KR102021558B1 - Novel lactic acid bacteria and composition and healthfunctional food comprising the same - Google Patents
Novel lactic acid bacteria and composition and healthfunctional food comprising the same Download PDFInfo
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- KR102021558B1 KR102021558B1 KR1020180152757A KR20180152757A KR102021558B1 KR 102021558 B1 KR102021558 B1 KR 102021558B1 KR 1020180152757 A KR1020180152757 A KR 1020180152757A KR 20180152757 A KR20180152757 A KR 20180152757A KR 102021558 B1 KR102021558 B1 KR 102021558B1
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Abstract
Description
본 발명은 신규한 유산균, 이를 포함하는 조성물 및 건강기능식품에 관한 것이다.The present invention relates to a novel lactic acid bacterium, a composition comprising the same and a dietary supplement.
유산균은 인간이 이용할 수 있는 가장 유익한 미생물의 한 종류로서 오랜 역사를 두고 인류의 생활에 직간접적으로 밀접한 관계를 맺고 있다. 유산균은 음식물뿐만 아니라 포유동물의 구강과 소화관, 토양 등 자연계에 널리 분포하고 있으며, 식품 보존 및 관능적 특성과 관련된 중요한 역할 등을 수행하는 인간생활에 유익한 균주이다.Lactobacillus is one of the most beneficial microorganisms available to human beings and has a long history of direct and indirect relationships with human life. Lactic acid bacteria are widely distributed not only in food but also in the natural world such as the oral cavity, digestive tract and soil of mammals, and are beneficial strains for human life that play important roles related to food preservation and sensory characteristics.
유산균이란 명칭은 개념적인 것이며 세균의 분류학적 학술명은 아니다. 보통 소비하는 당에 대하여 약 50% 이상의 유산을 대사산물로 생성하는 세균을 유산균이라고 한다. 대장균과 다르게 부패 산물을 생성하지 않는다. 따라서 당류로부터 다량의 유산을 생성하면서, 사람 및 동물의 장내에서 인체에 해로운 물질을 생성하지 않고 부패를 방지하는 등의 유익한 작용을 하는 세균을 개념적으로 유산균이라 한다. 통상적인 개념의 유산균은 그람(Gram) 양성 구균 또는 간균이며, 카탈라아제(catalase) 음성(혐기성발효), 포자(spore)를 형성하지 않으며, 대체적으로 운동성이 없다.The name lactic acid bacteria is conceptual and is not a taxonomy of bacteria. A bacterium that produces about 50% more lactic acid as a metabolite for sugars consumed is called lactic acid bacteria. Unlike E. coli, it does not produce rot products. Therefore, the bacterium that produces a large amount of lactic acid from sugars and has a beneficial effect such as preventing corruption without generating substances harmful to the human body in the intestines of humans and animals is conceptually referred to as lactic acid bacteria. Conventional concepts of lactic acid bacteria are Gram-positive cocci or bacilli, do not form catalase negative (anaerobic fermentation), spores, and are generally non-motile.
유산균은 다양한 항균물질과 항균활성이 있는 젖산과 초산과 같은 유기산들을 생산한다. 유산균은 산소 존재 하에서 과산화수소를 생산하고 카탈라아제(catalase) 활성을 가지고 있지 않으므로 과산화수소를 배지 내로 분비시켜 미생물 억제력을 가지는 농도까지 축적한다. 또한 유산균이 생산하는 비단백질성 항균물질의 하나인 디아세틸(diacetyl)은 유산균 발효제품의 대표적인 향미 성분중의 하나이며, pH와 상호작용하여 병원성 미생물에 대해 강한 억제작용을 가진다.Lactic acid bacteria produce various antimicrobial substances and organic acids such as lactic acid and acetic acid that have antimicrobial activity. Since lactic acid bacteria produce hydrogen peroxide in the presence of oxygen and do not have catalase activity, they secrete hydrogen peroxide into the medium and accumulate to concentrations having microbial inhibitory ability. In addition, diacetyl (diacetyl), which is a nonproteinaceous antibacterial substance produced by lactic acid bacteria, is one of the representative flavor components of lactic acid bacteria fermentation products, and has a strong inhibitory effect on pathogenic microorganisms by interacting with pH.
일반적으로 박테리오신(bacteriocin)은 비교적 작은 분자량을 가지는 단백질 또는 단백질복합체 물질로 세균 세포막의 양성자 구동력(proton motive force: ATP 합성의 원동력) 붕괴로 다른 미생물을 억제한다. 대부분 열에 안정하고, 낮은 pH에서도 안정하나 단백질 가수분해효소처리를 하면 대부분 불활성화 된다. 대표적인 유산균 유래 박테리오신 물질인 나이신(nisin)은 락토코커스 락티스(Lactococcus lactis)에서 생성되며 광범위한 항균 활성 때문에 오래 전부터 상용이 허용되어 왔다, 이외에도 유산균 유래 박테리오신으로는 락티신(Lacticin), 락토코신(Lactococcin), 페디오신(Pediocin) 등이 알려져 있다.In general, bacteriocin is a protein or protein complex material having a relatively small molecular weight that inhibits other microorganisms by disrupting the proton motive force of bacterial cell membranes. Most of them are stable to heat and stable even at low pH, but most of them are inactivated by proteolytic enzyme treatment. Nisin, a representative lactic acid bacteria-derived bacteriocin substance, is produced in Lactococcus lactis and has been commercially available for a long time because of its broad antibacterial activity. In addition, lacticin and lactocosin (Lacticin) and lactocosin ( Lactococcin), Pediocin (Pediocin) and the like are known.
본 발명은 신규한 유산균을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a novel lactic acid bacteria.
본 발명은 신규한 유산균의 다양한 활성, 기능성 등을 활용한 조성물, 건강기능식품을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a composition, health functional food utilizing various activities, functionalities, etc. of the novel lactic acid bacteria.
본 발명은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP를 제공한다.The present invention provides Lactobacillus plantarum L-14 KCTC13497BP.
본 발명은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양액 또는 그 추출물을 포함하는 조성물을 제공한다.The present invention provides a composition comprising Lactobacillus plantarum L-14 KCTC13497BP, a culture thereof or an extract thereof.
본 발명은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양액 또는 그 추출물을 포함하는 건강기능식품을 제공한다.The present invention provides a dietary supplement comprising Lactobacillus plantarum L-14 KCTC13497BP, its culture solution or its extract.
본 발명의 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP 균주는 항균, 항염, 항암, 지방세포 분화 억제, 항비만 등의 다양한 활성을 나타낸다. 이에, 의약 조성물, 건강기능식품, 화장료 조성물 등으로 바람직하게 활용될 수 있다.Lactobacillus plantarum L-14 KCTC13497BP strain of the present invention exhibits various activities such as antibacterial, anti-inflammatory, anti-cancer, adipocyte differentiation inhibition, anti-obesity, and the like. Thus, it can be preferably used as a pharmaceutical composition, health functional food, cosmetic composition and the like.
도 1 및 2는 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP의 유해 미생물 성장 억제 효과를 나타낸 것이다.
도 3은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP의 배양액의 유해 미생물 성장 억제 효과를 나타낸 것이다.
도 4 내지 6은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP의 구강 내 유해균 억제 효과를 나타낸 것이다.
도 7 내지 10은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP의 항암 효과를 나타낸 것이다.
도 11 내지 16은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP의 지방 세포 분화 억제 효과를 나타낸 것 이다.1 and 2 show the harmful microbial growth inhibitory effect of Lactobacillus plantarum L-14 KCTC13497BP.
Figure 3 shows the inhibitory effect of harmful microbial growth of the culture medium of Lactobacillus plantarum L-14 (Cactobacillus plantarum L-14) KCTC13497BP.
4 to 6 shows the inhibitory effect of oral bacteria in the oral cavity of Lactobacillus plantarum L-14 KCTC13497BP.
7 to 10 show the anticancer effect of Lactobacillus plantarum L-14 KCTC13497BP.
11 to 16 show the effect of inhibiting the adipocyte differentiation of Lactobacillus plantarum L-14 KCTC13497BP.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 신규한 유산균 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP에 관한 것이다.The present invention relates to a novel Lactobacillus plantarum L-14 KCTC13497BP.
