들을 분광광도계 (DS11, Thermo fisher scientific, USA)을 이용하여 정량한 후 각각 200 ng/μL의 농도로 균일화 하였다. 다음 cDNA 합성 키트 (High capacity cDNA reverse transcription kits, Applied biosystems, USA)를 이용하여 해당 제품의 매뉴얼에 따라 RNA, 프라이머, dNTP Mix, 역전사효소, 반응 완충액, DEPC water를 혼합하여 PCR 기기 (TP350, Takara, JPN)를 이용하여 하기 [표 1]의 조건으로 cDNA를 합성하였다.The RNAs recovered in Example 3-1 were quantified using a spectrophotometer (DS11, Thermo fisher scientific, USA) and then homogenized to a concentration of 200 ng/μL. Using the following cDNA synthesis kit (High capacity cDNA reverse transcription kits, Applied biosystems, USA), mix RNA, primer, dNTP Mix, reverse transcriptase, reaction buffer, and DEPC water according to the product's manual, using a PCR device (TP350, Takara). , JPN) was used to synthesize cDNA under the conditions shown in [Table 1] below.
[표 1][Table 1]
실시예 3-3. cDNA를 이용한 인간 유래 표피세포주인 HaCaT에서의 mRNA 발현량 조사Example 3-3. Investigation of mRNA expression level in HaCaT, a human-derived epidermal cell line, using cDNA
상기 실시예 3-2에서 합성한 cDNA와 하기 [표 2]에 나타낸 프라이머를 qRT-PCR 용 마스터 믹스 (2X SYBR green PCR master mix, Applied biosystems, USA)와 DEPC water에 혼합하여 하기 [표 3]의 조건으로 qRT-PCR 기기 (Quanstudio3, Thermo fisher scientific, USA)를 이용해 형광 신호 증폭으로 mRNA 발현량을 분석하였다.The cDNA synthesized in Example 3-2 and the primers shown in Table 2 below were mixed with a master mix for qRT-PCR (2X SYBR green PCR master mix, Applied biosystems, USA) and DEPC water as shown in Table 3 below. The mRNA expression level was analyzed by fluorescence signal amplification using a qRT-PCR device (Quanstudio3, Thermo fisher scientific, USA) under the following conditions.
도 3에서 보듯이, HaCaT 세포주에 페디오코쿠스 아시디락티시 LM1013의 사균체를 108 cells/mL의 농도로 처리 후 자외선 UVB를 조사하면 염증반응을 유도하는 사이토카인인 TNF-α, IL-6의 mRNA 발현량이 억제되는 것을 확인할 수 있다. 이를 통해, 본원의 페디오코쿠스 아시디락티시 LM1013의 사균체의 처리는 HaCaT 세포주에서 자외선 UVB에 의해 유도된 염증반응을 억제해주는 것을 확인하였다. As shown in Figure 3, when the HaCaT cell line is treated with dead cells of Pediococcus acidiractici LM1013 at a concentration of 10 8 cells/mL and then irradiated with ultraviolet UVB, TNF-α, IL-, which are cytokines that induce an inflammatory response, are produced. It can be confirmed that the mRNA expression level of 6 is suppressed. Through this, it was confirmed that the treatment of dead cells of Pediococcus acidilactici LM1013 suppresses the inflammatory response induced by ultraviolet UVB in the HaCaT cell line.
