KR20170090997A - Anti-fungal composition comprising lactobacillus plantarum - Google Patents
Anti-fungal composition comprising lactobacillus plantarum Download PDFInfo
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- KR20170090997A KR20170090997A KR1020160182201A KR20160182201A KR20170090997A KR 20170090997 A KR20170090997 A KR 20170090997A KR 1020160182201 A KR1020160182201 A KR 1020160182201A KR 20160182201 A KR20160182201 A KR 20160182201A KR 20170090997 A KR20170090997 A KR 20170090997A
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- South Korea
- Prior art keywords
- composition
- lactobacillus plantarum
- culture
- weight
- plantarum apsulloc
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Abstract
Description
본 명세서에는 락토바실러스 플란타룸의 항진균 용도가 개시된다.The antifungal use of Lactobacillus plantarum is disclosed herein.
피부염(Dermatitis)은 피부에 발생하는 염증을 의미하며 보통 습진(Eczema)와 같은 의미로 사용된다. 피부염은 크게 아토피성 피부염(Atopic dermatitis), 접촉성 피부염(Contact dermatitis), 지루성 피부염(Seborrhoeic dermatitis)으로 구분된다. 지루성 피부염은 머리, 이마 또는 겨드랑이 등 피지의 분비가 많은 부위에 잘 발생하는데, 홍반과 가는 인설, 즉 비듬을 동반하고 때로는 마른버짐이 일기도 한다.Dermatitis is inflammation that occurs in the skin and is usually used in the same way as eczema. Dermatitis is largely divided into atopic dermatitis, contact dermatitis, and seborrhoeic dermatitis. Seborrheic dermatitis occurs in areas of the sebaceous glands, such as the head, forehead or armpit, which can be accompanied by erythema, dandruff and sometimes dry skin.
지루성 피부염 및 비듬의 원인으로 다양한 내인성 요인 및 외인성 요인 등이 알려져 있으나, 최근 진균, 즉 효모균이 가장 큰 영향력이 있는 요인으로 알려졌다. 따라서, 이들의 증식을 억제할 수 물질에 대한 연구가 활발하다. 현재 시중에서 항진균제로 케토코나졸(Ketoconazole), 아연 피리치온(Zinc pyrithion), 이트라코나졸(Itraconazole) 등의 합성물과 타르, 유황, 살리실산 등이 복합적으로 사용되고 있으나 인체안전성에 관한 의문이 끊임없이 제기되고 있다. 따라서, 인체에 무해하면서 지루성 피부염과 비듬을 억제할 수 있는 물질에 대한 연구가 필요한 실정이다.Various endogenous and exogenous factors are known to cause seborrheic dermatitis and dandruff. However, recently, it has been known that fungi, yeast, are the most influential factors. Therefore, researches on substances capable of inhibiting their proliferation are actively conducted. Currently, commercial anti-fungal agents such as ketoconazole, zinc pyrithione, itraconazole, tar, sulfur and salicylic acid are used in combination, but there are constant questions about human safety. Therefore, it is necessary to study a substance which is harmless to the human body and can suppress seborrheic dermatitis and dandruff.
일 측면에서, 본 발명의 목적은, 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266(기탁번호: KCCM11181P)의 신규한 용도를 제공하는 것이다.In one aspect, an object of the present invention is to provide a novel use of Lactobacillus plantarum APsulloc 331266 (accession number: KCCM 11181P).
다른 측면에서, 본 발명의 목적은, 락토바실러스 플란타룸 APsulloc 331266을 유효성분으로 함유하는 조성물을 이용하여 지루성 두피염 또는 비듬을 예방, 개선 또는 치료하는 것이다.In another aspect, the object of the present invention is to prevent, ameliorate, or treat seborrhoeic dermatitis or dandruff using a composition containing Lactobacillus plantarum APsulloc 331266 as an active ingredient.
일 측면에서, 본 발명은, 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266(기탁번호: KCCM11181P) 균주, 그 파쇄물, 그 배양액 또는 그 추출물을 유효성분으로 포함하는 항진균용 조성물을 제공한다.In one aspect, the present invention provides a composition for an antifungal agent comprising Lactobacillus plantarum APsulloc 331266 (accession number: KCCM11181P) strain, a lysate thereof, a culture thereof or an extract thereof as an active ingredient.
일 측면에 있어서, 본 발명의 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266 균주, 그 파쇄물, 그 배양액 또는 그 추출물을 유효성분으로 포함하는 조성물은, 지루성 두피염 또는 비듬을 유발시키는 말라세지아 속 진균에 대한 항진균력이 있으므로, 지루성 두피염 또는 비듬을 예방, 개선 또는 치료하는데 사용할 수 있다. 또한, 상기 조성물은, 세포 독성이 없고, 피부 자극을 유발하지 않아 인체에 무해하며, 염증성 사이토카인의 발현양을 낮추므로 염증 진정 효과도 있다.In one aspect, a composition comprising the Lactobacillus plantarum APsulloc 331266 strain of the present invention, a lysate thereof, a culture thereof, or an extract thereof as an active ingredient may be used as an effective ingredient for preventing or treating seborrheic dermatitis or dandruff in Malassezia genus Because of the antifungal activity against fungi, it can be used to prevent, ameliorate or treat seborrheic dermatitis or dandruff. In addition, the composition has no cytotoxicity, does not induce skin irritation, is harmless to the human body, and lowers the expression level of inflammatory cytokines.
