KR20220081475A - Lactobacillus plantarum OKBL-L.PL 1 strain having anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganism and anti-thrombotic activity and uses thereof - Google Patents
Lactobacillus plantarum OKBL-L.PL 1 strain having anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganism and anti-thrombotic activity and uses thereof Download PDFInfo
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- KR20220081475A KR20220081475A KR1020200170977A KR20200170977A KR20220081475A KR 20220081475 A KR20220081475 A KR 20220081475A KR 1020200170977 A KR1020200170977 A KR 1020200170977A KR 20200170977 A KR20200170977 A KR 20200170977A KR 20220081475 A KR20220081475 A KR 20220081475A
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Abstract
본 발명은 항염 활성, 피부 미백 활성, 항균 활성 및 항혈전 활성을 가지는 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주 및 이의 용도에 관한 것이다. The present invention relates to Lactobacillus plantarum OKBL-L.PL 1 strain having an accession number of KCCM12792P having anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity, and to a use thereof.
Description
본 발명은 항염 활성, 피부 미백 활성, 유해 미생물에 대한 항균 활성 및 항혈전 활성을 가지는 락토바실러스 플란타룸 OKBL-L.PL 1 균주 및 이의 용도에 관한 것이다. The present invention relates to Lactobacillus plantarum OKBL-L.PL 1 strain having anti-inflammatory activity, skin whitening activity, antibacterial activity against harmful microorganisms and antithrombotic activity, and uses thereof.
유산균(Lactic acid bacteria)은 다양한 식품의 발효에 관여하여 독특한 풍미를 부여할 뿐 아니라, 항균성 물질을 생산하여 식품의 저장성 및 안정성을 증대시킨다. 이러한 유산균이 생성하는 항균성 물질로는 젖산 등의 유기산 이외에 디아세틸 및 박테리오신 등이 알려져 있다. Lactic acid bacteria are involved in the fermentation of various foods and not only impart a unique flavor, but also produce antibacterial substances to increase the shelf life and stability of foods. Examples of the antimicrobial substances produced by these lactic acid bacteria include diacetyl and bacteriocin in addition to organic acids such as lactic acid.
프로바이오틱스(probiotics)는 섭취되어 장에 도달하였을 때에 장내 환경에 유익한 작용을 하는 균주를 말하는 것으로, 건조 세포나 발효산물 형태로 사람이나 동물에 공급되어 장내 균총을 개선한다. 현재까지 알려진 대부분의 프로바이오틱스는 유산균이며 주로 락토바실러스(Lactobacillus) 속 균주를 이용하여 만들어진 발효유, 과립 또는 분말 등의 형태로 이용되고 있다. 유산균을 비롯한 균주들이 프로바이오틱스로 사용되기 위해서는 위산과 담즙산에서 살아남아 장까지 도달하여 증식 및 정착해야 하고 독성이 없으며 비병원성이어야 한다. 최근에는 기본적인 유산균의 특성 뿐만 아니라 다양한 유용 활성을 가지는 새로운 균주가 분리, 동정되어 보고되고 있다. Probiotics refer to strains that have a beneficial effect on the intestinal environment when ingested and reach the intestine, and are supplied to humans or animals in the form of dry cells or fermented products to improve the intestinal flora. Most probiotics known to date are lactic acid bacteria and are mainly used in the form of fermented milk, granules or powders made using Lactobacillus genus strains. In order for strains, including lactic acid bacteria, to be used as probiotics, they must survive gastric and bile acids to reach the intestine, multiply and settle, and must be non-toxic and non-pathogenic. Recently, new strains having various useful activities as well as basic characteristics of lactic acid bacteria have been isolated, identified and reported.
한편, 한국등록특허 제0986306호에서는 '김치유래 락토바실러스 플란타룸 BLS41 및 그 용도'가 개시되어 있고, 한국등록특허 제1348435호에서는 '항진균 활성을 가진 락토바실러스 플란타룸 YML007 및 이의 용도'가 개시되어 있으나, 본 발명의 '항염 활성, 피부 미백 활성, 유해 미생물에 대한 항균 활성 및 항혈전 활성을 가지는 락토바실러스 플란타룸 OKBL-L.PL 1 균주 및 이의 용도'에 대해서는 기재된 바가 없다.Meanwhile, Korean Patent No. 0986306 discloses 'Kimchi-derived Lactobacillus plantarum BLS41 and its use', and Korean Patent No. 1348435 discloses 'Lactobacillus plantarum YML007 with antifungal activity and uses thereof' However, there is no description of the 'Lactobacillus plantarum OKBL-L.PL 1 strain having anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity against harmful microorganisms and use thereof' of the present invention.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 김치로부터 락토바실러스 플란타룸 OKBL-L.PL 균주(기탁번호: KCCM12792P)를 분리하였고, 상기 분리된 균주의 우수한 항산화 활성, 항염 활성, 피부 미백 활성, 유해 미생물에 대한 항균 활성 및 항혈전 활성을 확인함으로써, 본 발명을 완성하였다. The present invention was derived from the above needs, and the present inventors isolated Lactobacillus plantarum OKBL-L.PL strain (Accession No.: KCCM12792P) from kimchi, and excellent antioxidant activity and anti-inflammatory activity of the isolated strain , skin whitening activity, antibacterial activity against harmful microorganisms, and antithrombotic activity were confirmed, thereby completing the present invention.
상기 과제를 해결하기 위해, 본 발명은 항염 활성, 피부 미백 활성, 항균 활성 및 항혈전 활성을 가지는 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주를 제공한다. In order to solve the above problems, the present invention provides an anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain having an accession number KCCM12792P.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 그람 음성균에 대한 항균용 조성물을 제공한다.In addition, the present invention provides a composition for antibacterial against Gram-negative bacteria containing the strain, its culture, the concentrate of the culture, or its dried product as an active ingredient.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 발효식품 제조용 스타터(starter) 조성물을 제공한다. In addition, the present invention provides a starter composition for producing a fermented food containing the strain, its culture, the concentrate of the culture or its dried product as an active ingredient.
또한, 본 발명은 상기 균주, 이의 균주 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 프로바이오틱 조성물을 제공한다. In addition, the present invention provides a probiotic composition containing the strain, its culture solution, the concentrate of the culture solution, or a dried product thereof as an active ingredient.
또한, 본 발명은 상기 균주, 이의 균주 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 혈전증의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for the prevention or improvement of thrombosis containing the strain, its culture solution, the concentrate of the culture solution, or a dried product thereof as an active ingredient.