[표 2][Table 2]
[표 3][Table 3]
실시예 4. MAPKs (Mitogen activated protein kinases) 신호전달경로를 통한 광노화 억제능 기전 조사 (Western blot)Example 4. Investigation of photoaging inhibition mechanism through MAPKs (Mitogen activated protein kinases) signaling pathway (Western blot)
페디오코쿠스 아시디락티시 LM1013의 사균체의 인간 유래 표피세포주인 HaCaT에 대한 광노화 개선능 기전을 조사하기 위해 세포 내 MAPK 신호전달경로를 웨스턴 블럿 방법으로 조사하였다. MAPKs (ERK, JNK) 신호전달경로는 세포 생존과 염증반응에 관여하는 대표적인 신호전달 경로로 알려져 있다. HaCaT 세포주를 1×105 cells/mL로 6-웰 플레이트에 첨가한 후 18 시간 동안 37℃, 5% CO2 인큐베이터에서 배양한 다음, 페디오코쿠스 아시디락티시 LM1013의 사균체를 각각 108 cells/mL의 농도로 제조하여 HaCaT 세포주 실험군에 처리하고 추가로 24 시간 동안 배양하였다. 그 다음, 양성대조군과 실험군에 UV crosslinker를 이용하여 18 mJ/cm2으로 자외선 UVB를 조사하고 24시간 배양 후 인산완충식염수로 세척하고 1000 rpm에서 5 분 동안 원심분리 하여 세포를 수득하였다. 각각 100 μl의 용해 버퍼를 첨가한 후에 1 시간 동안 얼음 상에서 반응시키고 12,000 rpm에서 20 분 동안 원심분리 하여 단백질을 수확하였다. 브래드포드 분석법을 이용하여 단백질을 정량하고 5Ⅹfluorescent master mix(cat.PS-FL05-8, Protein simple, USA)를 넣고 95℃에서 5 분 동안 변성 시킨 뒤에 JESS detection module (cat.PL3-0004, Protein simple, USA )의 메뉴얼에 따라 단백질 시료, 1차 항체, 2차항체, HRP (cat.042-414, Protein simple, USA), Luminol-peroxide mix (cat.043-311/379, Protein simple, USA), RePlex reagent mix (cat.043-827, Protein simple, USA)를 JESS 분석용 플레이트에 분주하였다. 다음, JESS 분석용 카트리지(cat.PS-CC01, Protein simple, USA)를 결합하고 JESS(Protein simple, USA) 기기를 가동하여 웨스턴 블럿을 수행하였다. To investigate the photoaging improvement mechanism of dead cells of Pediococcus acidilactici LM1013 against HaCaT, a human-derived epidermal cell line, the intracellular MAPK signaling pathway was examined using Western blotting. The MAPKs (ERK, JNK) signaling pathway is known to be a representative signaling pathway involved in cell survival and inflammatory responses. The HaCaT cell line was added to a 6-well plate at 1×10 5 cells/mL and cultured in a 37°C, 5% CO 2 incubator for 18 hours, and then 10 8 dead cells of Pediococcus acidilactici LM1013 were added to each well. It was prepared at a concentration of cells/mL, treated with the HaCaT cell line experimental group, and cultured for an additional 24 hours. Next, the positive control group and the experimental group were irradiated with ultraviolet UVB at 18 mJ/cm 2 using a UV crosslinker, and after culturing for 24 hours, cells were obtained by washing with phosphate-buffered saline and centrifuging at 1000 rpm for 5 minutes. After adding 100 μl of lysis buffer to each reaction, the mixture was reacted on ice for 1 hour and centrifuged at 12,000 rpm for 20 minutes to harvest the protein. Proteins were quantified using the Bradford analysis method, 5 , USA), protein sample, primary antibody, secondary antibody, HRP (cat.042-414, Protein simple, USA), Luminol-peroxide mix (cat.043-311/379, Protein simple, USA) according to the manual. , RePlex reagent mix (cat.043-827, Protein simple, USA) was dispensed onto the JESS analysis plate. Next, Western blot was performed by combining the JESS analysis cartridge (cat.PS-CC01, Protein simple, USA) and operating the JESS (Protein simple, USA) device.