도 1은, 디스크 확산법에 의해 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266 배양액의 말라세지아 퍼퍼(Malassezia furfur)에 대한 항진균력을 확인한 도이다.
도 2는, 락토바실러스 플란타룸 APsulloc 331266 배양액의 세포 독성을 확인한 도이다.
도 3은, 락토바실러스 플란타룸 APsulloc 331266 배양액의 피부 자극성 유무를 확인한 도이다.
도 4는, 락토바실러스 플란타룸 APsulloc 331266 배양액의 염증 억제 효과를 확인한 도이다.
도 5는, 락토바실러스 플란타룸 APsulloc 331266 배양액의 농도에 따른 말라세지아 퍼퍼(Malassezia furfur)에 대한 항진균력을 확인한 그래프이다.1 is a view showing antifungal activity against Malassezia furfur of a Lactobacillus plantarum APsulloc 331266 culture medium by a disk diffusion method.
FIG. 2 is a chart for confirming cytotoxicity of a Lactobacillus plantarum APsulloc 331266 culture medium. FIG.
Fig. 3 is a diagram showing the skin irritancy of Lactobacillus plantarum APsulloc 331266 culture. Fig.
Fig. 4 is a graph showing the inflammation inhibitory effect of the Lactobacillus plantarum APsulloc 331266 culture medium. Fig.
5 is a graph showing antifungal activity against Malassezia furfur according to the concentration of Lactobacillus plantarum APsulloc 331266 culture medium.
락토바실러스(Lactobacillus)는 대개 인간 또는 동물의 장관(Intestinal tract)에 존재하는 주요 유산균의 하나로 생리학적 활성에 대한 유리한 효과로 다양한 생균제(Probiotics)에 부가된다. 이 중 락토바실러스 플란타룸(Lactobacillus plantarum)은, 막대기 모양의 간균이며 그람양성균이다. 아라비노스, 글루코스, 프럭토스, 갈락토스, 말토스, 슈크로스, 덱스트란, 라피노스와 트레할로스 등을 발효시켜서 젖산을 생성한다. 주로 우유, 치즈, 버터, 케피어(kefir), 발효된 소시지, 발효된 감자, 곡물, 빵의 산성 반죽, 피클이나 김치와 같은 침채류, 토마토 등에서 분리되며, 자연계에서 가장 분포가 넓은 젖산균 중의 하나이다. 최근, 락토바실러스 플란타룸이 헬리코박터, 살모넬라, 에쉐리키아, 스타필로코커스, 칸디다 등의 병원성 균의 증식 또는 활성을 억제하는 물질을 함유하거나 배출한다는 연구가 상당수 진행된 바 있다. 그러나 락토바실러스 플란타룸을 이용하여 지루성 피부염, 두피염 및 비듬을 유발하는 말라세지아의 증식 또는 활성을 억제하고자 하는 연구는 전무하다. Lactobacillus bacteria (Lactobacillus) are usually added to a variety of probiotics (Probiotics) with favorable effects on the physiological activity as one of the main lactic acid bacteria present in the views of a human or animal (Intestinal tract). Among these, Lactobacillus plantarum is a rod-shaped bacterium and gram-positive bacteria. Lactic acid is produced by fermenting arabinose, glucose, fructose, galactose, maltose, sucrose, dextran, raffinose and trehalose. It is one of the most widely distributed lactic acid bacteria in nature, separated mainly from milk, cheese, butter, kefir, fermented sausages, fermented potatoes, grains, acid dough of bread, pickles and tomatoes such as pickles and kimchi . Recently, a lot of researches have been conducted to contain or release substances that inhibit the growth or activity of pathogenic bacteria such as Helicobacter, Salmonella, Escherichia, Staphylococcus, Candida, etc., in Lactobacillus plantarum. However, there have been no studies to inhibit proliferation or activity of malassezia causing seborrhoeic dermatitis, scalpitis and dandruff using Lactobacillus plantarum.
본 발명자들은, 제주 차나무(Camellia sinensis) 잎에서 분리한 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266 균주와 그 배양액이 지루성 피부염, 지루성 두피염 및 비듬을 유발하는 것으로 알려진 말라세지아(Malassezia sp.)에 대하여 항진균력이 있음을 발견하여, 본 발명을 완성하게 되었다.The present inventors have found that a strain of Lactobacillus plantarum APsulloc 331266 isolated from Jeju ( Camellia sinensis ) leaf and a culture thereof are effective against Malassezia sp., Which is known to cause seborrheic dermatitis, seborrheic dermatitis and dandruff. ), And thus the present invention has been completed.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은, 일 측면에서 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266(기탁번호: KCCM11181P) 균주, 그 파쇄물, 그 배양액 또는 그 추출물을 유효성분으로 포함하는 항진균용 조성물이다. In one aspect, the present invention is a composition for an antifungal agent comprising Lactobacillus plantarum APsulloc 331266 (accession number: KCCM11181P) strain, a lysate thereof, a culture thereof or an extract thereof as an active ingredient.