상기 균주는 한국생명공학연구원 생물자원센터에 2018.3.15자로 기탁되었고, 기탁번호는 KCTC13497BP이다.The strain was deposited with the Korea Biotechnology Research Institute Bioresource Center in 2018.3.15, accession number KCTC13497BP.
기탁기관명 : 한국생명공학연구원Depositary: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC13497BPAccession number: KCTC13497BP
수탁일자 : 20180315Deposit Date: 20180315
상기 균주는 글리코겐 대사능, L-아라비노즈(L-arabinose) 또는 α-메틸-D-만노사이드 대사능을 가질 수 있고, 상기 당들을 모두 대사할 수도 있다.The strain may have glycogen metabolism, L-arabinose or α-methyl-D-mannoside metabolism, and may metabolize all of the sugars.
상기 균주는 생존능이 우수하여, 예를 들면 배양액에서 배양시에 48시간까지 생균수가 감소하지 않을 수 있다.The strain is excellent in viability, for example, may not decrease the number of viable cells up to 48 hours when incubated in the culture.
상기 균주는 번식능이 현저히 빠른 성장 속도를 갖는다. 예를 들면 세대 시간(doubling time)이 20분 이내, 구체적으로는 15분 이내일 수 있다.The strain has a growth rate that is remarkably fast. For example, the doubling time may be within 20 minutes, specifically, within 15 minutes.
상기 균주 또는 그 배양물은 여러 우수한 활성을 가진다. 예를 들어, 유해 미생물 성장 억제 효과, 항염 효과, 항암 효과, 지방세포 분화 억제 효과 등이다. 이에, 상기 균주, 그 배양물 또는 이의 추출물은 항균용 조성물, 의약 조성물, 항염증용 조성물, 건강기능식품 등으로 활용될 수 있다. 상기 배양물은 상기 균주를 포함한 것이거나, 균주는 분리해낸 배양물일 수 있다.The strain or its culture has several excellent activities. For example, they are harmful microbial growth inhibitory effect, anti-inflammatory effect, anticancer effect, adipocyte differentiation inhibitory effect. Thus, the strain, the culture or extract thereof may be utilized as antimicrobial composition, pharmaceutical composition, anti-inflammatory composition, health functional food and the like. The culture may include the strain, or the strain may be an isolated culture.
또한, 본 발명은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양물 또는 그 추출물을 포함하는 조성물에 관한 것이다.The present invention also relates to a composition comprising Lactobacillus plantarum L-14 KCTC13497BP, its culture or its extract.
락토바실러스 플란타럼 L-14 KCTC13497BP 균주의 배양물은 상기 균주를 포함하는 배지(고체, 액체 등), 상기 균주를 배양하고 난 후, 균주가 분리된 배지(고체, 액체 등) 등을 포함할 수 있다.The culture of Lactobacillus plantarum L-14 KCTC13497BP strain may include a medium (solid, liquid, etc.) containing the strain, a medium (solid, liquid, etc.) from which the strain is isolated after culturing the strain. Can be.
상기 균주의 추출물은 예를 들면, 균주의 파쇄물일 수 있으며, 구체적으로 균주의 파쇄물을 동결건조하여 얻어진 파우더 또는 이의 용액 또는 분산액일 수 있다.The extract of the strain may be, for example, a lysate of the strain, specifically, a powder obtained by lyophilizing the lysate of the strain or a solution or dispersion thereof.
상기 조성물은 항균용 조성물일 수 있다.The composition may be an antimicrobial composition.
락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양물 또는 그 추출물은 다양한 유해균에 대해 항균 활성을 나타낼 수 있다.Lactobacillus plantarum L-14 KCTC13497BP, its culture or its extract may exhibit antimicrobial activity against various harmful bacteria.
유해균의 예를 들면, 스타필로코커스 속, 스트렙토코커스 속, 엔테로코커스 속, 슈도모나스 속, 에셰리키아 속 등에 속하는 미생물일 수 있고, 구체적인 예를 들면 스타필로코커스 아우레우스(Staphylococcus aureus, S.aureus), 스트렙토코커스 뮤탄스(Streptococcus mutans, S.mutans), 엔테로코커스 패칼리스(Enterococcus faecalis, E.faecalis), 슈도모나스 애루기노사(Pseudomonas aeruginosa; P.aeruginosa), 에셰리키아 콜라이(Escherichia coli, E.coli) 등일 수 있으나, 이에 제한되는 것은 아니다.Examples of harmful bacteria may be microorganisms belonging to the genus Staphylococcus, Streptococcus, Enterococcus, Pseudomonas, E. coli, etc. Specific examples include Staphylococcus aureus, S. aureus ), Streptococcus mutans (S.mutans), Enterococcus faecalis (E.faecalis), Pseudomonas aeruginosa (P.aeruginosa), Escherichia coli, Escherichia coli .coli) and the like, but is not limited thereto.
또한, 본 발명의 항균용 조성물은 유산균 등의 유익균이나 그외 세균들에 대해서는 별다른 영향을 미치지 않으면서, 상기와 같은 유해균들에 대해 우수한 항균 활성을 나타낼 수 있다.In addition, the antimicrobial composition of the present invention may exhibit excellent antimicrobial activity against the harmful bacteria as described above, without affecting beneficial bacteria such as lactic acid bacteria or other bacteria.
또한, 상기 조성물은 상기 균들이 유발하는 질환의 예방 또는 치료용 의약 조성물일 수 있다.In addition, the composition may be a pharmaceutical composition for the prevention or treatment of diseases caused by the bacteria.
앞서 예시한 스타필로코커스 속, 스트렙토코커스 속, 엔테로코커스 속, 슈도모나스 속, 에셰리키아 속 등의 유해균은 다양한 질병을 유발하는 것으로 알려져 있고, 구체적인 예를 들면 스타필로코커스 아우레우스는 식중독, 패혈증 등을 유발하고, 스트렙토코커스 뮤탄스, 엔테로코커스 패칼리스 등은 충치, 구강질환 등을 유발하고, 슈도모나스 애루기노사는 심내막염, 폐렴, 수막염 등을 유발하는 것으로 알려져 있으므로, 본 발명의 조성물은 상기 질환들의 예방 또는 치료용 의약 조성물로서 활용될 수 있다. 상기 예시한 것 외에도 상기 균들이 유발하는 것으로 알려져 있는 모든 질환에 대해 예방 또는 치료 효과를 나타낼 수 있다.The above-mentioned harmful bacteria such as the genus Staphylococcus, Streptococcus, Enterococcus, Pseudomonas, and Escherichia are known to cause various diseases.For example, Staphylococcus aureus is known as food poisoning and sepsis. And the like, Streptococcus mutans, Enterococcus faecalis and the like cause tooth decay, oral disease, and Pseudomonas aeruginosa are known to cause endocarditis, pneumonia, meningitis, and the like. It can be utilized as a pharmaceutical composition for prophylaxis or treatment. In addition to the above examples, it may have a prophylactic or therapeutic effect on all diseases known to be caused by the bacteria.
또한, 상기 조성물은 항염증용 조성물일 수 있다.In addition, the composition may be an anti-inflammatory composition.
락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양물 또는 그 추출물은 대식세포의 염증성 사이토카인 분비 억제 효과를 가져, 우수한 항염증 효과를 나타낼 수 있다.Lactobacillus plantarum L-14 KCTC13497BP, its culture or its extract may have an inhibitory effect on the inflammatory cytokine secretion of macrophages and may exhibit an excellent anti-inflammatory effect.
또한, 상기 조성물은 염증성 질환의 예방 또는 치료용 의약 조성물일 수 있다.In addition, the composition may be a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
상기 염증성 질환은 예를 들면, 아토피 피부염, 부종, 피부염, 알레르기, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 치질, 통풍, 간직성 척추염, 류마티스 열루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스관절염, 견관절주위염, 건염, 건초염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증 등을 들 수 있으나, 이에 제한되는 것은 아니다.The inflammatory diseases include, for example, atopic dermatitis, edema, dermatitis, allergies, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, spastic spondylitis, Rheumatoid fever, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, and multiple sclerosis. It is not.