페디오코쿠스 아시디락티시 LM1013의 사균체를 처리하고 자외선 UVB를 조사 후 MAPKs인 ERK와 JNK의 인산화를 측정한 결과, 해당 유산균의 존재 시 자외선 UVB만 조사한 대조군에 비해여 ERK와 JNK의 인산화가 감소됨을 확인하였다(도 4 참조). 따라서, 페디오코쿠스 아시디락티시 LM1013의 사균체의 인간 유래 표피세포주에서 나타내는 광노화 개선능이 MAPK (ERK) 신호전달경로의 비활성화로 인해 일어난다는 것을 알 수 있었다.As a result of measuring the phosphorylation of ERK and JNK, which are MAPKs, after treating dead cells of Pediococcus acidilactici LM1013 and irradiating them with ultraviolet UVB, phosphorylation of ERK and JNK was found in the presence of the lactic acid bacteria compared to the control group irradiated only with ultraviolet UVB. It was confirmed that it decreased (see Figure 4). Therefore, it was found that the photoaging improvement ability shown in the human-derived epidermal cell line of dead cells of Pediococcus acidilactici LM1013 occurs due to the inactivation of the MAPK (ERK) signaling pathway.
실시예 5. ECMs (Extracellular matrix)와 MMP (Matrix metalloproteinase) 신호전달 단백질을 통한 광노화 억제능 기전 조사 (Western blot)Example 5. Investigation of photoaging inhibition mechanism through ECMs (Extracellular matrix) and MMP (Matrix metalloproteinase) signaling protein (Western blot)
페디오코쿠스 아시디락티시 LM1013의 사균체의 인간 유래 표피세포주인 HaCaT에 대한 광노화 개선능 기전을 조사하기 위해 세포 내 ECMs 와 MMPs 신호전달 단백질들을 웨스턴 블럿 방법으로 조사하였다. ECMs (Involucrin, Loricrin, Filaggrin) 와 MMPs (MMP-1, MMP-2) 신호전달경로는 도 4에서 확인한 MAPKs 신호전달경로의 대표적인 하위 신호 단백질로서 세포외 기질의 구성요소 및 형성에 관여하는 것으로 알려져 있다. 활성화된 MAPKs 단백질은 MMPs 유전자의 과발현을 유도하고 비정상적으로 증가된 MMPs 단백질들은 세포외 기질인 Collagen을 분해하고 ECMs 단백질들의 감소를 유도하는 것으로 알려져 있다. 상기 실시예 4에서 얻은 단백질 시료를 실시예 4의 실험 분석법과 동일하게 JESS 기기를 이용하여 웨스턴블럿으로 단백질 양을 확인하였다. To investigate the photoaging improvement mechanism of dead cells of Pediococcus acidilactici LM1013 on HaCaT, a human-derived epidermal cell line, intracellular ECMs and MMPs signaling proteins were examined using Western blotting. ECMs (Involucrin, Loricrin, Filaggrin) and MMPs (MMP-1, MMP-2) signaling pathways are representative downstream signaling proteins of the MAPKs signaling pathway identified in Figure 4 and are known to be involved in the components and formation of the extracellular matrix. there is. Activated MAPKs proteins induce overexpression of MMPs genes, and abnormally increased MMPs proteins are known to decompose collagen, an extracellular matrix, and induce a decrease in ECMs proteins. The amount of the protein sample obtained in Example 4 was confirmed by Western blot using a JESS device in the same manner as in Example 4.