상기 균주는, 차나무(Camellia sinensis) 잎에서 분리한 균주로 락토바실러스 플란타룸(Lactobacillus plantarum)에 속한다. 구체적으로, APsulloc 331266은 차나무 잎을 차나무 잎 중량 대비 5 내지 15 중량%의 식염으로 절이는 단계; 절인 차나무 잎을 0.1% 내지 3%의 프락토 올리고당을 예로 들 수 있는 당용액과 혼합하여 25 내지 35에서 1 내지 5일간 배양하는 단계; 및 pH 5 미만으로 된 배양액을 취하여 25 내지 35 혐기 조건에서 1 내지 5일간 배양하는 단계를 포함하는 방법으로 분리할 수 있다. The strain is a strain isolated from the leaves of Camellia sinensis and belongs to Lactobacillus plantarum . Specifically, APsulloc 331266 includes the steps of ripening tea leaves with a salt of 5 to 15% by weight based on the weight of the tea leaves; Pickling the marinated tea leaves with a sugar solution, such as 0.1% to 3% fructooligosaccharide, and incubating at 25 to 35 for 1 to 5 days; And culturing the culture medium having a pH of less than 5, and culturing the culture medium for 1 to 5 days at 25 to 35 anaerobic conditions.
본 명세서에서, 상기 배양액 또는 그 추출물은, 균주, 예컨대, 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266를 배양하여 발생한 배양 산물을 함유하는 배양액 또는 그 배양액의 추출물을 의미할 수 있다. 배양액에는 균주 자체도 포함되어 있을 수 있다.In this specification, the culture broth or an extract thereof may mean a culture solution containing the culture product produced by culturing a strain, for example, Lactobacillus plantarum APsulloc 331266, or an extract of the culture solution. The culture medium may contain the strain itself.
상기와 같은 측면에서, 상기 항진균 대상인 균은 말라세지아(Malassezia sp.)일 수 있다. 말라세지아는, 피티로스포룸(Pityrosporum)이라고도 불리운다. 구체적으로, 상기 말라세지아는, 말라세지아 퍼퍼(Malassezia furfur), 말라세지아 글로보사(Malassezia globosa) 및 말라세지아 레스트릭타(Malassezia restricta)로 이루어진 군으로부터 선택된 하나 이상일 수 있다.In this aspect, the fungus of the antifungal agent may be Malassezia sp. Malasejia is also called Pityrosporum. Specifically, the Malassezia is Malassezia < RTI ID = 0.0 > furfur), dry years global Jia Bossa (Malassezia globosa), and do other tricks Les Jia three (Malassezia restricta ). < / RTI >
상기와 같은 측면에서, 상기 조성물은, 지루성 피부염 또는 비듬의 예방, 개선 또는 치료용 조성물일 수 있다. In the above aspect, the composition may be a composition for preventing, ameliorating or treating seborrheic dermatitis or dandruff.
상기와 같은 측면에서, 상기 지루성 피부염은 지루성 두피염일 수 있다.In the above aspect, the seborrheic dermatitis may be a seborrheic scalp salt.
구체적으로, 말라세지아 퍼퍼, 말라세지아 글로보사 및 말라세지아 레스트릭타는 지루성 피부염, 두피염 및 비듬을 유발하는 것으로 알려져 있으므로, 상기 균들의 증식, 생장을 억제하거나 상기 균들을 사멸시키면, 지루성 피부염, 두피염 및 비듬의 예방, 개선 또는 치료 효과를 기대할 수 있다.Specifically, it is known that Malasejia percha, Malasejia globosa and Malasejiaestrica cause seborrheic dermatitis, scalpitis and dandruff. Therefore, if the growth and growth of the above-mentioned fungi are suppressed or the fungi are killed, Dermatitis, scalpitis and dandruff can be expected.
상기와 같은 측면에서, 상기 락토바실러스 플란타룸 APsulloc 331266(기탁번호: KCCM11181P) 균주, 그 파쇄물, 그 배양액 또는 그 추출물은, 조성물 총 중량을 기준으로, 0.01중량% 내지 10중량%로 포함될 수 있다. 일 구체예에서, 상기 락토바실러스 플란타룸 APsulloc 331266(기탁번호: KCCM11181P) 균주, 그 파쇄물, 그 배양액 또는 그 추출물은, 조성물 총 중량을 기준으로, 0.1중량%~5중량%로 포함될 수 있다. 상기와 같은 측면에서, 상기 락토바실러스 플란타룸 APsulloc 331266 균주, 그 파쇄물, 그 배양액 또는 그 추출물은, 조성물 총 중량을 기준으로, 0.001중량% 이상, 0.005중량% 이상, 0.01중량% 이상, 0.1중량% 이상, 1중량% 이상, 3중량% 이상, 5중량% 이상, 7중량% 이상, 9중량% 이상, 10중량% 이상 또는 11중량% 이상 포함될 수 있고, 11중량% 이하, 10중량% 이하, 9중량% 이하, 7중량% 이하, 5중량% 이하, 3중량% 이하, 1중량% 이하, 0.1중량% 이하, 0.005중량% 이하 또는 0.003중량% 이하로 포함될 수 있다. In the above aspect, the Lactobacillus plantarum APsulloc 331266 (accession number: KCCM11181P) strain, the lysate thereof, the culture or the extract thereof may be contained in an amount of 0.01 to 10% by weight based on the total weight of the composition . In one embodiment, the Lactobacillus plantarum APsulloc 331266 (accession number: KCCM11181P) strain, the lysate thereof, the culture or the extract thereof may be contained in an amount of 0.1% to 5% by weight based on the total weight of the composition. In the above aspect, the Lactobacillus plantarum APsulloc 331266 strain, the lysate thereof, the culture solution thereof or the extract thereof may be added in an amount of 0.001 wt% or more, 0.005 wt% or more, 0.01 wt% or more, 0.1 wt% At least 1% by weight, at least 3% by weight, at least 5% by weight, at least 7% by weight, at least 9% by weight, at least 10% by weight or at least 11% by weight, , 9 wt% or less, 7 wt% or less, 5 wt% or less, 3 wt% or less, 1 wt% or less, 0.1 wt% or less, 0.005 wt% or less or 0.003 wt% or less.