또한, 본 발명은 상기 균주, 이의 균주 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 피부 미백용 화장료 조성물을 제공한다. In addition, the present invention provides a cosmetic composition for skin whitening containing the strain, its culture solution, the concentrate of the culture solution, or a dried product thereof as an active ingredient.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 사료첨가제 조성물을 제공한다.In addition, the present invention provides a feed additive composition containing the strain, its culture, the concentrate of the culture, or its dried product as an active ingredient.
본 발명의 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주는 항염 활성, 피부 미백 활성, 항균 활성 및 항혈전 활성이 우수하므로 항균용 조성물, 발효식품 제조용 스타터, 프로바이오틱, 혈전증의 예방용 조성물, 피부 미백용 조성물 및 사료첨가제 등으로 활용 가능하므로, 관련 산업 분야에 매우 유용하게 이용될 수 있다. Lactobacillus plantarum of the present invention ( Lactobacillus plantarum ) OKBL-L.PL 1 strain has excellent anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity, so antibacterial composition, starter for fermented food production, probiotic, thrombosis Since it can be used as a preventive composition, skin whitening composition, and feed additive, it can be very usefully used in related industrial fields.
도 1은 본 발명에서 분리한 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주의 리폭시게나아제 저해 활성을 측정한 결과이다. 0.1% IDM은 양성 대조군으로 사용한 인도메타신(indomethacin)이고, MRSB는 음성 대조군으로 사용한 MRS 배지이고, L.PL_O는 본 발명의 균주 배양액을 의미하고, L.PL_A는 본 발명의 균주 배양액+균체를 의미한다.
도 2는 본 발명에서 분리한 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주의 NO 생성 억제능을 측정한 결과이다. C는 증류수이고, LPS는 lipopolysaccharide이고, L.FE_O는 비교군으로 사용한 락토바실러스 퍼멘텀의 배양액이며, L.PL_A는 본 발명의 균주 배양액+균체를 의미한다.
도 3은 본 발명에서 분리한 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주의 티로시나아제 저해 활성을 분석한 결과이다. 0.05% ABT는 양성 대조군으로 사용한 티로시나아제 저해제이고, L.FE_O 비교군으로 사용한 락토바실러스 퍼멘텀의 배양액이며, L.PL_A는 본 발명의 균주 배양액+균체를 의미한다.
도 4는 본 발명에서 분리한 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주의 에스케리키아 콜리(Escherichia coli, E.coli)에 대한 항균 활성을 측정한 결과이다. MRSB는 음성 대조군으로 사용한 MRS 배지이고, L.FE_O 및 L.PE_O는 비교군으로 사용한 각각 락토바실러스 퍼멘텀 및 락토바실러스 펜토서스의 배양액이며, L.PL_O는 본 발명의 균주 배양액을 의미한다.
도 5는 본 발명에서 분리한 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주의 항혈전 활성을 측정한 결과이다. MRSB는 음성 대조군으로 사용한 MRS 배지이고, L.FE_O 및 L.PE_O는 비교군으로 사용한 각각 락토바실러스 퍼멘텀 및 락토바실러스 펜토서스의 배양액이며, L.PL_O는 본 발명의 균주 배양액을 의미한다.1 is a result of measuring the lipoxygenase inhibitory activity of Lactobacillus plantarum OKBL-L.PL 1 strain isolated in the present invention. 0.1% IDM is indomethacin used as a positive control, MRSB is MRS medium used as a negative control, L.PL_O means the strain culture of the present invention, and L.PL_A is the strain culture of the present invention + cells means
Figure 2 is a result of measuring the NO production inhibitory ability of the Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain isolated in the present invention. C is distilled water, LPS is lipopolysaccharide, L.FE_O is the culture solution of Lactobacillus permentum used as a comparison group, and L.PL_A means the culture medium of the present invention + cells.
3 is a result of analyzing the tyrosinase inhibitory activity of Lactobacillus plantarum OKBL-L.PL 1 strain isolated in the present invention. 0.05% ABT is a tyrosinase inhibitor used as a positive control, L.FE_O is a culture medium of Lactobacillus fermentum used as a comparison group, and L.PL_A means a culture medium of the present invention + cells.
Figure 4 is a result of measuring the antibacterial activity of Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain against Escherichia coli (E.coli ) isolated in the present invention. MRSB is the MRS medium used as a negative control, L.FE_O and L.PE_O are the cultures of Lactobacillus fermentum and Lactobacillus pentosus, respectively, used as a comparison group, and L.PL_O means the culture medium of the strain of the present invention.
Figure 5 is a result of measuring the antithrombotic activity of the Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain isolated in the present invention. MRSB is the MRS medium used as a negative control, L.FE_O and L.PE_O are the cultures of Lactobacillus fermentum and Lactobacillus pentosus, respectively, used as a comparison group, and L.PL_O means the culture medium of the strain of the present invention.
본 발명의 목적을 달성하기 위하여, 본 발명은 항염 활성, 피부 미백 활성, 항균 활성 및 항혈전 활성을 가지는 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주를 제공한다.In order to achieve the object of the present invention, the present invention provides a Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain whose deposit number is KCCM12792P having anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity do.
본 발명에서는 배추 김치로부터 항염 활성, 피부 미백 활성, 항균 활성 및 항혈전 활성을 가지는 균주를 분리하였으며, 최종 선별된 균주의 16S rRNA 염기서열 분석을 통해 동정한 결과, 락토바실러스 플란타룸(Lactobacillus plantarum) 균주임을 확인하였다. 본 발명자는 상기 최종 선별된 균주를 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주로 명명하고 한국미생물보존센터(KCCM)에 2020년 9월 18일자로 기탁하였다(수탁번호: KCCM12792P).In the present invention, a strain having anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity was isolated from Chinese cabbage kimchi, and as a result of identification through 16S rRNA sequencing analysis of the finally selected strain, Lactobacillus plantarum ( Lactobacillus plantarum ) ) was confirmed as a strain. The present inventor named the final selected strain Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain and deposited at the Korea Center for Microorganisms Conservation (KCCM) on September 18, 2020 (Accession No.: KCCM12792P) ).
본 발명은 또한, 상기 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 그람 음성균에 대한 항균용 조성물을 제공한다. The present invention also provides an antibacterial agent for Gram-negative bacteria containing the Lactobacillus plantarum OKBL-L.PL 1 strain, the culture solution thereof, the concentrate of the culture solution or the dried product thereof as an active ingredient, the accession number of which is KCCM12792P A composition is provided.