페디오코쿠스 아시디락티시 LM1013의 사균체를 처리한 후, 자외선 UVB를 조사하고 세포 내 ECMs와 MMPs 단백질 발현량들을 측정한 결과, 해당 유산균의 존재 시 자외선 UVB만 조사한 대조군에 비해여 MMPs (MMP-1, MMP-2) 단백질 발현량이 감소하고 이에 반해 ECMs (Involucrin, Loricrin, Filaggrin) 단백질 발현량은 증가하였다(도 5 참조). 따라서, 페디오코쿠스 아시디락티시 LM1013의 사균체가 인간 유래 표피세포주에서 나타내는 광노화 개선능은 실시예 4의 MAPK (ERK) 신호전달경로의 비활성화를 거쳐 MMPs의 단백질 발현량이 감소되어 세포외 기질인 ECMs의 단백질 발현량을 증가시킨다는 것을 알 수 있었다.After treating the dead cells of Pediococcus acidilactici LM1013, irradiating them with ultraviolet UVB and measuring the expression levels of ECMs and MMPs proteins in the cells, the results showed that in the presence of the lactic acid bacteria, MMPs (MMPs) were higher compared to the control group irradiated only with ultraviolet UVB. -1, MMP-2) protein expression decreased, whereas ECMs (Involucrin, Loricrin, Filaggrin) protein expression increased (see Figure 5). Therefore, the ability to improve photoaging shown by dead cells of Pediococcus acidilactici LM1013 in human-derived epidermal cell lines is due to a decrease in the protein expression of MMPs through inactivation of the MAPK (ERK) signaling pathway in Example 4, which is an extracellular matrix. It was found that the protein expression level of ECMs was increased.
실시예 6. 콜라겐 발현 변화에 따른 광노화 억제능 기전 확인 (ELISA)Example 6. Confirmation of photoaging inhibition mechanism according to changes in collagen expression (ELISA)
콜라겐 (Collagen)은 세포내에서 프로콜라겐 (Procollagen)으로 합성된 후 세포외로 분비되어 콜라겐 섬유로 중합되며, 세포의 형태를 유지하고 회복하는데 가장 중요한 물질이다. 이전 연구들에서 자외선 UVB에 의하여 프로콜라겐의 생성이 감소되는 것으로 알려져 있다. 페디오코쿠스 아시디락티시 LM1013의 사균체의 인간 유래 표피세포주에서 광노화 개선능 과정이 실시예 4와 실시예 5에서 수행한 신호전달과정을 거쳐 최종적으로 콜라겐의 발현을 조절하는 지 알아보고자 하였다. Collagen is synthesized intracellularly as procollagen and then secreted extracellularly to polymerize into collagen fibers, and is the most important substance in maintaining and recovering the shape of cells. In previous studies, it is known that the production of procollagen is reduced by ultraviolet UVB rays. We aimed to determine whether the photoaging improvement process in a human-derived epidermal cell line from dead cells of Pediococcus acidilactici LM1013 ultimately regulates collagen expression through the signal transduction process performed in Examples 4 and 5.
HaCaT 세포주를 1×105 cells/mL로 6-웰 플레이트에 첨가한 후 18 시간 동안 37℃, 5% CO2 인큐베이터에서 배양한 다음, 페디오코쿠스 아시디락티시 LM1013의 사균체를 각각 108 cells/mL의 농도로 제조하여 HaCaT 세포주 실험군에 처리하고 추가로 24 시간 동안 배양하였다. 그 다음, 양성대조군과 실험군에 UV 가교제(UV crosslinker)를 이용하여 18 mJ/cm2으로 자외선 UVB를 조사하고 24시간 배양 후 인산완충식염수로 세척하고 1000 rpm에서 5 분 동안 원심분리 하여 배양액을 수득하였다. 그 다음, 프로콜라겐(Procollagen) Type I C-peptide EIA kit (MK101, Takara, JPN)를 이용하여 ELISA를 매뉴얼에 따라 실험하였다. 항체가 코팅된 마이크로플레이트에 Antibody-POD conjugate solution을 결합시킨 후 대조군과 실험군의 배양액 시료 및 농도별로 희석된 표준 용액을 넣고 37℃, 2 시간 동안 반응시켰다. 반응 후 인산완충식염수로 세척하고 기질용액 (TMBZ; 3,3’,5,5’-tetramethylbenzidine)을 넣어 15분간 항원-항체 발색반응을 수행하고 정지용액 (stop solution)으로 반응종료 후 마이크로 플레이트 리더기로 450 nm에서 흡광도를 측정하였다. 