상기와 같은 측면에서, 상기 조성물은, 약학적 조성물, 화장료 조성물 또는 건강 식품 조성물일 수 있다.In this aspect, the composition may be a pharmaceutical composition, a cosmetic composition or a health food composition.
상기 화장료 조성물은 국소 적용에 적합한 모든 제형으로 제공될 수 있다. 예를 들면, 액, 수상에 유상을 분산시켜 얻은 에멀젼, 유상에 수상을 분산시켜 얻은 에멀젼, 현탁액, 고체, 겔, 분말, 페이스트, 포말(foam) 또는 에어로졸 조성물의 제형으로 제공될 수 있다. 이러한 제형의 조성물은 당해 분야의 통상적인 방법에 따라 제조될 수 있다.The cosmetic composition may be provided in all formulations suitable for topical application. For example, it may be provided as a liquid, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an oil phase in water, a suspension, a solid, a gel, a powder, a paste, a foam or an aerosol composition. Compositions of such formulations may be prepared according to conventional methods in the art.
상기 화장료 조성물은 상기한 물질 이외에 주 효과를 손상시키지 않는 범위 내에서, 바람직하게는 주 효과에 상승 효과를 줄 수 있는 다른 성분들을 함유할 수 있다. 본 발명에 따른 화장료 조성물은 비타민, 고분자 펩티드, 분자 다당 및 스핑고 지질로 이루어진 군에서 선택된 물질을 포함할 수 있다. 또한 본 발명에 따른 화장료 조성물은 보습제, 에몰리언트제, 계면 활성제, 자외선 흡수제, 방부제, 살균제, 산화 방지제, pH 조정제, 유기 및 무기 안료, 향료, 냉감제 또는 제한(制汗)제를 포함할 수 있다. 상기 성분의 배합량은 본 발명의 목적 및 효과를 손상시키지 않는 범위 내에서 당업자가 용이하게 선정 가능하며, 그 배합량은 조성물 전체 중량을 기준으로 0.01~5 중량%, 구체적으로 0.01~3 중량%일 수 있다.The cosmetic composition may contain, in addition to the above-mentioned substances, other ingredients which can give synergistic effects to the main effect, so long as they do not impair the main effect. The cosmetic composition according to the present invention may include a material selected from the group consisting of vitamins, high molecular peptides, molecular polysaccharides and sphingolipids. The cosmetic composition according to the present invention may also contain a moisturizing agent, an emollient agent, a surfactant, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, . The blending amount of the above components can be easily selected by those skilled in the art within a range not to impair the objects and effects of the present invention. The blending amount thereof can be 0.01 to 5% by weight, specifically 0.01 to 3% by weight based on the total weight of the composition have.
상기와 같은 측면에서, 상기 약학적 조성물 또는 화장료 조성물은 피부 외용제 조성물일 수 있다. 예컨대, 크림, 연고일 수 있다.In the above aspect, the pharmaceutical composition or cosmetic composition may be a composition for external application to the skin. For example, it may be cream or ointment.
상기 식품 조성물은 건강 식품 조성물일 수 있으며, 다(茶)류, 유제품류, 김치류, 양조 식품류를 예로 들 수 있는 발효가 필요한 발효 식품 조성물일 수 있다. 상기 식품 조성물의 제형은 특별히 한정되지 않으나, 예를 들어, 정제, 환제, 연질 및 경질 캅셀제, 과립제, 드링크제, 캐러멜, 다이어트바, 티백 등으로 제형화될 수 있다. 각 제형의 식품 조성물은 유효 성분 이외에 해당 분야에서 통상적으로 사용되는 성분들을 제형 또는 사용 목적에 따라 당업자가 어려움 없이 적의 선정하여 배합할 수 있으며, 다른 원료와 동시에 적용할 경우 상승 효과가 일어날 수 있다. 상기 유효 성분의 투여량 결정은 당업자의 수준 내에 있으며, 이의 1일 투여 용량은 예를 들어 락토바실러스 플란타룸 105내지 1013CFU/일, 보다 구체적으로는 106 내지 1010 CFU/일이 될 수 있으나, 이에 제한되지 않으며, 투여하고자 하는 대상의 연령, 건강 상태, 합병증 등의 다양한 요인에 따라 달라질 수 있다.The food composition may be a health food composition, and may be a fermented food composition requiring fermentation, such as tea, dairy, kimchi, and brewed foods. The formulation of the food composition is not particularly limited, but may be formulated into tablets, pills, soft and hard capsules, granules, drinks, caramels, diet bars, tea bags and the like. The food composition of each formulation can be blended with the ingredients commonly used in the field in addition to the active ingredient without difficulty by those skilled in the art depending on the purpose of formulation or use, and synergistic effect can be obtained when the composition is applied simultaneously with other ingredients. Determination of the dosage of the active ingredient is within the level of ordinary skill in the art, and its daily dose is, for example, 10 5 to 10 13 CFU / day, more specifically 10 6 to 10 10 CFU / day for Lactobacillus plantarum But may vary depending on various factors such as the age, health condition, complications, etc. of the subject to be administered.