본 발명의 일 구현 예에 따른 항균용 조성물에서, 상기 그람 음성균은 에스케리키아 콜리(Escherichia coli)일 수 있으나, 이에 제한되지 않는다. In the antimicrobial composition according to an embodiment of the present invention, the Gram-negative bacteria may be Escherichia coli , but is not limited thereto.
본 발명의 "항균(antimicrobial)"의 의미는 어떤 농도에서 미생물의 성장 또는 생존을 감소, 방지, 억제, 또는 제거하는 능력을 의미한다.By "antimicrobial" in the present invention is meant the ability to reduce, prevent, inhibit, or eliminate the growth or survival of microorganisms at certain concentrations.
본 발명의 "항균용 조성물"은 세균과 같은 미생물의 생육을 저해하는 활성을 가진 조성물로서, 항미생물제를 총칭하는 의미인 항생제와 같은 의미일 수 있고, 항균 효과가 요구되는 다양한 분야에 사용되는 모든 형태가 포함될 수 있으며, 예를 들어, 의약품, 의약외품, 식품 첨가제 또는 사료 첨가제 등의 형태일 수 있다.The "antibacterial composition" of the present invention is a composition having an activity to inhibit the growth of microorganisms such as bacteria, and may have the same meaning as antibiotics, which is a generic term for antimicrobial agents, and is used in various fields requiring antibacterial effect. The form may be included, for example, it may be in the form of pharmaceuticals, quasi-drugs, food additives or feed additives.
본 발명의 항균용 조성물은 상기 균주 또는 상기 균주의 배양액 또는 상기 균주의 배양액의 농축액 그 자체일 수 있고, 제형화에 필요한 부형제나 희석제 등의 통상적으로 사용되는 첨가제가 더 포함될 수 있으며, 본 발명의 균주 또는 이의 배양액 이외의 다른 항균 물질을 더 포함할 수도 있다. 유효성분의 혼합양은 그의 사용 목적에 따라 적합하게 결정될 수 있다.The antibacterial composition of the present invention may be the strain or the culture solution of the strain or the concentrate of the culture solution of the strain itself, and may further include commonly used additives such as excipients or diluents necessary for formulation, and of the present invention It may further include other antibacterial substances other than the strain or its culture solution. The mixing amount of the active ingredient may be suitably determined according to the purpose of its use.
상기 항균용 조성물에 포함되는 본 발명의 균주 또는 상기 균주의 배양액 또는 상기 균주의 배양액의 농축액의 함량은 사용목적 및 사용방법 등을 고려하여 적절하게 선택될 수 있으며, 예를 들어 전체 조성물을 기준으로 1 ~ 100중량%, 바람직하게는 10 ~ 100중량%, 더욱 바람직하게는 50 ~ 100중량%일 수 있다.The content of the strain of the present invention included in the antibacterial composition or the culture solution of the strain or the concentrate of the culture solution of the strain may be appropriately selected in consideration of the purpose and method of use, for example, based on the entire composition 1 to 100% by weight, preferably 10 to 100% by weight, more preferably 50 to 100% by weight.
본 발명의 "배양액"은 액체배지에서 배양한 배양액 자체, 상기 배양액을 여과 또는 원심분리하여 균주를 제거한 여액(원심분리한 상등액) 등의 배양액 자체로부터 파생되는 가공물을 포함한다. 배양액 중의 배양배지를 제거하고 농축된 균체만을 회수하기 위해 원심분리 또는 여과과정을 거칠 수 있으며, 이러한 단계는 당업자가 필요에 따라 수행할 수 있다. 농축된 균체는 통상적인 방법에 따라 냉동(frozen)하거나 또는 냉동건조(lyophilized)하여 그 활성을 잃지 않도록 보존할 수 있다.The "culture solution" of the present invention includes a processed product derived from the culture solution itself, such as the culture solution itself cultured in a liquid medium, and the filtrate (centrifuged supernatant) from which the strain is removed by filtration or centrifugation of the culture solution. In order to remove the culture medium in the culture medium and recover only the concentrated cells, centrifugation or filtration may be performed, and these steps may be performed by those skilled in the art as needed. The concentrated cells may be frozen or lyophilized according to a conventional method to preserve their activity so as not to lose their activity.
본 발명의 균주를 배양하는 방법은 당업계에 통상적으로 이용되는 방법에 따라 배양할 수 있으며, 특별한 방법에 한정되는 것은 아니다.The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a particular method.
본 발명은 또한, 상기 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 발효식품 제조용 스타터(starter) 조성물을 제공한다.The present invention also provides a starter for producing fermented food containing, as an active ingredient, the Lactobacillus plantarum OKBL-L.PL 1 strain, the culture medium thereof, the concentrate of the culture medium, or the dried product of the Lactobacillus plantarum having the accession number KCCM12792P as an active ingredient. ) to provide a composition.
본 발명에 있어서, 발효식품 제조용 스타터(starter)란 발효식품 제조를 위해 발효에 관여하는 미생물을 포함하는 제제 또는 조성물을 의미한다. 발효식품 제조 시에 첨가함으로써 발효된 식품에서 생장할 수 있는 미생물 또는 우점종으로 생장할 수 있는 미생물을 제공하기 위하여 사용된다. 상기 발효식품 제조용 스타터를 사용하여 식품을 제조하는 경우, 상기 발효식품 제조용 스타터에 포함된 미생물에 의하여, 식품의 품질을 일정하게 조절하거나, 특정한 목적을 달성할 수 있다. 본 발명에서는 락토바실러스 플란타룸 OKBL-L.PL 1 균주 또는 이의 배양액을 스타터 균주로 이용함으로써, 항염 활성, 피부 미백 활성, 항균 활성 및 항혈전 활성이 있는 발효식품을 제조할 수 있다. In the present invention, a starter for fermented food production means a preparation or composition containing microorganisms involved in fermentation for the production of fermented food. It is used to provide microorganisms that can grow in fermented foods or microorganisms that can grow as dominant species by adding them during the manufacture of fermented foods. When food is manufactured using the starter for manufacturing fermented food, the quality of the food may be uniformly controlled or a specific purpose may be achieved by the microorganisms included in the starter for manufacturing the fermented food. In the present invention, by using the Lactobacillus plantarum OKBL-L.PL 1 strain or a culture medium thereof as a starter strain, fermented food having anti-inflammatory activity, skin whitening activity, antibacterial activity and antithrombotic activity can be prepared.