그 결과, 페디오코쿠스 아시디락티시 LM1013의 사균체가 인간 유래 표피세포주에서의 광노화 개선 과정에서 콜라겐 재합성 유도과정으로 손상된 세포의 회복을 돕는 것을 알 수 있었다.HaCaT cell line was added to a 6-well plate at 1×10 5 cells/mL, cultured in a 37°C, 5% CO2 incubator for 18 hours, and then dead cells of Pediococcus acidilactici LM1013 were grown at 10 8 cells each. It was prepared at a concentration of /mL, treated with the HaCaT cell line experimental group, and cultured for an additional 24 hours. Next, the positive control group and the experimental group were irradiated with ultraviolet UVB at 18 mJ/cm 2 using a UV crosslinker, and after culturing for 24 hours, they were washed with phosphate-buffered saline and centrifuged at 1000 rpm for 5 minutes to obtain the culture medium. did. Next, ELISA was performed according to the manual using the Procollagen Type I C-peptide EIA kit (MK101, Takara, JPN). After binding the Antibody-POD conjugate solution to the antibody-coated microplate, culture samples from the control and experimental groups and standard solutions diluted by concentration were added and reacted at 37°C for 2 hours. After reaction, wash with phosphate-buffered saline solution, add substrate solution (TMBZ; 3,3',5,5'-tetramethylbenzidine), perform antigen-antibody color reaction for 15 minutes, terminate reaction with stop solution, and use microplate reader. Absorbance was measured at 450 nm. As a result, it was found that dead cells of Pediococcus acidilactici LM1013 help the recovery of damaged cells by inducing collagen resynthesis in the process of improving photoaging in human-derived epidermal cell lines.
전술한 본원의 설명은 예시를 위한 것이며, 본원이 속하는 기술분야의 통상의 지식을 가진 자는 본원의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The description of the present application described above is for illustrative purposes, and those skilled in the art will understand that the present application can be easily modified into other specific forms without changing its technical idea or essential features. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as single may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.
본원의 범위는 상기 상세한 설명보다는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본원의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present application is indicated by the claims described below rather than the detailed description above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present application.
Claims (15)
PEDIOCOCCUS ACIDILACTICI LM1013 (KCTC12039BP) dead cells.
상기 사균체는 콜라겐의 재생성을 유도하는 것인, 사균체.
According to clause 1,
The dead cells induce the regeneration of collagen.
상기 사균체는 피부 보습 및 주름 개선 효과가 있는 것인, 사균체.
According to clause 1,
The dead cells are those that have skin moisturizing and wrinkle-improving effects.
피부 광노화 개선용 식품 조성물.
Containing PEDIOCOCCUS ACIDILACTICI LM1013 (KCTC12039BP) as an active ingredient,
Food composition for improving skin photoaging.
상기 식품 조성물은 콜라겐의 재생성을 유도하는 것인,
피부 광노화 개선용 식품 조성물.
According to clause 4,
The food composition induces the regeneration of collagen,
Food composition for improving skin photoaging.
상기 식품 조성물은 피부 보습 및 주름 개선 효과가 있는 것인,
피부 광노화 개선용 식품 조성물.
According to clause 4,
The food composition has skin moisturizing and wrinkle improvement effects,
Food composition for improving skin photoaging.
피부 광노화 관련 질환 예방 또는 치료용 약학 조성물.
Containing PEDIOCOCCUS ACIDILACTICI LM1013 as an active ingredient,
Pharmaceutical composition for preventing or treating diseases related to skin photoaging.
상기 약학 조성물은 콜라겐의 재생성을 유도하는 것인,
피부 광노화 관련 질환 예방 또는 치료용 약학 조성물.
According to clause 7,
The pharmaceutical composition induces the regeneration of collagen,
Pharmaceutical composition for preventing or treating diseases related to skin photoaging.