또한, 상기 조성물은 암의 예방 또는 치료용 의약 조성물일 수 있다.In addition, the composition may be a pharmaceutical composition for the prevention or treatment of cancer.
락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양물 또는 그 추출물은 우수한 암세포 저하 효과, 이에 따른 종양 크기 감소 효과를 가진다.Lactobacillus plantarum L-14 KCTC13497BP, its culture, or its extract, has excellent cancer cell lowering effect, thereby reducing tumor size.
본 발명의 의약 조성물은 당 분야에 공지된 암에 제한없이 적용될 수 있으며, 예를 들면 뇌종양, 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 식도암, 췌장암, 방광암, 전립선암, 대장암, 두경부암, 피부암, 흑생종, 결장암 및 자궁경부암 등일 수 있으나, 이에 제한되는 것은 아니다. 구체적인 예를 들면 흑생종일 수 있다.The pharmaceutical composition of the present invention can be applied to cancers known in the art without limitation, for example, brain tumors, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, bladder cancer, prostate Cancer, colorectal cancer, head and neck cancer, skin cancer, melanoma, colon cancer, cervical cancer, and the like, but is not limited thereto. Specific examples may be melanoma.
또한, 상기 조성물은 지방 세포 분화 억제용 조성물 또는 항비만용 조성물일 수 있다.In addition, the composition may be a composition for inhibiting adipocyte differentiation or a composition for anti-obesity.
락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양물 또는 그 추출물은 지방전구세포의 지방세포로의 분화, 줄기세포의 지방세포로의 분화를 억제할 수 있다.Lactobacillus plantarum L-14 KCTC13497BP, its culture or its extract can inhibit the differentiation of adipocytes into adipocytes and the differentiation of stem cells into adipocytes.
상기 줄기세포는 예를 들면 배아줄기세포, 성체줄기세포 또는 유도만능줄기세포일 수 있고, 성체줄기세포는 중간엽줄기세포일 수 있으나, 이에 제한되는 것은 아니다.The stem cells may be, for example, embryonic stem cells, adult stem cells or induced pluripotent stem cells, adult stem cells may be mesenchymal stem cells, but is not limited thereto.
본 발명의 조성물은 경구, 비경구, 직장, 국소, 경피, 정맥 내, 근육 내, 복강 내, 피하 등으로 투여될 수 있다. 경구 투여를 위한 제형은 정제, 환제, 연질 및 경질 캅셀제, 과립제, 산제, 세립제, 액제, 유탁제 또는 펠렛제일 수 있으나, 이에 제한되는 것은 아니다. 비경구 투여를 위한 제형은 용액제, 현탁제, 유액제, 겔, 주사제, 점적제, 좌제(坐劑), 패취, 연고제 또는 분무제일 수 있으나, 이에 제한되는 것은 아니다. 상기 제형은 당해 분야의 통상적인 방법에 따라 용이하게 제조될 수 있으며, 계면 활성제, 부형제, 수화제, 유화 촉진제, 현탁제, 삼투압 조절을 위한 염 또는 완충제, 착색제, 향신료, 안정화제, 방부제, 보존제 또는 기타 상용하는 보조제를 추가로 포함할 수 있다.The compositions of the present invention can be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, and the like. Formulations for oral administration may be, but are not limited to, tablets, pills, soft and hard capsules, granules, powders, granules, solutions, emulsions or pellets. Formulations for parenteral administration may be, but are not limited to, solutions, suspensions, emulsions, gels, injections, drops, suppositories, patches, ointments or sprays. The formulations can be readily prepared according to conventional methods in the art and include surfactants, excipients, hydrating agents, emulsifiers, suspending agents, salts or buffers for controlling osmotic pressure, colorants, spices, stabilizers, preservatives, preservatives or Other commercially available auxiliaries may further be included.
본 발명의 조성물의 적용량 또는 투여량은 투여 받을 대상의 연령, 성별, 체중, 병리 상태 및 그 심각도, 투여 경로 또는 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 적용량 결정은 당업자의 수준 내에 있으며, 유효성분의 1일 투여 용량은 예를 들어 0.0001μg/kg/일 내지 5000mg/kg/일, 보다 구체적으로는 0.00001mg/kg/일 내지 15 mg/kg/일이 될 수 있으나, 이에 제한되는 것은 아니다.The dosage or dosage of the composition of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. Application dose determination based on these factors is within the level of those skilled in the art, and the daily dose of the active ingredient is for example 0.0001 μg / kg / day to 5000 mg / kg / day, more specifically 0.00001 mg / kg / day to 15 mg / kg / day, but is not limited thereto.
또한, 본 발명은 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양물 또는 그 추출물을 포함하는 건강기능식품에 관한 것이다.The present invention also relates to a dietary supplement comprising Lactobacillus plantarum L-14 KCTC13497BP, its culture or its extract.
본 발명의 건강기능식품은 앞서의 항균, 항염, 항암, 지방세포 분화 억제 또는 항비만 등의 건강기능성(예방 또는 개선)을 가질 수 있다.The health functional food of the present invention may have health functionalities (prevention or improvement), such as antibacterial, anti-inflammatory, anti-cancer, adipocyte differentiation inhibition or anti-obesity.
본 발명의 건강기능식품의 제형은 특별히 한정되지 않으나, 예를 들어, 정제, 과립제, 환제, 분말제, 캡슐제, 드링크제와 같은 액제, 캐러멜, 겔, 바, 티백 등으로 제형화될 수 있다. 각 제형의 식품 조성물 또는 건강식품 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.The formulation of the health functional food of the present invention is not particularly limited, but may be, for example, formulated into a liquid such as tablets, granules, pills, powders, capsules, drinks, caramels, gels, bars, tea bags, and the like. The food composition or health food composition of each formulation can be appropriately selected and formulated by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredients, depending on the formulation or purpose of use, and synergistic effect when applied simultaneously with other raw materials Can happen.
본 발명의 건강기능식품은 단순 섭취, 음용, 주사 투여, 스프레이 투여 또는 스퀴즈 투여 등 다양한 방법으로 투여될 수 있다.The health functional food of the present invention can be administered by various methods such as simple intake, drinking, injection, spray or squeeze.
본 발명의 건강기능식품에 있어서, 상기 유효 성분의 투여량 결정은 당업자의 수준 내에 있으며, 이의 1일 투여 용량은 예를 들어 0.00001mg/kg/일 내지 15 mg/kg/일이 될 수 있으나, 이에 제한되지 않으며, 투여하고자 하는 대상의 연령, 건강 상태, 합병증 등 다양한 요인에 따라 달라질 수 있다.In the dietary supplement of the present invention, the dosage determination of the active ingredient is within the level of those skilled in the art, and its daily dosage may be, for example, 0.00001 mg / kg / day to 15 mg / kg / day, The present invention is not limited thereto and may vary depending on various factors such as age, health condition, and complications of the subject to be administered.
본 발명의 건강기능식품은 예를 들어, 츄잉껌, 캐러멜 제품, 캔디류, 빙과류, 과자류 등의 각종 식품류, 청량 음료, 미네랄 워터, 알코올 음료 등의 음료 제품, 비타민이나 미네랄 등을 포함한 건강기능성 식품류일 수 있다.The health functional food of the present invention may be, for example, various functional foods such as chewing gum, caramel products, candy, ice cream, confectionery, beverage products such as soft drinks, mineral water, alcoholic drinks, vitamins and minerals, and the like. have.
본 발명의 건강기능식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 증진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 포함할 수 있다.The health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
본 발명의 건강기능식품은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않으나 본 발명의 일측면에 따른 조성물 100 중량부 당 0 내지 약 50 중량부의 범위에서 포함되는 것이 일반적이다.The health functional food of the present invention may include a pulp for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is typically included in the range of 0 to about 50 parts by weight per 100 parts by weight of the composition according to one aspect of the invention.
또한, 본 발명은 상기 락토바실러스 플란타럼 L-14 (Lactobacillus plantarum L-14) KCTC13497BP, 그 배양물 또는 그 추출물을 포함하는 화장료 조성물에 관한 것이다.In addition, the present invention relates to a cosmetic composition comprising the Lactobacillus plantarum L-14 KCTC13497BP, its culture or its extract.