본 발명의 일 구현 예에 따른 발효식품 제조용 스타터 조성물에서, 상기 발효식품은 전통 김치류일 수 있으나, 이에 제한되지 않는다.In the starter composition for manufacturing fermented food according to an embodiment of the present invention, the fermented food may be traditional kimchi, but is not limited thereto.
본 발명은 또한, 상기 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 프로바이오틱(probiotic) 조성물을 제공한다.The present invention also provides that the accession number is KCCM12792P Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain, its culture medium, a concentrate of the culture medium or a probiotic containing a dried product thereof as an active ingredient. A composition is provided.
본 발명의 프로바이오틱 조성물은 상기 유효성분 외에 통상적인 약학적 담체 및 부형제를 추가로 포함할 수 있으며, 이러한 조성물은 통상적인 프로바이오틱 조성물 제조방법에 따라 열건조 또는 동결-건조하여 생균제 형태로 제조하여 이용할 수 있다.The probiotic composition of the present invention may further include conventional pharmaceutical carriers and excipients in addition to the above active ingredients, and these compositions are heat-dried or freeze-dried according to a conventional probiotic composition preparation method to form a probiotic. can be manufactured and used.
본 발명의 균주는 상기 전술한 바와 같이 다양한 활성을 가지고 있으므로 식품, 의약품, 화장품, 건강식품 등의 프로바이오틱 조성물 제조에 이용될 수 있다.Since the strain of the present invention has various activities as described above, it can be used in the manufacture of probiotic compositions such as food, medicine, cosmetics, and health food.
본 발명은 또한, 상기 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 혈전증의 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention also provides for the prevention or improvement of thrombosis containing, as an active ingredient, the Lactobacillus plantarum OKBL-L.PL 1 strain, the culture medium thereof, the concentrate of the culture medium, or the dried product thereof, the deposit number of which is KCCM12792P It provides a health functional food composition.
본 발명의 용어, "혈전증"이란, 혈전에 의해 발생되는 질환으로, 혈전색전증이라고도 하며, 특히 혈전에 의해 혈관이 막힌 질환을 일컫는다.As used herein, the term "thrombosis" refers to a disease caused by a thrombus, also called thromboembolism, and in particular refers to a disease in which blood vessels are blocked by a thrombus.
용어 "혈전"은 주로 혈소판과 세포성분을 둘러싼 섬유소로 구성된 혈액성분의 응집을 말하며, 혈액의 기타 형성된 혈액 요소와 뒤섞인 것으로 주로 혈소판 및 피브린의 응집물로 이루어진다.The term "thrombus" refers to the aggregation of blood components mainly composed of platelets and fibrin surrounding cellular components, intermingled with other formed blood components of the blood and mainly composed of aggregates of platelets and fibrin.
본 발명의 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주, 이의 배양액을 유효성분으로 함유하는 항혈전 건강기능식품 조성물이 예방 또는 개선 효과를 가지는 질병은 다양하고, 예를 들어 고혈압, 심장병, 뇌졸증, 심근경색 등이 있으나, 이에 제한되지 않으며, 혈전 및 혈소판의 응집으로 유발되는 질병은 모두 이에 포함될 수 있다.Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain of the present invention, the antithrombotic health functional food composition containing the culture medium as an active ingredient has a preventive or ameliorating effect on various diseases, for example, hypertension , heart disease, stroke, myocardial infarction, etc., but is not limited thereto, and diseases caused by blood clots and platelet aggregation may all be included therein.
본 발명의 건강기능식품 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽, 발포정 및 음료 중에서 선택된 어느 하나의 제형으로 제조될 수 있으나, 이에 제한되지 않는다. The health functional food composition of the present invention may be prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, effervescent tablet and beverage, but is not limited thereto.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분은 그의 사용 목적(예방 또는 개선)에 따라 적절하게 사용될 수 있다. 그러나 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 안전성 면에서 아무런 문제가 없는 범위의 양으로 사용될 수 있다. 상기 식품의 종류에는 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The active ingredient may be used appropriately depending on the purpose of its use (prevention or improvement). However, in the case of long-term intake for the purpose of health control, it can be used in an amount within a range that does not cause any problems in terms of safety. There is no particular limitation on the type of the food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the ordinary sense.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함시킬 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로오스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다. 상기 건강기능식품 조성물 외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다.In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols (eg, , xylitol, sorbitol, erythritol, etc.) is preferable. As the flavoring agent, natural flavoring agents (eg, taumartin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used. In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent and the like used in beverages.
본 발명은 또한, 상기 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 피부 미백용 화장료 조성물을 제공한다.The present invention also provides a cosmetic composition for skin whitening containing, as an active ingredient, the Lactobacillus plantarum OKBL-L.PL 1 strain, the culture solution thereof, the concentrate of the culture solution, or the dried product of the Lactobacillus plantarum having the accession number KCCM12792P as an active ingredient to provide.
본 발명의 일 구현 예에 따른 피부 미백용 화장료 조성물에서, 상기 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주는 티로시나아제 저해 활성이 우수하므로, 피부 미백 효과가 있다.In the cosmetic composition for skin whitening according to an embodiment of the present invention, the Lactobacillus plantarum ( Lactobacillus plantarum ) OKBL-L.PL 1 strain having the deposit number KCCM12792P has excellent tyrosinase inhibitory activity, so the skin whitening effect there is
피부 미백은 티로시나아제를 저해하는 것을 특징으로 할 수 있으나, 이에 제한되지 않는다. Skin lightening may be characterized by inhibiting tyrosinase, but is not limited thereto.
본 발명의 일 구현 예에 따른 화장료 조성물에 있어서, 상기 피부 미백용 화장료 조성물은 스킨, 스킨 소프트너, 스킨 토너, 로션, 밀크로션, 모이스쳐로션, 영양로션, 마사지크림, 영양크림, 아이크림, 모이스쳐크림, 핸드크림, 에센스, 영양에센스, 팩, 클렌징폼, 클렌징워터, 클렌징크림, 바디로션, 바디클렌져, 비누 및 파우더 중에서 선택된 어느 하나의 제형을 가질 수 있으나, 이에 제한되지 않는다. 이들 각 제형으로 이루어진 화장료 조성물은 그 제형의 제제화에 필요하고 적절한 각종의 기제와 첨가물을 함유할 수 있으며, 이들 성분의 종류와 양은 당업자에 의해 용이하게 선정될 수 있다.In the cosmetic composition according to an embodiment of the present invention, the cosmetic composition for skin whitening is a skin, skin softener, skin toner, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, eye cream, moisture It may have any one formulation selected from cream, hand cream, essence, nutritional essence, pack, cleansing foam, cleansing water, cleansing cream, body lotion, body cleanser, soap and powder, but is not limited thereto. The cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.