이하, 본 발명을 하기의 실시예 및 실험예를 통하여 설명한다. 하기 실시예 및 실험예는 본 발명에 대한 이해를 돕기 위해 예시의 목적으로만 제공된 것일 뿐 본 발명의 범주 및 범위가 그에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described with reference to the following examples and experimental examples. The following Examples and Experimental Examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, but the scope and scope of the present invention are not limited thereto.
[실시예 1]차나무 잎에서 분리한 락토바실러스 플란타룸 APsulloc 331266의 준비[Example 1] Preparation of Lactobacillus planta APsulloc 331266 isolated from tea leaves
[실시예 1-1]락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266의 분리[Example 1-1] Isolation of Lactobacillus plantarum APsulloc 331266
아래에서 본 발명의 일 실시예에 따른 유산균 중 락토바실러스 플란타룸 APsulloc 331266의 분리 과정에 대해 설명한다.Hereinafter, the separation process of Lactobacillus plantarum APsulloc 331266 among lactic acid bacteria according to an embodiment of the present invention will be described.
차나무 잎 200g을 1차 증류수에 2회 세척하여 이물질을 제거하였다. 세척한 차나무 잎의 물기를 털어내고 차나무 잎 중량의 8%에 해당하는 식염과 혼합한 후 3시간 동안 실온에 방치하였다. 식염에 절여진 차나무 잎을 1% 프락토 올리고당 용액 1000 mL에 혼합한 후 3일간 32 인큐베이터에서 배양하였다. 3일 후 배양액의 pH가 5 미만으로 떨어졌는지 확인하고 pH 5 미만인 경우 이를 취해 디프코 락토바실리 MRS 아가 (Difco Lactobacilli MRS Agar®) 배지에 배양하였다. 이때 배양은 32℃, 혐기 조건의 챔버에서 2일간 배양한 후 백색 집락을 보이는 콜로니를 취했다.200 g of tea leaves were washed twice with primary distilled water to remove foreign matter. The water from the washed tea leaves was bled out and mixed with the salt equivalent to 8% of the weight of the tea leaves and left at room temperature for 3 hours. The edible tea leaves were mixed in 1000 mL of 1% fructooligosaccharide solution and cultured in 32 incubator for 3 days. After 3 days, it was confirmed whether the pH of the culture broth was lowered to less than 5. When the pH was less than 5, it was taken and cultured in Difco Lactobacilli MRS Agar (R) medium. At this time, the culture was carried out in a chamber of anaerobic condition at 32 ° C for 2 days, and then a colony showing white colonies was taken.
위와 같은 방법으로 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266을 차나무 잎으로부터 분리하였다. Lactobacillus plantarum APsulloc 331266 was isolated from tea leaves in the same manner as above.
[실시예 1-2] 락토바실러스 플란타룸 APsulloc 331266 배양액의 제조[Example 1-2] Preparation of Lactobacillus flutarium APsulloc 331266 culture medium
실시예 1-1에서 분리 및 동정한 락토바실러스 플란타룸 APsulloc 331266을 배지(MRS Broth)에서 37℃ 환경에서 24시간 동안 배양하였다. 이 락토바실러스 플란타룸 APsulloc 331266은 탈지유(Skim milk)에 10중량%로 크라이오튜브(Cryotube)에서 보관한 것을 접종한 것이다. 액체 배지 내 균수가 5 X 108 ~ 1 X 109 가 되었을 때, 4℃에서, 4000 rpm 원심 분리를 통하여 영양세포를 제거하고, 포어사이즈가 0.2 mm인 실린지 필터링을 통해 기타 부유물을 제거하였다.Lactobacillus plantarum APsulloc 331266 isolated and identified in Example 1-1 was cultured in a medium (MRS Broth) for 24 hours at 37 ° C. The Lactobacillus plantarum APsulloc 331266 was inoculated with 10% by weight of skim milk stored in a cryotube. When the number of bacteria in the liquid medium reached 5 × 10 8 to 1 × 10 9 , the nutrient cells were removed by centrifugation at 4000 rpm at 4 ° C. and other floating matters were removed by syringe filtration with a pore size of 0.2 mm .
[실험예 1-1] 항진균력의 측정[Experimental Example 1-1] Measurement of antifungal activity
락토바실러스 플란타룸 APsulloc 331266 균주의 배양액의 항진균력을 측정하기 위하여 Agar well diffusion assay를 실시하였다. 구체적으로, 90 mm 플라스틱 평판 가운데에 페니실린더를 세우고 멸균된 Agar를 20 mL 부어 굳혔다. MLNA 배지에 말라세지아(Malassezia sp.)를 5 X 108 내지 1 X 109 cell/mL 농도로 접종하고, 굳은 Agar 위에 5 mL을 붓고 굳혔다. 상기 두 겹의 배지가 모두 굳으면 페니실린더를 조심스럽게 제거하고 생긴 홀(Hall)에 미량(약 50 - 100 uL)의 Agar를 채워 넣어 웰을 만들었다. 그 후, 실시예 1-2에서 제조된 배양액이 웰을 가득 채울 때까지 채우고 플레이트의 뚜껑을 덮은 후, 37℃에서 3일 배양하여 억제대의 지름을 자로 재어 결과를 판독하였다. 그 결과는 도 1에 나타나 있다.Agar well diffusion assay was performed to measure the antifungal activity of Lactobacillus plantarum APsulloc 331266. Specifically, a penicillin disc was placed in a 90 mm plastic plate, and 20 mL of sterilized Agar was poured and hardened. Malassezia sp. Was inoculated to the MLNA medium at a concentration of 5 × 10 8 to 1 × 10 9 cells / mL, and 5 mL was poured over the hard agar and allowed to harden. When the two-fold culture medium was completely hardened, the penicillin was carefully removed and a small amount (about 50-100 uL) of Agar was filled into the formed holes to form wells. Thereafter, the culture solution prepared in Example 1-2 was filled until the wells were filled, the plate was covered with the lid, and the plate was incubated at 37 DEG C for 3 days. The results are shown in Fig.