본 발명의 화장료 조성물은 앞서의 항염 활성을 가질 수 있다.The cosmetic composition of the present invention may have the above anti-inflammatory activity.
본 발명의 화장료 조성물은 유효 성분으로서 상기 목서 발효추출물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있으며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.The cosmetic composition of the present invention may include, as an active ingredient, ingredients commonly used in cosmetic compositions in addition to the wood fermentation extract, and include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and flavors, and And a carrier.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제 함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있다. 다만, 이에 반드시 한정되는 것은 아니다. 더욱 상세하게는, 유연 화장수, 수렴 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포옴, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.Cosmetic compositions of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, Formulated as oils, powder foundations, emulsion foundations, wax foundations, sprays, and the like. However, the present invention is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, astringent lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.In case the formulation of the cosmetic composition of the present invention is a paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. This can 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 the carrier component, and in particular, in the case of a spray, additionally chlorofluorohydro Propellant such as carbon, propane / butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜오일, 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, solubilizing agent or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. Fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화이소스테아릴 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, a liquid diluent such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester as carrier components , Microcrystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.
본 발명의 화장료 조성물의 제형이 계면활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, the carrier component may be an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, methyl taurate, sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다. Hereinafter, the present invention will be described in detail with reference to Examples.
실시예Example
1. 미생물학적 특성1. Microbiological Characteristics
(1) 물질 대사(1) metabolism
API 동정 kit를 이용하여 L14의 물질대사 특성을 확인하였다. 유산균 동정을 위해 사용한 API 50 CHL kit의 실험 결과와 kit 매뉴얼에 적혀 있는 표준 L. plantarum의 대사능력 표와 대조하였을 때, L14가 L. plantarum과 가장 가까운 대사 능력을 가지고 있음을 확인하였다(도 1 참조).The metabolic properties of L14 were determined using the API identification kit. Compared with the experimental results of the
API kit의 원리는 50가지 종류의 당이 들어있는 kit strip에 균을 키워 당을 대사할 수 있는지 확인하는 것으로서, 특정 당을 대사할 수 있으면 pH 변화에 의해 색이 보라색에서 노란색으로 바뀌게 된다.The principle of the API kit is to check whether the metabolism can be metabolized by growing bacteria on a kit strip containing 50 kinds of sugars. If a specific sugar can be metabolized, the color changes from purple to yellow due to pH change.
도 1을 참조하면, L14는 A사 L. plantarum ML PRIMETM (LALLEMAND, South Australia, Australia, 이하 LS)과 다르게 Glycogen 대사 능력을 가지고 있고, B사 L. plantarum 락토피 김치유산균 (㈜프로바이오닉, 전라북도, 전주, 이하 LP)과는 다르게 L-arabinose 대사 능력을 가지고 있음을 확인할 수 있다.Referring to FIG. 1, L14 has Glycogen metabolism ability unlike A company L. plantarum ML PRIMETM (LALLEMAND, South Australia, Australia, hereinafter LS), and B company L. plantarum lactopy Kimchi Lactobacillus (Probiotic, Jeollabuk-do) , Jeonju, hereinafter LP) can be seen to have the ability to metabolize L-arabinose.
(2) doubling time 확인(2) Checking doubling time
L14의 doubling time을 분석하기 위해 시간에 따른 배양액의 탁도(O.D.)와, 생균수 변화(colony forming unit; CFU)를 측정하였다(도 2).In order to analyze the doubling time of L14, the turbidity (O.D.) and the number of colony forming units (CFU) of the culture medium were measured over time (FIG. 2).
탁도와 생균수 변화 그래프는 일치하는 향상을 보였으며, 약 48시간까지 생균수가 감소하지 않은 것을 확인하였다.The turbidity and viable cell number change graph showed a consistent improvement, and the viable cell number did not decrease until about 48 hours.
L14의 doubling time을 분석한 결과 약 14.9분으로 확인되었고, 이는 기존에 알려진 유산균 L. acidophilus의 약 70분, 대장균 20분, 녹농균 35분, B. megaterium 25분에 비해 유의적으로 빠른 성장 속도로서, 이는 생산 효율에 긍정적인 영향을 줄 수 있으므로 타 유산균보다 시장성이 있다고 볼 수 있다.As a result of analyzing the doubling time of L14, it was found to be about 14.9 minutes, which is significantly faster than the known L. acidophilus, about 70 minutes,
2. 유해미생물 성장 억제2. Suppresses harmful microbial growth
(1) 황색 포도상구균 (Staphylococcus aureus; S.aureus)(1) Staphylococcus aureus; S.aureus
L14 생균의 식중독 및 패혈증을 유발할 수 있는 황색 포도상구균 (Staphylococcus aureus; S.aureus)에 대한 성장 억제 능력을 확인하였다.The growth inhibitory ability against Staphylococcus aureus (S. aureus), which can cause food poisoning and sepsis, was confirmed.
멸균된 disk paper에 증류수 및 농축시킨 Lactobacillus reuteri(L.reuteri, 양성대조군), L14 생균을 20 μL 접종하여 Staphylococcus aureus (S.aureus) 균이 이미 도말된 고체 배지에 올려 S.aureus 성장 억제 효과를 확인하였다.20 μL of Lactobacillus reuteri (L.reuteri, positive control) and L14 live bacteria were inoculated on sterilized disk paper and placed on a solid medium where Staphylococcus aureus (S.aureus) was already smeared to inhibit S.aureus growth. Confirmed.
구체적으로, L14 및 Lactobacillus reuteri 균은 각각 MRS 액체 배지 5 mL에 S.aureus 균은 TSB 액체 배지 5 mL에 접종하여 37℃, 18시간 배양하였다. 이후, 증류수 100 mL에 시중에서 판매하는 MRS 배지 파우더 3.66 mg, TSB 배지 파우더 1 mg, agar 1.5 mg을 첨가하여 autoclave로 멸균시켰다. 배지가 충분히 식었을 때, 위의 TSB 액체 배지에서 배양된 S.aureus 균을 각각 1 mL씩 첨가하고 충분히 섞어준 뒤 petri dish에 부어 고체 배지를 만들었다. 배양된 L14 균 및 L.reuteri 균을 12,000g, 3 min 원심분리한 뒤 약 500 μL의 배지만 남겨두고 상등액을 제거한 뒤 남은 액체 배지로 재부유 시켰다(1/10 농축). 멸균된 disk paper에 증류수 및 농축 시킨 L14 생균을 20 μL 접종하여 고체 배지에 올리고, 37℃에서 18시간 배양하였다.Specifically, L14 and Lactobacillus reuteri bacteria were inoculated in 5 mL of MRS liquid medium and 5 mL of S. aureus in TSB liquid medium, respectively, and cultured at 37 ° C. for 18 hours. Thereafter, 3.66 mg of commercially available MRS medium powder, 1 mg of TSB medium powder, and 1.5 mg of agar were added to 100 mL of distilled water and sterilized by autoclave. When the medium had cooled sufficiently, 1 mL of each of S. aureus cultured in the above TSB liquid medium was added, mixed well, and poured into a petri dish to make a solid medium. The cultured L14 and L.reuteri bacteria were centrifuged at 12,000 g for 3 min, and left only about 500 μL of medium, and the supernatant was removed and resuspended with the remaining liquid medium (1/10 concentration). 20 μL of the L14 viable cells inoculated with distilled water and concentrated on sterile disk paper was inoculated in a solid medium and incubated at 37 ° C. for 18 hours.
도 3을 참조하면 증류수(음성 대조군)를 접종한 그룹 및 시중에 이미 유해균 억제 효과로 유통되고 있는 생균 L.reuteri(양성 대조군)에 비해 L14 생균이 보다 효과적으로 주위의 S.aureus 균의 성장을 억제하는 것을 확인할 수 있다.Referring to FIG. 3, L14 live bacteria more effectively inhibit the growth of surrounding S. aureus bacteria than the group inoculated with distilled water (negative control) and live bacteria L.reuteri (positive control), which are already distributed in the market with a harmful inhibitory effect. You can see that.