본 발명의 화장료 조성물은 유효성분 이외에 추가로 동일 또는 유사한 기능을 나타내는 피부 미백 활성 성분을 1종 이상 함유할 수 있다. 피부 미백 활성 성분으로는 코지산 및 이의 유도체, 알부틴, 아스코르브산 및 이의 유도체, 하이드로퀴논 및 이의 유도체, 레조르시놀, 사이클로알카논, 메틸렌디옥시페닐 알칸올, 2,7-디니트로인다졸 또는 덩굴귤 추출물, 쌀 추출물, 감초 추출물과 같은 식물 추출물 등이 있으나, 이에 제한되는 것은 아니다.In addition to the active ingredient, the cosmetic composition of the present invention may contain one or more skin whitening active ingredients that exhibit the same or similar function. Skin lightening active ingredients include kojic acid and its derivatives, arbutin, ascorbic acid and its derivatives, hydroquinone and its derivatives, resorcinol, cycloalkanone, methylenedioxyphenyl alkanol, 2,7-dinitroindazole, or Plant extracts such as tendril extract, rice extract, and licorice extract, but are not limited thereto.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as a carrier component etc. may be used.
본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판-부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro It may contain a propellant such as carbon, propane-butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용매화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butylglycol oil, glycerol fatty esters, polyethylene glycol or sorbitan.
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.
본 발명의 화장료 조성물의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 아세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 리놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acetionate, imidazolinium derivative, methyl taurate, sarcosinate as carrier components , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolin derivative or ethoxylated glycerol fatty acid ester, and the like can be used.
본 발명은 또한, 상기 기탁번호가 KCCM12792P인 락토바실러스 플란타룸(Lactobacillus plantarum) OKBL-L.PL 1 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 사료첨가제 조성물을 제공한다.The present invention also provides a feed additive composition containing, as an active ingredient, the Lactobacillus plantarum OKBL-L.PL 1 strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof, the accession number of which is KCCM12792P. .
상기 균주는 전술한 바와 같으며, 항염, 항균 및 항혈전을 목적으로 사료첨가제 조성물로 첨가할 수 있다. 본 발명의 사료첨가제는 사료관리법상의 보조사료에 해당한다.The strain is as described above, and may be added as a feed additive composition for the purpose of anti-inflammatory, antibacterial and antithrombotic. The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act.
본 발명의 사료첨가제 조성물은 사료에 첨가될 수 있으며, "사료"는 동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한끼식의 성분을 의미할 수 있다. 상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다.The feed additive composition of the present invention may be added to a feed, and "feed" means any natural or artificial diet, one meal, etc., or a component of the one meal meal suitable for or suitable for the animal to eat, ingest, and digest. can do. The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 이에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.
실시예 1. 균주의 분리 및 동정 Example 1. Isolation and identification of strains
배추 김치 시료를 생리식염수에 희석한 후 LB 배지(1% 수크로즈, 1% 효모 추출물 및 0.15% NaH2PO4)에 접종하고 배지가 든 플라스크를 30℃에서 24시간 동안 배양하였다. 이후, 배양액을 고체 배지에 도말하여 형성된 콜로니를 선별하여 순수 분리하였다. 선별 균주의 16S rRNA 유전자의 염기서열(서열번호 1)을 분석한 결과, 락토바실러스 플란타룸(Lactobacillus plantarum)으로 판명되었으며, Lactobacillus plantarum OKBL-L.PL 1 균주로 명명하고 한국미생물보존센터(KCCM)에 2020년 9월 18일자로 기탁하였다(기탁번호 : KCCM12792P). After diluting the cabbage kimchi sample in physiological saline, it was inoculated in LB medium (1% sucrose, 1% yeast extract and 0.15% NaH 2 PO 4 ), and the flask containing the medium was cultured at 30° C. for 24 hours. Thereafter, colonies formed by smearing the culture medium on a solid medium were selected and purified. As a result of analyzing the nucleotide sequence (SEQ ID NO: 1) of the 16S rRNA gene of the selected strain, Lactobacillus It was identified as a plantarum ( Lactobacillus plantarum ), named Lactobacillus plantarum OKBL-L.PL 1 strain, and deposited at the Korea Microorganism Conservation Center (KCCM) on September 18, 2020 (Accession No.: KCCM12792P).
실시예Example 2. 선별 균주의 특성 분석2. Characterization of selected strains
선별 균주를 MRS 액체 배지에 접종하여 37℃에서 18시간 동안 배양하고, 3,000 rpm으로 10분 동안 원심분리한 후 상층액을 취하여 균주 배양액(L.PL_O)으로 명명하였고, 균주를 배양한 후 초음파 분해(sonication)하여 3,000 rpm으로 10분 동안 원심분리 후 상층액을 취하여 균주 배양액+균체(L.PL_A)로 명명하였다. 본 발명의 균주의 특성을 분석하기 위하여 균주 배양액(L.PL_O) 및 균주 배양액+균체(L.PL_A)의 당도와 pH를 각각 측정하였다. 당도는 총 가용성 고형물(total soluble solids, Brix) 함량을 당도계(Hand refractormeter, Germany)로 측정하여 나타내었다. pH는 pH 미터기(SevenCompactTM pH/Ion S220, Switzerland)를 이용하여 3회 측정한 평균값으로 나타내었다.Selected strains were inoculated into MRS liquid medium, incubated at 37°C for 18 hours, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was taken and named strain culture solution (L.PL_O), followed by sonication after culturing the strain (sonication), centrifuged at 3,000 rpm for 10 minutes, and then the supernatant was taken and named as the strain culture medium + cells (L.PL_A). In order to analyze the characteristics of the strain of the present invention, the sugar content and pH of the strain culture medium (L.PL_O) and the strain culture medium + cells (L.PL_A) were measured, respectively. Sugar content was expressed by measuring the total soluble solids (Brix) content with a saccharometer (Hand refractormeter, Germany). The pH was expressed as an average value measured three times using a pH meter (SevenCompact™ pH/Ion S220, Switzerland).