[실험예 1-2] 항진균력의 측정[Experimental Example 1-2] Measurement of Antifungal Activity
락토바실러스 플란타룸 APsulloc 331266 균주의 배양액의 항진균력을 측정하기 위하여 시료 농도와 배양 시간에 따른 말라세지아 퍼퍼(Malassezia furfur)의 억제 정도를 Agar plate counting을 통하여 확인하였다.In order to measure the antifungal activity of Lactobacillus plantarum APsulloc 331266, the inhibitory effect of Malassezia furfur on the sample concentration and incubation time was confirmed by agar plate counting.
4개의 50 mL 튜브에 락토바실러스 플란타룸 APsulloc 331266 균주 배양액이 0%, 10%, 20%, 30%가 되도록 시료 농도를 구배하였다. 구체적으로는 MLNA 배지를 21 mL 부은 후, 1) MRS 배지 9 mL, 2) MRS 배지 6 mL과 배양액 3 mL, 3) MRS 배지 3 mL과 배양액 6 mL, 4) 배양액 9 mL을 추가로 혼합하여 시료 농도를 정확히 구배할 수 있다. 각 튜브에 말라세지아 퍼퍼를 1 X 106 cell/mL 농도가 되도록 접종 후 35 인큐베이터에서 배양하였다. 배양 즉시, 24 시간 후, 48 시간 후, 72 시간 후 각 시험 샘플로부터 1 mL를 취하여 생리식염수에 1/10 연속 희석을 실시하고 이를 MLNA agar에 스프레딩한 후 35에서 3일 배양하여 생성된 콜로니 수를 확인하므로써 생균수 결과를 판독하였다(도 5). 그 결과, 락토바실러스 플란타룸 APsulloc 331266의 배양액은 농도가 증가할수록 말라세지아 퍼퍼에 대한 항진균력이 증가함을 알수 있었다.The sample concentration was graded so that the culture broth of Lactobacillus plantarum APsulloc 331266 was 0%, 10%, 20%, and 30% in four 50-mL tubes. Specifically, 1 mL of MRS medium, 6 mL of MRS medium, 3 mL of MRS medium, 3 mL of MRS medium, 6 mL of culture medium and 9 mL of culture medium were further mixed The sample concentration can be accurately graded. Malassezia pupae were inoculated into each tube at a concentration of 1 × 10 6 cells / mL and then cultured in a 35 incubator. Immediately after culturing, 1 mL of each test sample was taken after 24 hours, 48 hours, and 72 hours, and 1/10 serial dilution was performed in physiological saline, spread on MLNA agar, and cultured for 35 days for 3 days. The number of living cells was read by confirming the number (Fig. 5). As a result, it was found that the antifungal activity against Lactobacillus plantarum APsulloc 331266 increased with increasing concentration.
[실험예 2] 세포 독성 측정[Experimental Example 2] Cytotoxicity measurement
실시예 1-2의 락토바실러스 플란타룸 APsulloc 331266 균주 배양액의 세포독성 유무를 확인하기 위하여 하기 실험을 수행하였다. 인간 각질 형성 세포주인 HaCaT 세포(Invitrogen)를 96 웰 플레이트에 2X104 cells/well 농도로 부착시키고, FBS를 포함하는 DMEM 배지에서 24시간 배양하였다. PBS(phosphate buffered saline)로 인간 각질 형성 세포를 세척한 후 락토바실러스 플란타룸 APsulloc 331266의 배양액을 농도별(1%, 2%, 5%)로 처리하여 24시간 동안 반응시켰다. 그 후, 세포 배양액 100 ㎕ 당 10 ㎕의 WST-1 용액을 넣고 30분 내지 2시간 동안 37℃에서 반응 시키고 450 nm에서 흡광도를 측정하였다. 흡광도는 살아있는 세포 수에 비례한다. 무처리군의 흡광도를 100%로 하였을 때 락토바실러스 플란타룸 APsulloc 331266의 배양액을 농도별로 처리하였을 때의 흡광도를 상대적으로 비교하였다(도2). 그 결과, 락토바실러스 플란타룸 APsulloc 331266의 배양액은 세포 독성이 없음을 알 수 있었다.The following experiment was conducted to confirm the cytotoxicity of the Lactobacillus plantarum APsulloc 331266 culture medium of Example 1-2. HaCaT cells (Invitrogen), a human keratinocyte cell line, were attached to a 96-well plate at a concentration of 2 × 10 4 cells / well and cultured in DMEM medium containing FBS for 24 hours. Human keratinocytes were washed with PBS (phosphate buffered saline) and treated with Lactobacillus plantarum APsulloc 331266 (1%, 2%, 5%) for 24 hours. Thereafter, 10 占 퐇 of WST-1 solution was added per 100 占 of the cell culture solution, reacted at 37 占 폚 for 30 minutes to 2 hours, and the absorbance at 450 nm was measured. Absorbance is proportional to the number of living cells. When the absorbance of the untreated group was taken as 100%, the absorbance of the culture solution of Lactobacillus plantarum APsulloc 331266 was comparatively compared (FIG. 2). As a result, it was found that the culture solution of Lactobacillus plantarum APsulloc 331266 was not cytotoxic.