(2) 대장균 (Escherichia coli; E. coli), 황색 포도상구균 (Staphylococcus aureus; S.aureus), 녹농균 (Pseudomonas aeruginosa; P. aeruginosa)(2) Escherichia coli (E. coli), Staphylococcus aureus (S.aureus), Pseudomonas aeruginosa (P. aeruginosa)
L14 생균은 대장균 (Escherichia coli; E. coli), 식중독 및 패혈증을 유발할 수 있는 황색 포도상구균 (Staphylococcus aureus; S.aureus), 녹농균 (Pseudomonas aeruginosa; P. aeruginosa) 성장 억제 능력을 가지고 있다. 다음과 같은 방법으로 항균 효과를 확인하였다.Probiotics L14 have the ability to inhibit the growth of Escherichia coli (E. coli), Staphylococcus aureus (S.aureus), and Pseudomonas aeruginosa (P. aeruginosa), which can cause food poisoning and sepsis. The antimicrobial effect was confirmed by the following method.
L14균은 MRS 액체 배지 5 mL에 E.coli균은 LB 액체 배지 5 mL에, S. aureus, P. aeruginosa는 TSB 액체 배지 5mL에 각각 접종하여 37℃, 18시간 배양하였다. 증류수 100 mL에 시중에서 판매하는 MRS 배지 파우더 3.66 mg, LB 배지 파우더 1 mg, TSB 배지 파우더 1 mg, agar 1.5 mg을 첨가하여 autoclave로 멸균시켰다. 이후, 배지가 충분히 식었을 때, 위의 LB, TSB 액체 배지에서 배양된 E.coli, S. aureus, P. aeruginosa균을 각각 1 mL 씩 첨가하고 충분히 섞어준 뒤 petri dish에 부어 고체 배지를 만들었다. 배양된 L14 균을 12,000g, 3 min 원심분리한 뒤 약 500 μL의 배지만 남겨두고 상등액을 제거한 뒤 남은 액체 배지로 재부유시켰다(1/10 농축). 멸균된 disk paper에 PBS (Negative control; NC), 항생제 ampicillin (1 mg/mL), 농축 시킨 L14 생균을 20 μL 접종하여 고체 배지에 올리고, 37℃에서 18시간 배양하였다.L14 bacteria were inoculated in 5 mL of MRS liquid medium and 5 mL of E. coli in LB liquid medium, and S. aureus and P. aeruginosa were inoculated in 5 mL of TSB liquid medium, respectively. 3.66 mg of commercially available MRS medium powder, 1 mg of LB medium powder, 1 mg of TSB medium powder, and 1.5 mg of agar were added to 100 mL of distilled water and sterilized by autoclave. Then, when the medium was sufficiently cooled, 1 mL of E. coli, S. aureus, and P. aeruginosa cultured in the above LB and TSB liquid medium were added to each of 1 mL, mixed well, and poured into a petri dish to prepare a solid medium. . The cultured L14 bacteria were centrifuged for 12,000 g, 3 min, and left only about 500 μL of medium, and the supernatant was removed and resuspended with the remaining liquid medium (1/10 concentration). PBS (Negative Control; NC), antibiotic ampicillin (1 mg / mL), concentrated L14 viable cells were inoculated on sterile disk paper, inoculated in solid medium, and incubated at 37 ° C. for 18 hours.
도 4를 참조하면, PBS(음성 대조군)을 접종한 그룹과 대비하여 L14 생균은 주위의 E.coli, S.aureus 균의 성장을 억제하는 것을 확인할 수 있다. 또한 기존 실험실 사용 농도 (0.1 mg/mL)의 10배 높은 농도 ampicillin과 같은 유해균 억제 효과가 있음을 확인할 수 있다. 또한 항생제 ampicillin에 대한 효과가 없는 녹농균에 대해서도 L14는 항균 효과를 보임을 확인할 수 있다.Referring to Figure 4, in contrast to the group inoculated with PBS (negative control) L14 live bacteria can be seen to inhibit the growth of the surrounding E. coli, S. aureus bacteria. In addition, it can be confirmed that there is an inhibitory effect against harmful bacteria such as ampicillin, which is 10 times higher than the existing laboratory concentration (0.1 mg / mL). In addition, L14 shows antimicrobial effect against Pseudomonas aeruginosa without the antibiotic ampicillin.
(3) Streptococcus mutans (S.mutans)와 Enterococcus faecalis (E.faecalis)(3) Streptococcus mutans (S.mutans) and Enterococcus faecalis (E.faecalis)
L14 생균은 충치 및 구강질환을 유발한다고 알려진 유해 미생물 Streptococcus mutans (S.mutans)와 Enterococcus faecalis (E.faecalis)에 대한 성장 억제 능력을 확인하였다.L14 probiotics have been shown to inhibit growth against the harmful microorganisms Streptococcus mutans (S.mutans) and Enterococcus faecalis (E.faecalis), which are known to cause caries and oral disease.
멸균된 disk paper에 증류수 및 농축시킨 L14 생균을 20 μL 접종하여 S.mutans와 E.faecalis 균이 이미 도말된 고체 배지에 올려 S.mutans와 E.faecalis 성장 억제 효과를 확인하였다.S.mutans and E.faecalis bacteria were inoculated in 20 μL of distilled water and concentrated L14 viable bacteria on a sterile disk paper to confirm the effect of S.mutans and E.faecalis growth.
구체적으로, L14균은 MRS 액체 배지 5 mL에 S.mutans, E.faecalis 균은 TSB 액체 배지 5 mL에 각각 접종하여 37℃, 18시간 배양하였다. 증류수 100 mL에 시중에서 판매하는 MRS 배지 파우더 3.66 mg, TSB 배지 파우더 1 mg, agar 1.5 mg을 첨가하여 autoclave로 멸균시켰다. 배지가 충분히 식었을 때, 위의 TSB 액체 배지에서 배양된 S.mutans와 E.faecalis 균을 각각 1 mL씩 첨가하고 충분히 섞어준 뒤 petri dish에 부어 고체 배지를 만들었다. 배양된 L14 균을 12,000g, 3min 원심분리한 뒤 약 500 μL의 배지만 남겨두고 상등액을 제거한 뒤 남은 액체 배지로 재부유시켰다(1/10 농축). 멸균된 disk paper에 PBS (Negative control; NC), 항생제 ampicillin (1 mg/mL), 농축 시킨 L14 생균을 20 μL 접종하여 고체 배지에 올리고, 37℃에서 18시간 배양하였다.Specifically, L14 bacteria were inoculated into 5 mL of S.mutans and E. faecalis in 5 mL of MRS liquid medium, and then cultured at 37 ° C. for 18 hours. 3.66 mg of commercially available MRS medium powder, 1 mg of TSB medium powder, and 1.5 mg of agar were added to 100 mL of distilled water and sterilized by autoclave. When the medium had cooled sufficiently, 1 mL of S.mutans and E. faecalis cultured in the TSB liquid medium were added, mixed well, and poured into a petri dish to prepare a solid medium. The cultured L14 bacteria were centrifuged at 12,000g, 3min, and only about 500 μL of the medium was removed, and the supernatant was removed and resuspended with the remaining liquid medium (1/10 concentration). PBS (Negative Control; NC), antibiotic ampicillin (1 mg / mL), concentrated L14 viable cells were inoculated on sterile disk paper, inoculated in solid medium, and incubated at 37 ° C. for 18 hours.
도 5를 참조하면, PBS(음성 대조군)를 접종한 그룹과 대비하여 L14 생균은 주위의 S.mutans와 E.faecalis 균의 성장을 억제하는 것을 확인할 수 있다. 또한 기존 실험실 사용 농도(0.1 mg/mL)의 10배 높은 농도 ampicillin과 같은 유해균 억제 효과가 있음을 확인할 수 있다.Referring to Figure 5, compared with the group inoculated with PBS (negative control) L14 live bacteria can be seen to inhibit the growth of the surrounding S.mutans and E. faecalis bacteria. In addition, it can be confirmed that there is an inhibitory effect on harmful bacteria such as ampicillin, which is 10 times higher than the existing laboratory concentration (0.1 mg / mL).