그 결과, 본 발명의 락토바실러스 플란타룸 OKBL-L.PL 1 균주의 pH 및 당도는 대조군 대비 낮은 값을 나타내었고, 균주 배양액(L.PL_O) 및 배양액+균체(L.PL_A)는 유사한 특성을 나타내는 것을 확인하였다(표 1).As a result, the pH and sugar content of the Lactobacillus plantarum OKBL-L.PL 1 strain of the present invention showed lower values compared to the control, and the strain culture medium (L.PL_O) and the culture medium + cells (L.PL_A) had similar characteristics. was confirmed to represent (Table 1).
실시예Example 3. 항염 활성 분석3. Anti-inflammatory activity assay
(1) 리폭시게나아제 저해 활성 측정(1) Measurement of lipoxygenase inhibitory activity
리폭시게나아제(lipoxygenase)는 정상적인 세포에서는 활성이 미비하지만, 염증이 유발되면 류코트리엔(leukotrienes) 물질의 증가로 인해 활성이 증가한다. OD600=1.0의 균주 배양액 또는 배양액+균체 100 ㎕에 200 mM 보레이트 완충액(borate buffer, pH 9.0) 1 ㎖을 넣은 후, 기질인 리놀렌산(linoleic acid, 0.6 mM/㎖ in ethanol) 2 ㎖을 추가하여 혼합한 후 234 nm에서 보정값을 측정하였다. 이후, 상기 혼합물에 10,000 unit/㎖의 리폭시게나아제 100 ㎕를 넣고 상온에서 10분간 반응시킨 후 234 nm에서 흡광도를 측정하였다. 양성 대조군은 인도메타신(indomethacin, IDM)을 0.1%(w/v)로 에탄올에 녹여서 사용하였다. 리폭시게나아제 억제 활성(%)은 다음과 같은 식에 의해 산출되었다. Lipoxygenase has insufficient activity in normal cells, but increases in activity due to an increase in leukotrienes when inflammation is induced. After adding 1 ml of 200 mM borate buffer (pH 9.0) to 100 μl of the strain culture medium or culture medium + cells with an OD 600 = 1.0, 2 ml of linoleic acid (0.6 mM/ml in ethanol) as a substrate was added. After mixing, the correction value was measured at 234 nm. Thereafter, 100 μl of 10,000 unit/ml lipoxygenase was added to the mixture, and the mixture was reacted at room temperature for 10 minutes, and then absorbance was measured at 234 nm. The positive control was used by dissolving indomethacin (IDM) in ethanol at 0.1% (w/v). Lipoxygenase inhibitory activity (%) was calculated by the following formula.
리폭시게나아제 억제 활성(%)={1-(b'-b)/(a'-a)}x100Lipoxygenase inhibitory activity (%) = {1-(b'-b)/(a'-a)}x100
a: 효소 무첨가군a: enzyme-free group
a': 효소 첨가군a': enzyme addition group
b: 효소 무첨가 및 시료 첨가군b: enzyme-free and sample-added group
b': 효소 및 시료 첨가군b': enzyme and sample addition group
실험으로부터 얻어진 결과는 one-way ANOVA 검정에 의한 평균치와 표준 오차(mean±SE)로 표시하였다.The results obtained from the experiment were expressed as the mean value and standard error (mean±SE) by one-way ANOVA test.
그 결과, 본 발명의 락토바실러스 플란타룸 OKBL-L.PL 1 균주는 음성 대조군(MRS 배지) 대비 현저한 리폭시게나아제 저해 활성을 가지고 있음을 확인하였으며, 균주 배양액+균체(L.PL_A)가 배양액(L.PL_O)보다 저해 활성이 우수함을 확인하였다(도 1).As a result, it was confirmed that the Lactobacillus plantarum OKBL-L.PL 1 strain of the present invention had a significant lipoxygenase inhibitory activity compared to the negative control (MRS medium), and the strain culture medium + bacteria (L.PL_A) was the culture medium. It was confirmed that the inhibitory activity was superior to (L.PL_O) (FIG. 1).
(2) NO 생성 억제율 측정(2) NO production inhibition rate measurement
락토바실러스 플란타룸 OKBL-L.PL 1 균주의 항염 활성을 분석하기 위하여 LPS(lipopolysaccharide)로 염증이 유도된 RAW 264.7 세포에서의 NO 생성 억제율을 측정하였다. RAW 264.7 세포는 한국세포주은행(KCLB, Korea)에서 구입하여 사용하였으며 10% FBS 및 페니실린-스트렙토마이신을 첨가한 DMEM 배지에서 37℃, 5% CO2 의 조건으로 배양하여 이후 실험에 사용하였다. RAW 264.7 세포를 24웰 플레이트에 1x104 세포/웰로 24시간 동안 배양한 후, 본 발명의 균주 배양액+균체(L.PL_A)를 10%로 전처리한 다음 LPS(1 ㎍/㎖)를 처리하고 24시간 동안 반응시켰다. 이후, 세포 배양액 100 ㎕를 취하여 그리스 시약(1% 설폰산, 0.1% N-1-나프틸 에틸렌 디아민이염산염, 2.5% 인산)과 반응시켜 마이크로플레이트 리더로 546 nm에서 흡광도를 측정하였다. In order to analyze the anti-inflammatory activity of Lactobacillus plantarum OKBL-L.PL 1 strain, the inhibition rate of NO production in RAW 264.7 cells induced by lipopolysaccharide (LPS) was measured. RAW 264.7 cells were purchased from Korea Cell Line Bank (KCLB, Korea) and cultured in DMEM medium supplemented with 10% FBS and penicillin-streptomycin at 37° C., 5% CO 2 conditions and used for subsequent experiments. After culturing RAW 264.7 cells in a 24-well plate at 1x10 4 cells/well for 24 hours, the strain culture solution of the present invention + cells (L.PL_A) were pretreated with 10% and then treated with LPS (1 μg/ml) and 24 reacted for an hour. Then, 100 μl of the cell culture solution was taken and reacted with a grease reagent (1% sulfonic acid, 0.1% N-1-naphthyl ethylene diamine dihydrochloride, 2.5% phosphoric acid), and absorbance was measured at 546 nm with a microplate reader.
그 결과, 본 발명의 락토바실러스 플란타룸 OKBL-L.PL 1 균주의 배양액+균체(L.PL_A)는 LPS 처리에 의한 NO의 생성을 억제하는 효과가 있음을 확인하였다.As a result, it was confirmed that the culture medium + cells (L.PL_A) of the Lactobacillus plantarum OKBL-L.PL 1 strain of the present invention had an effect of inhibiting the production of NO by the LPS treatment.