[실험예 3] 피부 자극성 유무 확인[Experimental Example 3] Confirmation of skin irritancy
상기 실시예 1-2의 배양액이 피부에 자극을 일으키는지 확인하기 위해 하기 실험을 수행하였다. 인간 각질 형성 세포 라인 HaCaT 세포를 96 well cell culture plate에 2X104 cells/well 농도로 부착시키고 FBS를 포함하는 DMEM 배지에서 24시간 배양하여 준비하였다. PBS(phosphate buffered saline)로 세포를 세척한 후 DMEM 배지 및 10μM PMA(12-ο-tetradecanoylphorbol-13-acetate) 또는 상기 실시예 1-2의 배양액을 20% 농도로 처리하여 세포에서 분비되는 염증성 사이토카인인 IL-1α와 TNF-α의 양을 비교하였다. 그 결과, 락토바실러스 플란타룸 APsulloc 331266 배양액은 피부 자극성이 없음을 알 수 있었다(도 3).The following experiment was conducted to confirm whether the culture medium of Example 1-2 was stimulating the skin. Human keratinocyte line HaCaT cells were attached to a 96 well cell culture plate at a concentration of 2 × 10 4 cells / well and cultured in DMEM medium containing FBS for 24 hours. Cells were washed with PBS (phosphate buffered saline) and treated with DMEM medium and 10 μM PMA (12-o-tetradecanoylphorbol-13-acetate) or the culture medium of Example 1-2 at a concentration of 20% The amounts of IL-1α and TNF-α, which are kinases, were compared. As a result, it was found that the culture of Lactobacillus plantarum APsulloc 331266 had no skin irritation (Fig. 3).
[실험예 4] 염증 억제 효과 확인 [Experimental Example 4] Confirmation of inflammation inhibitory effect
상기 실시예 1-2의 배양액이 염증 억제 효과가 있는지 확인하기 위해 하기 실험을 수행하였다. 인간 각질 형성 세포 라인 HaCaT 세포를 96 well cell culture plate에 2X104 cells/well 농도로 부착시키고 FBS를 포함하는 DMEM 배지에서 24시간 배양하여 준비하였다. PBS(phosphate buffered saline)로 세포를 세척한 후 DMEM 배지 및 10μM PMA(12-ο-tetradecanoylphorbol-13-acetate)를 처리하여 세포에 염증성 사이토카인의 분비를 유도하였다. 그 후, 락토바실러스 플란타룸 APsulloc 331266의 배양액을 처리하고 염증성 사이토카인의 분비량을 확인하였다. 그 결과, 락토바실러스 플란타룸 APsulloc 331266의 배양액을 처리한 군에서 IL-1α의 분비량이 감소하는 것을 확인하였고, 상기 균주 배양액이 염증 억제 효과가 있음을 알 수 있었다(도 4). The following experiment was conducted to confirm whether the culture solution of Example 1-2 had an inflammation-inhibiting effect. Human keratinocyte line HaCaT cells were attached to a 96 well cell culture plate at a concentration of 2 × 10 4 cells / well and cultured in DMEM medium containing FBS for 24 hours. Cells were washed with PBS (phosphate buffered saline) and treated with DMEM medium and 10 μM PMA (12-ο-tetradecanoylphorbol-13-acetate) to induce the secretion of inflammatory cytokines into the cells. After that, the culture medium of Lactobacillus plantarum APsulloc 331266 was treated and the secretion amount of inflammatory cytokine was confirmed. As a result, it was confirmed that the secretion amount of IL-1α was decreased in the group treated with the culture solution of Lactobacillus plantarum APsulloc 331266, and the culture medium of the strain showed an inflammation-inhibiting effect (FIG. 4).
본 발명의 일 실시예에 따른 조성물의 제형예를 아래에서 설명하나, 다른 여러 가지 제형으로도 응용 가능하며, 이는 본 발명을 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다Formulation examples of compositions according to one embodiment of the present invention are described below, but may be applied to various other formulations, which are not intended to be limiting but merely illustrative of the present invention
[제형예 1] 영양화장수[Formulation Example 1] Nutritional lotion
하기 표 1에 기재된 조성에 따라 통상적인 방법으로 영양화장수를 제조하였다.Nutrition lotion was prepared according to a conventional method according to the composition shown in Table 1 below.
[제형예 2] 영양로션[Formulation Example 2] Nutrition lotion
하기 표 2에 기재된 조성에 따라 통상적인 방법으로 영양로션을 제조할 수 있다.Nutrition lotions can be prepared in a conventional manner according to the composition shown in Table 2 below.
[제형예 3] 영양크림[Formulation Example 3] Nourishing cream
하기 표 3에 기재된 조성에 따라 통상적인 방법으로 영양크림을 제조할 수 있다.Nutrition creams can be prepared in a conventional manner according to the composition shown in Table 3 below.
[제형예 4] 헤어 팩[Formulation Example 4] Hair pack
하기 표 4에 기재된 조성에 따라 통상적인 방법으로 팩을 제조할 수 있다.The pack can be manufactured in a conventional manner according to the composition shown in Table 4 below.
