KR20220077603A - Composition for preventing, treating or improving bone diseases containing extracellular vesicles derived from Lactobacillus sakei CVL001 strain culture medium - Google Patents
Composition for preventing, treating or improving bone diseases containing extracellular vesicles derived from Lactobacillus sakei CVL001 strain culture medium Download PDFInfo
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- KR20220077603A KR20220077603A KR1020200166710A KR20200166710A KR20220077603A KR 20220077603 A KR20220077603 A KR 20220077603A KR 1020200166710 A KR1020200166710 A KR 1020200166710A KR 20200166710 A KR20200166710 A KR 20200166710A KR 20220077603 A KR20220077603 A KR 20220077603A
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Abstract
본 발명은 락토바실러스 사케이(Lactobacillus sakei) 배양액 유래 세포외소포체(Extracellular Vesicle; EV)를 포함하는 골 질환 예방, 치료 또는 개선용 조성물에 관한 것으로서, 락토바실러스 사케이 CVL-001 균주의 배양액으로부터 분리된 세포외소포체가 파골세포 분화 억제 효과를 나타내므로, 이를 효과적으로 골 질환의 예방, 치료 또는 개선에 이용할 수 있다.The present invention relates to a composition for preventing, treating, or improving bone diseases, including Lactobacillus sakei culture-derived extracellular vesicles (EV), and is isolated from the culture solution of Lactobacillus sakei CVL-001 strain. Since the endoplasmic reticulum exhibits an inhibitory effect on osteoclast differentiation, it can be effectively used for prevention, treatment or improvement of bone diseases.
Description
본 발명은 락토바실러스 사케이(Lactobacillus sakei) 배양액 유래 세포외소포체를 포함하는 골 질환 예방, 치료 또는 개선용 조성물에 관한 것으로서, 더욱 상세하게는 락토바실러스 사케이 CVL-001 균주의 배양액으로부터 분리된 세포외소포체를 포함하는 골 질환 예방, 치료 또는 개선용 조성물에 관한 것이다.The present invention relates to a composition for preventing, treating or ameliorating a bone disease comprising an extracellular vesicle derived from a Lactobacillus sakei culture medium, and more particularly, cells isolated from the culture medium of the Lactobacillus sakei CVL-001 strain. It relates to a composition for preventing, treating or ameliorating a bone disease comprising an exosome.
갱년기의 여성에게서는 여러 폐경 증후들이 나타나는데, 특히 에스트로겐의 감소로 인하여 뼈에서 칼슘이 빠져나가 뼈의 질량이 감소하고, 구멍이 많아지며, 골 손실이 증가함으로 인하여 골다공증 질환의 발병률이 높아지게 되었다. 폐경기 이후 후기변화는 증상이 나타나기까지 다소 시간이 걸릴 수 있으므로 문제를 쉽게 자각하지 못하는 경우가 많다. 보건복지부 국민 건강 통계치에 따르면 30세 이상에서 골다공증 유병률이 남성에게서는 1%, 여성에게서는 9%로 여성 유병률이 남성대비 9배나 높게 나타난 것으로 보고되었다.In menopausal women, various symptoms of menopause appear. In particular, due to the decrease in estrogen, calcium is lost from the bones, the bone mass is reduced, holes are increased, and the incidence of osteoporosis disease is increased due to the increase in bone loss. Post-menopausal changes may take some time for symptoms to appear, so the problem is often not easily recognized. According to the National Health Statistics of the Ministry of Health and Welfare, the prevalence of osteoporosis among men over 30 years of age was 1% in men and 9% in women, and it was reported that the prevalence in women was 9 times higher than in men.
골다공증에 의해 야기되는 골절은 주된 건강 문제를 구성하며 건강 관리 시스템 상에 막대한 경제적 부담을 준다. 골다공증으로 인한 골절의 위험은 서양에서 높으며(여성에서 약 50% 및 남성에서 약 20%), 골절은 현저한 치사율 및 이환율과 연관된다. 피질골(cortical bone)은 신체에서 뼈의 약 80%를 구성하며, 여러 연구들은 피질골이 뼈 강도의 주요 결정인자이며, 이에 따라 골절 민감성의 주요 결정인자임을 보여주었다. 65세 이후에 뼈 손실은 해면질골이 아니라 주로 피질골에서의 손실에 기인한다.Fractures caused by osteoporosis constitute a major health problem and place a huge economic burden on the health care system. The risk of fractures due to osteoporosis is high in the West (about 50% in women and about 20% in men), and fractures are associated with significant mortality and morbidity. Cortical bone constitutes about 80% of bone in the body, and several studies have shown that cortical bone is a major determinant of bone strength and, thus, a major determinant of fracture susceptibility. Bone loss after age 65 is primarily attributable to the loss of cortical bone and not cancellous bone.