실시예 4. 피부 미백 활성 분석Example 4. Analysis of skin whitening activity
락토바실러스 플란타룸 OKBL-L.PL 1 균주의 피부 미백 활성을 분석하기 위하여 in vitro 머쉬룸 티로시나아제(mushroom tyrosinase) 저해 활성을 측정하였다. 1.5 ㎖ 플라스틱 큐벳(plastic cuvette)에 2.5 mM L-DOPA(3,4-dihydroxyphenylalanine) 0.3㎖, 본 발명의 균주 배양액+균체(L.PL_A) 0.1 ㎖ 및 0.1 M 인산완충용액(pH 6.8)을 혼합하여 25℃에서 반응시켰다. 이후, 1,380 units/㎖ 머쉬룸 티로시나아제(Sigma, USA) 0.05 ㎖을 넣고 25℃에서 2분간 반응시켜 분광광도계를 이용하여 470 nm에서 흡광도를 측정한 후 하기 식에 대입하여 티로시나아제 저해 활성을 산출하였다. 대조구는 시료가 들어있지 않는 시료 용해 용액을 사용하였고, 양성 대조군으로는 티로시나아제 저해제로 알려진 0.05% 알부틴(Arbutin)을 사용하였다. In order to analyze the skin whitening activity of the Lactobacillus plantarum OKBL-L.PL 1 strain, the mushroom tyrosinase inhibitory activity was measured in vitro . In a 1.5 ml plastic cuvette, 0.3 ml of 2.5 mM L-DOPA (3,4-dihydroxyphenylalanine), 0.1 ml of the strain culture solution of the present invention + cells (L.PL_A), and 0.1 M phosphate buffer solution (pH 6.8) were mixed. and reacted at 25°C. Then, add 0.05 ml of 1,380 units/ml mushroom tyrosinase (Sigma, USA) and react at 25° C. for 2 minutes to measure the absorbance at 470 nm using a spectrophotometer. calculated. As a control, a sample dissolution solution containing no sample was used, and as a positive control, 0.05% arbutin, known as a tyrosinase inhibitor, was used.
티로시나아제 저해 활성(%)=100-[(A-B)/A]x100Tyrosinase inhibitory activity (%)=100-[(A-B)/A]x100
A : 시료가 들어있지 않은 반응액의 0.5~1분 사이의 흡광도 차이A: The difference in absorbance between 0.5 and 1 minute of the reaction solution without sample
B : 시료가 들어있는 반응액의 0.5~1분 사이의 흡광도 차이B: The difference in absorbance between 0.5 and 1 minute of the reaction solution containing the sample
그 결과, 본 발명의 락토바실러스 플란타룸 OKBL-L.PL 1 균주 배양액+균체(L.PL_A)는 비교군인 락토바실러스 퍼멘텀의 배양액보다 티로시나아제 저해 활성이 우수함을 확인함으로써 락토바실러스 플란타룸 OKBL-L.PL 1 균주 배양액+균체(L.PL_A)가 피부 미백 활성이 있음을 알 수 있었다. As a result, it was confirmed that the Lactobacillus plantarum OKBL-L.PL 1 strain culture solution of the present invention + the cells (L.PL_A) has superior tyrosinase inhibitory activity than the culture solution of Lactobacillus permentum, which is a control group. Room OKBL-L.PL 1 strain culture medium + cells (L.PL_A) were found to have skin whitening activity.
실시예Example 5. 항균 활성 분석5. Antimicrobial Activity Assay
본 발명의 락토바실러스 플란타룸 OKBL-L.PL 1 균주의 항균 활성을 분석하기 위하여 장출혈성 대장균인 에스케리키아 콜리(Escherichia coli, E.coli) O157:H7(ATCC 43894)를 American TyPL Culture Collection으로부터 분양받아 디스크 확산법을 이용하여 항균 활성을 측정하였다. E.coli를 LB(Luria-Bertani) 액체 배지에서 37℃, 24시간 동안 배양한 후, 배양액 200 ㎕를 LB 고체 배지에 도말하고 멸균된 10 ㎜ 페이퍼 디스크를 고체 배지위에 올려놓았다. 이후, 락토바실러스 플란타룸 OKBL-L.PL 1 균주 배양액(L.PL_O)을 상기 페이퍼 디스크에 처리하고 37℃에서 24시간 동안 배양한 다음 페이퍼 디스크 주변의 억제환(clear zone)을 관찰하였다.To analyze the antibacterial activity of the Lactobacillus plantarum OKBL-L.PL 1 strain of the present invention, Escherichia coli, E. coli O157:H7 (ATCC 43894), an intestinal hemorrhagic E. coli, was used in the American TyPL Culture Collection. Antibacterial activity was measured using the disk diffusion method. After culturing E. coli in LB (Luria-Bertani) liquid medium at 37° C. for 24 hours, 200 μl of the culture solution was spread on LB solid medium, and a sterilized 10 mm paper disk was placed on the solid medium. Then, the Lactobacillus plantarum OKBL-L.PL 1 strain culture solution (L.PL_O) was treated on the paper disk and cultured at 37° C. for 24 hours, and then a clear zone around the paper disk was observed.
그 결과, 본 발명의 락토바실러스 플란타룸 OKBL-L.PL 1 균주 배양액(L.PL_O)이 E.coli에 대한 항균 활성이 있음을 확인하였다(도 4).As a result, it was confirmed that the Lactobacillus plantarum OKBL-L.PL 1 strain culture solution (L.PL_O) of the present invention had antibacterial activity against E. coli (FIG. 4).