(4) 황색 포도상구균 (Staphylococcus aureus; S.aureus)과 녹농균 (Pseudomonas aeruginosa; P.aeruginosa)(4) Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa)
L14를 배양한 배양액의 식중독 및 패혈증을 유발할 수 있는 황색 포도상구균 (Staphylococcus aureus; S.aureus)과 녹농균 (Pseudomonas aeruginosa; P.aeruginosa)에 대한 성장 억제 능력을 확인하였다.The growth inhibitory ability of Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa), which may cause food poisoning and sepsis, was confirmed in the culture medium of L14.
배양된 L14 균을 원심분리한 뒤 배양액을 분리하고, 멸균된 disk paper에 증류수 및 L14 배양액 20 μL 접종하여 S.aureus와 P.aeruginosa가 이미 접종된 고체 배지에 올려 S.aureus와 P.aeruginosa 성장 억제 능력을 확인하였다.After centrifugation of cultured L14 bacteria, the culture medium was separated, and 20 μL of distilled water and L14 culture medium were inoculated on sterile disk paper and grown on solid medium already inoculated with S.aureus and P.aeruginosa to grow S.aureus and P.aeruginosa. Inhibition ability was confirmed.
구체적으로, L14균은 MRS 액체 배지 5 mL에 S.aureus, P.aeruginosa 균은 TSB 액체 배지 5 mL에 각각 접종하여 37℃, 18시간 배양하였다. 증류수 100 mL에 시중에서 판매하는 MRS 배지 파우더 3.66 mg, TSB 배지 파우더 1 mg, agar 1.5 mg을 첨가하여 autoclave로 멸균시켰다. 배지가 충분히 식었을 때, 위의 TSB 액체 배지에서 배양된 S.aureus 와 P.aeruginosa 균을 각각 1 mL씩 첨가하고 충분히 섞어준 뒤 petri dish에 부어 고체 배지를 만들었다. 배양된 L14 균을 12,000g, 3 min 원심분리한 뒤 상등액(배양액)만 분리하였다. 멸균된 disk paper에 증류수 및 L14 배양액 20 μL 접종하여 고체 배지에 올리고 37℃에서 18시간 배양하였다.Specifically, L14 bacteria were inoculated into 5 mL of S.aureus and P.aeruginosa in 5 mL of MRS liquid medium, and then incubated at 37 ° C. for 18 hours. 3.66 mg of commercially available MRS medium powder, 1 mg of TSB medium powder, and 1.5 mg of agar were added to 100 mL of distilled water and sterilized by autoclave. When the medium cooled enough, 1 mL of S.aureus and P.aeruginosa cultured in the above TSB liquid medium were added and mixed well, and then poured into a petri dish to prepare a solid medium. 12,000 g of cultured L14 bacteria were centrifuged for 3 min, and only the supernatant (culture solution) was separated. 20 μL of distilled water and L14 culture solution were inoculated on sterilized disk paper, and the resultant was incubated at 37 ° C. for 18 hours.
도 6을 참조하면 증류수(대조군)을 접종한 disk paper와 대비하여 L14 배양액은 주위의 S.aureus 와 P.aeruginosa 균의 성장을 억제하는 것을 확인할 수 있다.Referring to Figure 6 it can be seen that in contrast to the disk paper inoculated with distilled water (control) L14 culture medium inhibits the growth of the surrounding S. aureus and P.aeruginosa bacteria.
(5) 실제 환경에서의 대장균과 황색 포도상구균의 억제 효과(5) Inhibitory Effects of Escherichia Coli and Staphylococcus Aureus in Real Environments
실험실 환경이 아닌 실제적 환경에서의 항균 효과를 확인하기 위해 L14와 유해균 (E. coli와 S. aureus)이 함께 자랄 수 있는 액체 배지에서 둘을 공동 배양하였다.In order to confirm the antimicrobial effect in the real environment, not in the laboratory environment, both were co-cultured in a liquid medium in which L14 and harmful bacteria (E. coli and S. aureus) can grow together.
L14균은 MRS 액체 배지 5 mL에 E.coli균은 LB 액체 배지 5 mL에, S. aureus는 TSB 액체 배지 5mL에 각각 접종하여 37℃, 18시간 배양하였다. 증류수 100 mL에 시중에서 판매하는 MRS 배지 파우더 3.66 mg, LB 배지 파우더 1 mg, TSB 배지 파우더 1 mg를 첨가하여 autoclave로 멸균시켰다. 배지가 충분히 식었을 때, 위의 미리 배양된 E. coli, S. aureus, L14 균을 다양한 CFU로 접종하고, 37℃, 18시간 배양하였다. 이후, E. coli와 S. aureus만 성장하여 색을 띠게 하는 film media에 1mL, MRS agar plate에 100 uL씩 접종하고, 37℃, 18시간 배양하였다.L14 bacteria were inoculated in 5 mL of MRS liquid medium and 5 mL of E. coli in LB liquid medium, and S. aureus in 5 mL of TSB liquid medium, respectively. 3.66 mg of commercially available MRS medium powder, 1 mg of LB medium powder, and 1 mg of TSB medium powder were added to 100 mL of distilled water and sterilized by autoclave. When the medium sufficiently cooled, the above-pre-cultured E. coli, S. aureus, L14 bacteria were inoculated with various CFU, and cultured at 37 ° C. for 18 hours. Thereafter, only 1 mL of E. coli and S. aureus grow and inoculate 100 μL of MRS agar plate on film media, and incubated at 37 ° C. for 18 hours.
도 7을 참조하면, L14를 함께 접종하였을 때, CFU와 관계 없이 E. coli 및 S. aureus 성장 억제효과를 가지고, 특히 L14가 E. coli의 1/100, S. aureus의 1/10을 접종하였을 때도 성장을 억제함을 보임을 확인할 수 있다.Referring to Figure 7, when inoculated together with L14, regardless of the CFU has E. coli and S. aureus growth inhibitory effect, in particular L14 inoculated 1/100 of E. coli, 1/10 of S. aureus It can be seen that even when suppressed growth.
그리고, 초기 접종양과 관계없이 MRS 배지에 비슷한 수의 L14의 colony가 생겨, 적은 수의 L14로도 유해균의 성장을 억제하고 우세해질 수 있음을 확인하였다.Regardless of the initial inoculation amount, a similar number of colonies of L14 were generated in the MRS medium, and it was confirmed that even a small number of L14 could inhibit the growth of harmful bacteria and predominate.
3. 항염 효과3. anti-inflammatory effect
L14는 면역세포로 알려진 마우스 대식세포 (ATCC RAW 264.7)에 직접적으로 작용하여 염증 유도 싸이토카인의 분비를 억제시킴을 확인하였다.L14 was found to act directly on mouse macrophages known as immune cells (ATCC RAW 264.7) to inhibit the secretion of inflammation-induced cytokines.
RAW264.7를 12 well plate에 1.0 × 106 cells/well씩 접종하여 하루 동안 안정화시켰다. 다음날 미리 배양한 L14를 1.0 × 106 CFU/mL로 접종하고 6시간 동안 배양하였다. 새로운 배지로 교체해주면서 염증 유도물질인 내독소 lipopolysaccharide (LPS)를 10~100 ug/mL 접종하였다. 6시간 배양 후, 배양액을 분리하고, 분리된 배양액에 들어있는 염증 싸이토카인을 효소면역측정법 (ELISA)를 통하여 정량화하였다.RAW264.7 was inoculated in a 12 well plate at 1.0 × 10 6 cells / well to stabilize for one day. The next day, the incubated L14 was inoculated at 1.0 × 10 6 CFU / mL and incubated for 6 hours. Inflammatory inducer endotoxin lipopolysaccharide (LPS) was inoculated with 10-100 ug / mL while replacing with fresh medium. After 6 hours of incubation, the cultures were separated and inflammatory cytokines contained in the isolated cultures were quantified by enzyme-linked immunosorbent assay (ELISA).
도 8을 참조하면, L14와 RAW 264.7을 공동 배양하였을 때, LPS에 의한 염증 싸이토카인 IL-6, TNFa, MCP1의 분비가 감소함을 확인할 수 있다.Referring to FIG. 8, when co-culture of L14 and RAW 264.7, the secretion of inflammatory cytokines IL-6, TNFa, and MCP1 by LPS is reduced.