골격은 뼈 형성 골아세포(OBs) 및 뼈흡수 파골 세포(OCLs)에 의해 리모델링된다. 대식세포 콜로니 자극인자(MCSF)는 OCLs 전구세포의 증식 및 생존을 증가시킬뿐만 아니라 OCL에서 핵인자-κB(RANK)의 리셉터 활성제의 발현을 상향조절한다. 이는 RANK 리간드(RANKL)가 바인딩하고 OCL 형성을 이끄는 시그널링 캐스케이드를 개시하도록 한다. RANKL의 영향은 RANKL에 대한 유인 리셉터인 오스테오프로테게린(OPG)에 의해 저해될 수 있다.The skeleton is remodeled by osteogenic osteoblasts (OBs) and bone resorbing osteoclasts (OCLs). Macrophage colony stimulating factor (MCSF) not only increases proliferation and survival of OCLs progenitor cells, but also upregulates the expression of receptor activators of nuclear factor-κB (RANK) in OCLs. This allows the RANK ligand (RANKL) to bind and initiate a signaling cascade leading to OCL formation. The effect of RANKL can be inhibited by osteoprotegerin (OPG), an attractive receptor for RANKL.
최근에, 건강 및 질병 모두를 위해 장내 미생물(gut microbiota; 이하 GM)의 중요성이 집중적으로 연구되어 왔다. GM은 총괄적으로 인간 게놈보다 150배 더 많은 유전자를 함유하는 엄청난 양의 박테리아로 구성된다. 이는 출생시 얻어지며, 구별되는 실재(distinct entity)임에도 불구하고, 분명히 인간 게놈과 함께 공진화하고, 다양한 방식으로 이의 숙주와 소통하고 영향을 미치는 다세포 생물로 간주될 수 있다.In recent years, the importance of gut microbiota (hereinafter GM) for both health and disease has been intensively studied. GM is made up of huge numbers of bacteria that collectively contain 150 times more genes than the human genome. Although it is acquired at birth and is a distinct entity, it can be considered as a multicellular organism that apparently co-evolves with the human genome and communicates and affects its host in various ways.
GM의 구성은 음식 및 항생제 치료와 같은 다수의 환경적 요인에 의해 조절된다. 장내 미생물에 의해 생산된 분자들은 유익할 수도 있고 해로울 수도 있으며, 장 내의 내분비 세포, 장 신경계, 장 투과성 및 면역 시스템에 영향을 미치는 것으로 알려져 있다. 동요된 미생물 구성은 크론병, 궤양성 대장염, 류마티스성 관절염, 다발성 경화증, 당뇨, 음식 알레르기, 습진 및 천식뿐만 아니라 비만 및 대사 증후군을 포함하여 장 내외에서 다양한 염증 조건에 연루되는 것으로 상정되어 왔다.The composition of GM is regulated by a number of environmental factors, such as diet and antibiotic treatment. Molecules produced by the gut microbiota can be beneficial or harmful and are known to affect the endocrine cells in the gut, the enteric nervous system, gut permeability and the immune system. Perturbed microbial makeup has been postulated to be implicated in a variety of inflammatory conditions in and out of the gut, including Crohn's disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, diabetes, food allergies, eczema and asthma, as well as obesity and metabolic syndrome.
한편, 락토바실러스 속 미생물은 동형 또는 이형발효를 하는 유산간균으로서, 유제품이나 채소의 발효과정에서 흔히 볼 수 있는 균이다. 최근 락토바실러스를 포함한 다양한 발효식품 유래 미생물에서 분비하는 세포외소포체에 대하여 건강기능식품, 식품 첨가제 등으로 개발하고자 하는 효능 평가 연구가 활발히 진행되고 있다. 세포외소포체는 다양한 생물학적 활성을 나타내고, 세포들 사이에 유전 물질과 단백질을 옮기면서 세포 간 상호작용에 중요한 역할을 한다.On the other hand, microorganisms of the genus Lactobacillus are lactobacilli that perform homozygous or heterozygous fermentation, and are commonly seen in the fermentation process of dairy products or vegetables. Recently, efficacy evaluation studies to develop extracellular vesicles secreted by microorganisms derived from various fermented foods, including Lactobacillus, as health functional foods, food additives, etc. are being actively conducted. The extracellular vesicles exhibit a variety of biological activities and play an important role in intercellular interactions by transferring genetic material and proteins between cells.