실시예Example 6. 항혈전 활성 분석6. Antithrombotic Activity Assay
본 발명의 락토바실러스 플란타룸 OKBL-L.PL 1 균주의 항혈전 활성을 분석하기 위하여 혈액을 응고시키는 피브린노겐(fibrinogen) 용해능을 측정하였다. 0.6% 피브리노겐(Sigma, USA)을 0.05 M 붕산나트륨(sodium borate) 버퍼(pH 7.5)에 첨가하여 37℃에서 1시간 동안 용해시킨 후 페트리 디쉬에 10 ㎖씩 분주하고, 1,000 units의 트롬빈(Sigma, USA)을 40 units/㎖의 농도로 희석한 후 총 20 unit이 되도록 페트리 디쉬에 처리하여 균일하게 혼합되도록 잘 흔들어주었다. 이후, 약 15분 동안 굳혀 피브린 응고물(fibrin clot)을 형성시켜 실험에 사용하였다. 상기 피브린 응고물이 형성된 페트리 디쉬에 페이퍼 디스크를 올려두고, OD600=1.0의 균주 배양액(L.PL_O) 50 ㎕를 점적하여 37℃ 배양기에서 반응시킨 후 피브린이 용해되어 생기는 투명대(clear zone)을 관찰하였다.In order to analyze the antithrombotic activity of the Lactobacillus plantarum OKBL-L.PL 1 strain of the present invention, the fibrinogen dissolving ability to coagulate blood was measured. 0.6% fibrinogen (Sigma, USA) was added to 0.05 M sodium borate buffer (pH 7.5) and dissolved at 37° C. for 1 hour. Then, 10 ml each was dispensed in a Petri dish, and 1,000 units of thrombin (Sigma, USA) was diluted to a concentration of 40 units/ml, treated in a Petri dish to make a total of 20 units, and shaken well to mix evenly. Then, it was hardened for about 15 minutes to form a fibrin clot and used in the experiment. A paper disk is placed on a Petri dish in which the fibrin clot is formed, and 50 μl of a strain culture solution (L.PL_O) having an OD 600 = 1.0 is added dropwise and reacted in an incubator at 37° C., followed by dissolution of fibrin (clear zone) observed.
그 결과, 본 발명의 락토바실러스 플란타룸 OKBL-L.PL 1 균주 배양액(L.PL_O)은 항혈전 활성이 우수한 것을 확인하였다(도 5).As a result, it was confirmed that the Lactobacillus plantarum OKBL-L.PL 1 strain culture solution (L.PL_O) of the present invention had excellent antithrombotic activity (FIG. 5).
<110> OKBIOLAND Co., Ltd. <120> Lactobacillus plantarum OKBL-L.PL 1 strain having anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganism and anti-thrombotic activity and uses thereof <130> PN20311 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1452 <212> DNA <213> Lactobacillus plantarum <400> 1 cccatcgggc tatctgcagt cgacgactct ggtattgatt ggtgcttgca tcatgattta 60 catttgagtg agtggcgaac tggtgagtaa cacgtgggaa acctgcccag aagcggggga 120 taacacctgg aaacagatgc taataccgca taacaacttg gaccgcatgg tccgagcttg 180 aaagatggct tcggctatca cttttggatg gtcccgcggc gtattagcta gatggtgggg 240 taacggctca ccatggcaat gatacgtagc cgacctgaga gggtaatcgg ccacattggg 300 actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg 360 aaagtctgat ggagcaacgc cgcgtgagtg aagaagggtt tcggctcgta aaactctgtt 420 gttaaagaag aacatatctg agagtaactg ttcaggtatt gacggtattt aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggat 540 ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag ccttcggctc 600 aaccgaagaa gtgcatcgga aactgggaaa cttgagtgca gaagaggaca gtggaactcc 660 atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct 720 ggtctgtaac tgacgctgag gctcgaaagt atgggtagca aacaggatta gataccctgg 780 tagtccatac cgtaaacgat gaatgctaag tgttggaggg tttccgccct tcagtgctgc 840 agctaacgca ttaagcattc cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac 960 cttaccaggt cttgacatac tatgcaaatc taagagatta gacgttccct tcggggacat 1020 ggatacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttatt atcagttgcc agcattaagt tgggcactct ggtgagactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggatggtaca acgagttgcg aactcgcgag agtaagctaa 1260 tctcttaaag ccattctcag ttcggattgt aggctgcaac tcgcctacat gaagtcggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccatgagagt ttgaacaccc aaagtcggtg gggaaccttt agaaccgccg 1440 ctaaggacga gg 1452 <110> OKBIOLAND Co., Ltd. <120> Lactobacillus plantarum OKBL-L.PL 1 strain having anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganisms and anti-thrombotic activity and uses thereof <130> PN20311 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1452 <212> DNA <213> Lactobacillus plantarum <400> 1 cccatcgggc tatctgcagt cgacgactct ggtattgatt ggtgcttgca tcatgattta 60 catttgagtg agtggcgaac tggtgagtaa cacgtgggaa acctgcccag aagcggggga 120 taacacctgg aaacagatgc taataccgca taacaacttg gaccgcatgg tccgagcttg 180 aaagatggct tcggctatca cttttggatg gtcccgcggc gtattagcta gatggtgggg 240 taacggctca ccatggcaat gatacgtagc cgacctgaga gggtaatcgg ccacattggg 300 actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg 360 aaagtctgat ggagcaacgc cgcgtgagtg aagaagggtt tcggctcgta aaactctgtt 420 gttaaagaag aacatatctg agagtaactg ttcaggtatt gacggtattt aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggat 540 ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag ccttcggctc 600 aaccgaagaa gtgcatcgga aactgggaaa cttgagtgca gaagaggaca gtggaactcc 660 atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct 720 ggtctgtaac tgacgctgag gctcgaaagt atgggtagca aacaggatta gataccctgg 780 tagtccatac cgtaaacgat gaatgctaag tgttggaggg tttccgccct tcagtgctgc 840 agctaacgca ttaagcattc cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac 960 cttaccaggt cttgacatac tatgcaaatc taagagatta gacgttccct tcggggacat 1020 ggatacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttatt atcagttgcc agcattaagt tgggcactct ggtgagactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggatggtaca acgagttgcg aactcgcgag agtaagctaa 1260 tctcttaaag ccattctcag ttcggattgt aggctgcaac tcgcctacat gaagtcggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccatgagagt ttgaacaccc aaagtcggtg gggaaccttt agaaccgccg 1440 ctaaggacga gg 1452
Claims (8)
The Lactobacillus plantarum of claim 1 ( Lactobacillus plantarum ) OKBL-L.PL 1 strain, a culture solution thereof, a concentrate of the culture solution, or a feed additive composition containing a dried product thereof as an active ingredient.
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| KR102424598B1 (en) * | 2020-11-30 | 2022-07-25 | 파이토지노믹스 주식회사 | Lactobacillus pentosus OKBL-L.PE 1 strain having antioxidant activity, anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganism and anti-thrombotic activity and uses thereof |
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| KR102424598B1 (en) * | 2020-11-30 | 2022-07-25 | 파이토지노믹스 주식회사 | Lactobacillus pentosus OKBL-L.PE 1 strain having antioxidant activity, anti-inflammatory activity, skin whitening activity, antimicrobial activity against pathogenic microorganism and anti-thrombotic activity and uses thereof |
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