4. 항암 효과4. Anticancer effect
L14 유산균 추출물의 인간 흑색종 세포주 (human malignant melanomas; A375P & A375SP)의 in vitro 및 in vivo에서의 억제 효과를 확인하였다.In vitro and in vivo inhibitory effects of L14 lactobacillus extract on human malignant melanomas (A375P & A375SP) were confirmed.
L14 유산균 추출물은 다음과 같이 제작하였다. 먼저, L14 유산균을 멸균된 MRS 액체 배지 10 mL에 접종하여 35℃, 18시간 배양하였다(전배양). 배양된 L14 유산균을 멸균된 MRS 액체 배지 1 L에 접종하여 35℃, 18시간 배양하였다(본배양). 본배양된 L14 균을 4℃, 12,000g, 15 min 원심분리한 뒤 상등액 제거하고 L14 균만 얻었다. PBS로 재부유 시킨 후, 4℃, 12,000g, 15 min 원심분리한 뒤 상등액 제거하는 공정을 2회 반복하였다. 멸균 증류수로 재부유시킨 후, 4℃, 12,000g, 15 min 원심분리한 뒤 상등액 제거한 뒤, 100 mL 증류수로 재부유시켰다. 초음파 파쇄기를 이용하여 증류수로 재부유시킨 L14 유산균을 충분히 초음파 파쇄시켰다. 4℃, 12,000g, 15 min 원심분리한 뒤 상등액만 얻어내어 0.45 μm 필터로 걸러주었고, 얻어진 파쇄 추출물을 3일간 동결건조시켰다. 동결건조 된 L14 유산균 추출물 파우더를 PBS에 적정 농도로 녹인 뒤, 0.45 μm 필터로 걸러서 추출물을 얻었다.L14 lactic acid bacteria extract was prepared as follows. First, L14 lactic acid bacteria were inoculated in 10 mL of sterile MRS liquid medium and incubated at 35 ° C. for 18 hours (preculture). Cultured L14 lactic acid bacteria were inoculated in 1 L of sterile MRS liquid medium and cultured at 35 ° C. for 18 hours (main culture). The cultured L14 bacteria were centrifuged at 4 ° C., 12,000 g for 15 min, and then the supernatant was removed. Only L14 bacteria were obtained. After resuspending with PBS, centrifugation was performed at 4 ° C., 12,000 g for 15 min, and the supernatant was removed twice. After resuspended in sterile distilled water, the supernatant was removed after centrifugation at 4 ℃, 12,000g, 15 min, and resuspended in 100 mL distilled water. L14 lactic acid bacteria resuspended in distilled water were sufficiently ultrasonically crushed using an ultrasonic crusher. After centrifugation at 4 ° C., 12,000 g and 15 min, only the supernatant was collected and filtered through a 0.45 μm filter, and the obtained crushed extract was lyophilized for 3 days. The lyophilized L14 lactic acid bacteria extract powder was dissolved in PBS at an appropriate concentration, and then filtered through a 0.45 μm filter to obtain an extract.
(1) in vitro(1) in vitro
L14 유산균 추출물의 흑색종 세포주에 대한 in vitro에서의 항암 효과를 다음과 같이 확인하였다. 인간 피부 섬유아세포 (human dermal fibroblast; HDF)와 인간 흑색종 세포주 A375P와 A375SM을 12 well plate에는 10,000 cells/well로, 96 well plate에는 1,000 cells/well로 접종하여 24시간 배양하였다. 배지를 갈아줌과 동시에 L14 유산균 추출물을 농도를 달리하여 함께 처리하였다. 3일 후, L14 유산균 추출물에 의한 항암 효과를 12 well plate에서는 현미경을 통해 관찰하고, 96 well plate에서는 WST-1 viability assay를 통해 확인하였다.The anticancer effect of L14 lactic acid bacteria extract on melanoma cell line in vitro was confirmed as follows. Human dermal fibroblast (HDF) and human melanoma cell lines A375P and A375SM were inoculated at 10,000 cells / well in 12 well plates and 1,000 cells / well in 96 well plates and incubated for 24 hours. At the same time the medium was changed and L14 lactic acid bacteria extract was treated with different concentrations. After 3 days, the anticancer effect of L14 lactobacillus extract was observed through a microscope in a 12 well plate and confirmed by a WST-1 viability assay in a 96 well plate.
도 9를 참조하면 L14 유산균 추출물의 처리 농도가 증가하였을 때, 인간 피부 섬유아세포 (HDF)에는 영향이 없는 반면, A375P와 A375SM에서는 뚜렷한 항암효과가 나타나는 것을 확인할 수 있고, 도 10을 참조하면 L14 유산균 추출물의 처리 농도가 증가하였을 때 A375P와 A375SM에서는 뚜렷한 항암효과가 나타남을 확인할 수 있다.Referring to FIG. 9, when the treatment concentration of the L14 lactobacillus extract was increased, it did not affect human skin fibroblasts (HDF), whereas A375P and A375SM showed a clear anticancer effect. Referring to FIG. As the concentration of the extract increased, A375P and A375SM showed distinct anticancer effects.
(2) in vivo(2) in vivo
L14 유산균 추출물의 인간 흑색종 세포주 A375SM의 in vivo에서의 항암 능력을 확인하였다.The anticancer activity of the human melanoma cell line A375SM of L14 lactic acid bacteria extract was confirmed in vivo.
면역이 결핍된 nude mouse에 A375SM 세포주를 피하에 접종함으로써 종양을 인위적으로 생성하였고, 그룹을 무작위로 나누어 PBS와 L14 유산균 추출물을 2일 간격으로 22일간 복강주사 하였다. 도 11을 참조하면, 대조군에 비해 L14 유산균 추출물을 처리한 그룹 nude mouse의 종양 크기가 작음을 확인할 수 있다.Tumors were artificially generated by subcutaneously inoculating subcutaneous A375SM cell lines into immune-deficient nude mice. Groups were randomly divided into 22-day intraperitoneal injections of PBS and L14 lactobacillus extracts. Referring to Figure 11, it can be seen that the tumor size of the group nude mice treated with L14 lactic acid bacteria extract compared to the control.
In vivo에서의 L14 유산균 추출물의 항암효과를 정량적으로 나타내기 위해 절제한 종양의 무게와 부피를 측정하여 분석하였다. 도 12를 참조하면, 유의적으로 L14 유산균 추출물에 의해 종양의 무게와 부피가 감소함을 확인할 수 있다. (Control group n;6, L14-treated group n;9)In order to quantitatively demonstrate the anticancer effect of L14 lactic acid bacteria extract in vivo, the weight and volume of the excised tumors were measured and analyzed. Referring to Figure 12, it can be seen that significantly reduced tumor weight and volume by the L14 lactic acid bacteria extract. (Control group n; 6, L14-treated group n; 9)
5. 지방세포 분화 억제 효과5. Inhibitory effect of adipocyte differentiation
L14 유산균 추출물은 마우스 지방전구세포주 (3T3-L1)와 인간 중간엽줄기세포 (human mesenchymal stem cells; hMSCs)의 지방세포로의 분화 억제 효과를 확인하였다.L14 Lactobacillus extract was confirmed to inhibit the differentiation of mouse adipose progenitor cell lines (3T3-L1) and human mesenchymal stem cells (hMSCs) into adipocytes.
(1) L14 유산균 추출물의 마우스 지방전구세포주 (3T3-L1)의 지방세포 분화 억제 효과를 다음과 같이 확인하였다.(1) The effect of inhibiting the adipocyte differentiation of the mouse adipocyte cell line (3T3-L1) of L14 lactic acid bacteria extract was confirmed as follows.