상기 피부 광노화 관련 질환은 기미, 주근깨, 색소침착, 흑자, 만성 광선피부염, 다형광발진, 피부 노화, 주름, 안면홍조, 피부암, 광선각화증 및 지루각화증으로 이루어진 군에서 선택되는 하나 이상인 것인, 피부 광노화 관련 질환 예방 또는 치료용 약학 조성물.
According to clause 7,
The skin photoaging-related disease is one or more selected from the group consisting of spots, freckles, pigmentation, lentigo, chronic actinic dermatitis, polymorphic photorash, skin aging, wrinkles, facial flushing, skin cancer, actinic keratosis, and seborrheic keratosis. Pharmaceutical composition for preventing or treating photoaging-related diseases.
피부 광노화 개선용 화장료 조성물.
Containing PEDIOCOCCUS ACIDILACTICI LM1013 as an active ingredient,
Cosmetic composition for improving skin photoaging.
상기 화장료 조성물은 콜라겐의 재생성을 유도하는 것인,
피부 광노화 개선용 화장료 조성물.
According to clause 10,
The cosmetic composition induces the regeneration of collagen,
Cosmetic composition for improving skin photoaging.
상기 화장료 조성물은 피부 보습 및 주름 개선 효과가 있는 것인,
피부 광노화 개선용 화장료 조성물.
According to clause 10,
The cosmetic composition has skin moisturizing and wrinkle improvement effects,
Cosmetic composition for improving skin photoaging.
반려동물 피부 광노화 개선용 사료 조성물.
Containing PEDIOCOCCUS ACIDILACTICI LM1013 as an active ingredient,
Feed composition for improving photoaging of pet skin.
상기 사료 조성물은 콜라겐의 재생성을 유도하는 것인,
반려동물 피부 광노화 개선용 사료 조성물.
According to clause 13,
The feed composition induces the regeneration of collagen,
Feed composition for improving photoaging of pet skin.
상기 사료 조성물은 피부 보습 및 주름 개선 효과가 있는 것인,
반려동물 피부 광노화 개선용 사료 조성물.According to clause 13,
The feed composition has skin moisturizing and wrinkle improvement effects,
Feed composition for improving photoaging of pet skin.
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| KR1020220039668A KR20230140872A (en) | 2022-03-30 | 2022-03-30 | Composition for preventing or improving uv-induced skin damage comprising pediococcus acidilactici lm1013 |
| PCT/KR2022/012153 WO2023191196A1 (en) | 2022-03-30 | 2022-08-16 | Composition for preventing or reducing uv-induced skin damage, comprising pediococcus acidilactici lm1013 as active ingredient |
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| KR1020220039668A KR20230140872A (en) | 2022-03-30 | 2022-03-30 | Composition for preventing or improving uv-induced skin damage comprising pediococcus acidilactici lm1013 |
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| KR101420355B1 (en) | 2012-03-13 | 2014-07-16 | 농림수산식품기술기획평가원 | Novel strain of Pediococcus acidilactici M76 and exopolysaccharide produced from the same |
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| KR101349692B1 (en) * | 2011-12-06 | 2014-01-16 | 에이엠바이오 (주) | The Alcohol resistant strain of lactic acid bacteria, Pediococcus acidilactici and its use |
| JP6090854B2 (en) * | 2013-11-15 | 2017-03-08 | 株式会社東洋新薬 | Composition for promoting collagen production |
| WO2019160115A1 (en) * | 2018-02-16 | 2019-08-22 | イチビキ株式会社 | Lactic acid bacterium, interleukin-22 production inducing agent, skin barrier function enhancing agent |
| KR102169087B1 (en) * | 2019-05-02 | 2020-10-22 | 롯데푸드 주식회사 | Novel Pediococcus acidilactici LRCC5296 having reduction of hypersensitivity skin disorder |
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| KR101420355B1 (en) | 2012-03-13 | 2014-07-16 | 농림수산식품기술기획평가원 | Novel strain of Pediococcus acidilactici M76 and exopolysaccharide produced from the same |
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