[제형예 5] 샴푸[Formulation Example 5] Shampoo
하기 표 5에 기재된 조성에 따라 통상적인 방법으로 샴푸를 제조할 수 있다.The shampoos can be prepared by a conventional method according to the composition shown in Table 5 below.
[제형예 6] 린스[Formulation Example 6] Rinse
하기 표 6에 기재된 조성에 따라 통상적인 방법으로 린스를 제조할 수 있다.Rinse can be prepared according to a conventional method according to the composition shown in Table 6 below.
[제형예 7] 헤어 스프레이[Formulation Example 7] Hair spray
하기 표 7에 기재된 조성에 따라 통상적인 방법으로 스프레이를 제조할 수 있다.Spray can be prepared by a conventional method according to the composition shown in Table 7 below.
[제형예 8] 헤어토닉[Formulation Example 8] Hair tonic
하기 표 8에 기재된 조성에 따라 통상적인 방법으로 헤어토닉을 제조할 수 있다.Hair tonics can be prepared by a conventional method according to the composition shown in Table 8 below.
상기 락토바실러스 플란타룸(Lactobacillus plantarum) APsulloc 331266은 2011년 3월 28일 한국미생물보존센터(Korean Culture Center of Microorganisms)에 미생물 기탁번호 KCCM11181P로 기탁하였다.The Lactobacillus plantarum APsulloc 331266 was deposited with the Korean Culture Center of Microorganisms on March 28, 2011 as microorganism deposit number KCCM 11181P.
Claims (8)
상기 항진균 대상인 균은, 말라세지아(Malassezia sp.)인, 항진균용 조성물.The method according to claim 1,
Wherein the bacterium which is a target of the antifungal agent is Malassezia sp.
상기 말레세지아는,
말라세지아 퍼퍼(Malassezia furfur), 말라세지아 글로보사(Malassezia globosa) 및 말라세지아 레스트릭타(Malassezia restricta)로 이루어진 군으로부터 선택된 하나 이상인, 항진균용 조성물.3. The method of claim 2,
The above-
Malassezia furfur), dry years global Jia Bossa (Malassezia globosa), and do other tricks Les Jia three (Malassezia restricta ). < / RTI >
상기 조성물은, 지루성 피부염 또는 비듬의 예방, 개선 또는 치료용인, 항진균용 조성물.The method according to claim 1,
Wherein said composition is for preventing, ameliorating or treating seborrheic dermatitis or dandruff.
상기 지루성 피부염은, 지루성 두피염인, 항진균용 조성물. 5. The method of claim 4,
Wherein said seborrheic dermatitis is a seborrheic dermatitis.
상기 락토바실러스 플란타룸 APsulloc 331266(기탁번호: KCCM11181P) 균주, 그 파쇄물, 그 배양액 또는 그 추출물은,
조성물 총 중량을 기준으로, 0.01중량% 내지 10중량%로 포함되는, 항진균용 조성물.The method according to claim 1,
The Lactobacillus plantarum APsulloc 331266 (Accession No .: KCCM11181P) strain, the lysate thereof, the culture or the extract thereof,
Wherein the composition comprises from 0.01% to 10% by weight, based on the total weight of the composition.
상기 락토바실러스 플란타룸 APsulloc 331266(기탁번호: KCCM11181P) 균주, 그 파쇄물, 그 배양액 또는 그 추출물은,
조성물 총 중량을 기준으로, 0.1중량% 내지 5중량%로 포함되는, 항진균용 조성물.The method according to claim 1,
The Lactobacillus plantarum APsulloc 331266 (Accession No .: KCCM11181P) strain, the lysate thereof, the culture or the extract thereof,
Wherein the composition comprises from 0.1% to 5% by weight, based on the total weight of the composition.
상기 조성물은,
화장료 조성물 또는 건강 식품 조성물인, 항진균용 조성물.The method according to claim 1,
The composition may comprise,
A composition for an antifungal agent, which is a cosmetic composition or a health food composition.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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Cited By (4)
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KR20190002079A (en) * | 2017-06-29 | 2019-01-08 | (주)아모레퍼시픽 | Anti-aging composition comprising extracellular vesicles derived from lactic acid bacteria |
KR101966184B1 (en) | 2018-09-21 | 2019-04-05 | 주식회사 제이투케이바이오 | NTI-FUNGAL Cosmetic Composition of Malassezia furfur |
WO2020208253A1 (en) * | 2019-04-12 | 2020-10-15 | Biodermic Health & Beauty Gmbh & Co. Kg | Composition for a topical application on the skin |
KR102606325B1 (en) * | 2022-11-15 | 2023-12-04 | 주식회사 큐옴바이오 | Composition for antibacterial and skin disease improvement against skin harmful bacteria containing lactobacillus plantarum q1 strain |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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KR20190002079A (en) * | 2017-06-29 | 2019-01-08 | (주)아모레퍼시픽 | Anti-aging composition comprising extracellular vesicles derived from lactic acid bacteria |
KR101966184B1 (en) | 2018-09-21 | 2019-04-05 | 주식회사 제이투케이바이오 | NTI-FUNGAL Cosmetic Composition of Malassezia furfur |
WO2020208253A1 (en) * | 2019-04-12 | 2020-10-15 | Biodermic Health & Beauty Gmbh & Co. Kg | Composition for a topical application on the skin |
KR102606325B1 (en) * | 2022-11-15 | 2023-12-04 | 주식회사 큐옴바이오 | Composition for antibacterial and skin disease improvement against skin harmful bacteria containing lactobacillus plantarum q1 strain |
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