3T3-L1을 24 well plate에 100,000 cells/well씩 접종하고 수 일 동안 배양하였다. Confluency가 95~100%에 도달하였을 때, 지방세포 분화 유도 배지 (MDI medium)로 배지를 갈아줌과 동시에 L14 유산균 추출물을 농도별로 함께 처리하였다(Day 0). 2일 후(Day 2), 지방세포 분화 유도 배지로 배지를 갈아주며 L14 유산균 추출물을 다시 처리하였다. Day 4부터 L14 유산균 추출물과 함께 지방세포 분화 유지 배지로 갈아주고 Day 12까지 2일 간격으로 배지를 갈아주었다. 지방세포로 분화가 완료된 Day 12의 세포를 이용하여 Oil Red O 염색, 유전자 분석, Triacylglycerol (TAG) assay 등을 통해 지방세포 분화 억제 효과를 확인하였다.3T3-L1 was inoculated in 100,000 cells / well in a 24 well plate and incubated for several days. When the confluency reached 95 to 100%, the medium was changed to an adipocyte differentiation induction medium (MDI medium) and L14 lactic acid bacteria extract was treated with each concentration (Day 0). After 2 days (Day 2), the medium was changed to adipocyte differentiation induction medium and retreated with L14 lactic acid bacteria extract.
도 13을 참조하면, L14 유산균 추출물의 처리 농도가 증가함에 따라 억제효과가 커지는 것을 확인할 수 있다.Referring to Figure 13, it can be seen that the inhibitory effect increases as the treatment concentration of L14 lactic acid bacteria extract increases.
그리고 L14 유산균 추출물의 지방세포 억제효과가 세포에 대한 독성 때문인지 확인하기 위해 WST-1 viability assay를 통해 확인하였으나, 도 14를 참조하면 고농도에서도 3T3-L1에 부정적인 영향이 없음을 확인할 수 있다.And to confirm whether the fat cell inhibitory effect of L14 lactic acid bacteria extract is due to the toxicity to the cell, but confirmed through WST-1 viability assay, referring to Figure 14 it can be seen that there is no negative effect on 3T3-L1 even at high concentrations.
그리고, 염색된 지방세포의 oil red o를 isopropanol로 다시 녹여내어 그 함량을 500 nm 파장의 O.D. 값을 측정하여 분석하였다. 도 15를 참조하면, L14 유산균 추출물의 처리 농도가 증가함에 따라 oil red o로 염색된 정도가 유의적으로 감소한 것을 확인할 수 있다.In addition, the oil red o of the dyed fat cells was dissolved again with isopropanol and the content of O.D. The value was measured and analyzed. Referring to Figure 15, it can be seen that as the treatment concentration of L14 lactic acid bacteria extract increased the degree of staining with oil red o significantly.
그리고, TAG assay를 통하여 지방세포의 지방이 축적된 양을 정확하게 정량하여 L14 유산균 추출물의 지방세포 분화 억제 효과를 재확인하였다. 도 16을 참조하면, L14 유산균 추출물의 처리 농도가 증가함에 따라 지방세포 분화 억제 효과가 증가하는 것을 확인할 수 있다.Accurately quantified the amount of fat accumulation of adipocytes by TAG assay, and reconfirmed the effect of inhibiting adipocyte differentiation of L14 lactic acid bacteria extract. Referring to Figure 16, it can be seen that the effect of inhibiting adipocyte differentiation increases as the treatment concentration of L14 lactic acid bacteria extract increases.
그리고, L14 유산균 추출물의 마우스 지방세포 분화 억제 효과를 유전자 수준에서 확인하였다. 지방세포 분화 핵심 유전자 마커인 C/EBPα, PPARγ, FABP4의 발현을 확인하였다. 도 17을 참조하면, L14 유산균 추출물 처리 농도가 높아질수록 대조군(control)에 비해 세 유전자의 발현양이 현저히 감소함을 확인할 수 있다. NC: negative control (지방세포로 분화되지 않은 일반 3T3-L1 세포).In addition, the mouse adipocyte differentiation inhibitory effect of L14 lactic acid bacteria extract was confirmed at the gene level. The expression of adipocyte differentiation key gene markers C / EBPa, PPARγ, FABP4 was confirmed. Referring to FIG. 17, it can be seen that the expression level of the three genes is significantly reduced as compared with the control (control) as the concentration of L14 lactic acid bacteria extract treatment. NC: negative control (normal 3T3-L1 cells that did not differentiate into adipocytes).
또한, 타사 유산균 L. plantarum (LS, LP)과 L14 추출물의 마우스 지방세포 분화 억제 효과를 유전자 수준에서 비교하기 위해 지방세포 분화 핵심 유전자 마커인 PPARγ, C/EBPα, FABP4의 발현을 확인하였다. 도 18을 참조하면, L14 추출물이 LS, LP 추출물보다 분자적 수준에서 지방세포 분화 관련 유전자의 발현을 효과적으로 억제함을 확인할 수 있다(도 13의 결과와 비교).In addition, the expression of PPARγ, C / EBPa, and FABP4, which are key gene markers for adipocyte differentiation, were compared to compare the mouse adipocyte differentiation inhibitory effects of L. plantarum (LS, LP) and L14 extracts at the gene level. Referring to FIG. 18, it can be seen that L14 extract effectively inhibits the expression of adipocyte differentiation-related genes on a molecular level than LS and LP extracts (compare with the results of FIG. 13).
(2) L14 유산균 추출물의 인간 중간엽줄기세포(hMSCs)의 지방세포 분화 억제 효과를 다음과 같이 확인하였다.(2) The inhibitory effect of L14 lactic acid bacteria extract on adipocyte differentiation of human mesenchymal stem cells (hMSCs) was confirmed as follows.
hMSCs을 24 well plate에 100,000 cells/well 씩 접종하고 수 일 동안 배양하였다. Confluency가 95~100%에 도달하였을 때, 지방세포 분화 유도 배지 (MDI medium)로 배지를 갈아줌과 동시에 L14 유산균 추출물을 농도별로 함께 처리하였다(Day 0). 2일 후 (Day 2), 지방세포 분화 유도 배지로 배지를 갈아주며 L14 유산균 추출물을 다시 처리하였다. Day 4부터 L14 유산균 추출물과 함께 지방세포 분화 유지 배지로 갈아주고 Day 7까지 2일 간격으로 배지를 갈아주었다. 지방세포로 분화가 완료된 Day 7의 세포를 이용하여 oil red o 염색 방법을 통해 지방세포 분화 억제 효과를 확인하였다.hMSCs were inoculated at 100,000 cells / well in 24 well plates and incubated for several days. When the confluency reached 95 to 100%, the medium was changed to an adipocyte differentiation induction medium (MDI medium) and L14 lactic acid bacteria extract was treated with each concentration (Day 0). After 2 days (Day 2), the medium was changed to adipocyte differentiation induction medium and retreated with L14 lactic acid bacteria extract. From
도 19를 참조하면, L14 유산균 추출물 처리 농도가 높아질수록 지방세포 분화 억제 효과가 증가함을 확인할 수 있다.Referring to Figure 19, it can be seen that the higher the L14 Lactobacillus extract treatment concentration increases the effect of adipocyte differentiation.
Claims (8)
Lactobacillus plantarum L-14 KCTC13497BP.
The strain according to claim 1, which has glycogen metabolism and L-arabinose metabolism.
스타필로코커스 아우레우스(Staphylococcus aureus, S.aureus), 스트렙토코커스 뮤탄스(Streptococcus mutans, S.mutans), 엔테로코커스 패칼리스(Enterococcus faecalis, E.faecalis), 슈도모나스 애루기노사(Pseudomonas aeruginosa; P.aeruginosa) 또는 에셰리키아 콜라이(Escherichia coli, E.coli)에 대한 항균용 조성물.
Including the strain of claim 1, its culture or strain extract,
Staphylococcus aureus (S.aureus), Streptococcus mutans (S.mutans), Enterococcus faecalis (E.faecalis), Pseudomonas aeruginosa; .aeruginosa) or antimicrobial composition against Escherichia coli (E. coli).
상기 감염성 질환은 식중독, 패혈증, 충치, 심내막염, 페렴 및 수막염으로 이루어진 군에서 선택된 1종 이상인 조성물.
A pharmaceutical composition for preventing or treating an infectious disease comprising the strain of claim 1, a culture thereof, or a strain extract,
The infectious disease is at least one composition selected from the group consisting of food poisoning, sepsis, caries, endocarditis, pneumonia and meningitis.
Anti-inflammatory composition comprising the strain of claim 1, its culture or strain extract.
A pharmaceutical composition for preventing or treating melanoma, comprising the strain of claim 1, a culture thereof, or a strain extract.
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