이에 본 발명자들은 락토바실러스 사케이(Lactobacillus sakei) 미생물에 대한 연구를 계속한 결과, 김치로부터 분리한 락토바실러스 사케이 균주 유래 세포외소포체(Extracellular Vesicle; EV)의 파골세포 분화 억제 효과가 월등히 우수한 것을 확인하였다.Accordingly, the present inventors continued their research on Lactobacillus sakei microbes, and as a result, it was found that the Lactobacillus sakei strain-derived Extracellular Vesicle (EV) isolated from kimchi had an exceptionally excellent inhibitory effect on osteoclast differentiation. Confirmed.
이에, 본 발명의 목적은 락토바실러스 사케이 배양액 유래 세포외소포체를 포함하는 골 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating bone disease, comprising the Lactobacillus Sake culture medium-derived extracellular vesicles.
본 발명의 다른 목적은 락토바실러스 사케이 배양액 유래 세포외소포체를 포함하는 골 질환 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for improving bone disease, comprising the Lactobacillus Sake culture medium-derived extracellular vesicles.
본 발명의 또 다른 목적은 락토바실러스 사케이 배양액 유래 세포외소포체의골 질환 예방, 치료 또는 개선 용도에 관한 것이다.Another object of the present invention relates to the use of the Lactobacillus Sake culture-derived extracellular vesicles for preventing, treating or improving bone disease.
본 발명은 락토바실러스 사케이(Lactobacillus sakei) 배양액 유래 세포외소포체(Extracellular Vesicle; EV)를 포함하는 골 질환 예방, 치료 또는 개선용 조성물에 관한 것으로, 본 발명에 따른 조성물은 우수한 파골세포 분화 억제 효과를 나타낸다. The present invention relates to a composition for preventing, treating or ameliorating a bone disease comprising Lactobacillus sakei culture medium-derived Extracellular Vesicle (EV), and the composition according to the present invention has an excellent inhibitory effect on osteoclast differentiation indicates
본 발명자들은 락토바실러스 사케이 CVL-001 균주의 배양액으로부터 세포외소포체를 분리하였고, 이를 마우스 대식세포에 처리함으로써 파골세포 분화를 억제하는 효과가 있음을 확인하였다. The present inventors isolated the extracellular vesicles from the culture medium of the Lactobacillus sacai CVL-001 strain, and confirmed that there is an effect of inhibiting osteoclast differentiation by treatment with mouse macrophages.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 락토바실러스 사케이 배양액 유래 세포외소포체를 포함하는 골 질환 예방 또는 치료용 약학적 조성물이다.One aspect of the present invention is a pharmaceutical composition for preventing or treating bone disease, comprising the Lactobacillus Sake culture medium-derived extracellular vesicles.
본 명세서상의 용어 “세포외소포체”는 세포 간 정보교환을 위해 모든 세포들이 외부 환경으로 분비하는 나노 크기의 소포체를 의미한다. 단백질, 지질, 핵산, 대사물질(metabolites) 등 생물학적 활성을 보이는 다양한 물질을 포함하고 있고, 이들의 유래하는 세포들의 상태를 반영하고 있다.As used herein, the term “extracellular vesicle” refers to a nano-sized ER secreted by all cells to the external environment for information exchange between cells. It contains various substances showing biological activity, such as proteins, lipids, nucleic acids, and metabolites, and reflects the state of cells from which they are derived.
본 발명에 있어서 락토바실러스 사케이는 수탁번호 KCTC13816BP로 기탁된 락토바실러스 사케이 CVL-001 균주인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the Lactobacillus saccharis may be the Lactobacillus sacillus CVL-001 strain deposited under the accession number KCTC13816BP, but is not limited thereto.
본 발명에 있어서 골 질환은 골다공증(osteoporosis), 뼈전이암(bone metastatic cancer), 골연화증(osteomalacia), 구루병, 섬유성 골염, 무형성 골질환, 대사성 골질환 및 치주질환으로 이루어진 군으로부터 선택되는 것일 수 있고, 예를 들어, 골다공증인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the bone disease may be selected from the group consisting of osteoporosis, bone metastatic cancer, osteomalacia, rickets, fibrous osteomyelitis, aplastic bone disease, metabolic bone disease, and periodontal disease. and, for example, may be osteoporosis, but is not limited thereto.
본 발명에 있어서 배양액은 락토바실러스 사케이 CVL-001 균주를 배양하고, 균주를 제거한 후 수득된 배양 상층액, 이의 농축물, 이의 분획물 또는 이의 동결건조물인 것일 수 있다.In the present invention, the culture medium may be a culture supernatant obtained after culturing the Lactobacillus Sakei CVL-001 strain and removing the strain, a concentrate thereof, a fraction thereof, or a freeze-dried product thereof.
본 발명에 있어서 배양액은 락토바실러스 사케이 CVL-001 균주를 12시간 내지 30시간, 12시간 내지 24시간, 18시간 내지 30시간 또는 18시간 내지 24시간, 예를 들어, 22시간 내지 26시간 동안 배양하여 수득한 것일 수 있으나, 이에 한정되는 것은 아니다. 배양 시간은 24시간을 기점으로 하여 그 이상 배양하는 것이 배양 시간 의존적으로 파골세포 분화 억제 활성에 대하여 현저한 상승효과를 나타내는 것은 아니다.In the present invention, the culture medium is the Lactobacillus Sake CVL-001 strain for 12 hours to 30 hours, 12 hours to 24 hours, 18 hours to 30 hours or 18 hours to 24 hours, for example, culturing for 22 hours to 26 hours. It may be obtained by doing, but is not limited thereto. As for the culture time, culturing longer than 24 hours does not show a significant synergistic effect on the osteoclast differentiation inhibitory activity in a culture time-dependent manner.
본 발명에 있어서 배양액은 pH 6.5 내지 8.5, 6.5 내지 8.0, 6.5 내지 7.5, 7.0 내지 8.5 또는 7.0 내지 8.0, 예를 들어, pH 7.0 내지 7.5인 것일 수 있으나, 이에 한정되는 것은 아니다. 배양액에서는 유산균이 자라면서 젖산 등의 대사체를 생산하게 되어 산성화될 수 있고, 이는 세포 배양에 적절하지 않은 pH 범위에 해당한다. 이러한 점에 근거하여, 세포 배양에 있어서 가장 적절한 pH 값은 7.4이다.In the present invention, the culture medium may have a pH of 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5 or 7.0 to 8.0, for example, pH 7.0 to 7.5, but is not limited thereto. In the culture medium, as lactic acid bacteria grow, they produce metabolites such as lactic acid and may be acidified, which corresponds to a pH range that is not suitable for cell culture. Based on this point, the most appropriate pH value for cell culture is 7.4.
본 발명의 약학적 조성물은 락토바실러스 사케이 CVL-001 균주 배양액의 약학적 유효량 및/또는 약학적으로 허용되는 담체를 포함하는 약학적 조성물로 이용될 수 있다.The pharmaceutical composition of the present invention may be used as a pharmaceutical composition comprising a pharmaceutically effective amount of a culture medium of Lactobacillus Sake CVL-001 strain and/or a pharmaceutically acceptable carrier.
본 명세서에서 용어 "약학적 유효량"은 상술한 락토바실러스 사케이 CVL-001 균주 배양액의 효능 또는 활성을 달성하는 데 충분한 양을 의미한다.As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the above-described Lactobacillus Sakei CVL-001 strain culture.
본 발명의 약학적 조성물에 포함되는 약학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
본 발명에 따른 약학적 조성물은 인간을 포함하는 포유동물에 다양한 경로로 투여될 수 있다. 투여 방식은 통상적으로 사용되는 모든 방식일 수 있으며, 예컨대, 경구, 피부, 정맥, 근육, 피하 등의 경로로 투여될 수 있으며, 바람직하게는 경구로 투여될 수 있다. The pharmaceutical composition according to the present invention may be administered to mammals including humans by various routes. The administration method may be any method commonly used, for example, may be administered by routes such as oral, skin, intravenous, intramuscular, subcutaneous, etc., and preferably orally.
본 발명의 약학적 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.
본 발명의 약학적 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제, 캅셀제 또는 젤(예컨대, 하이드로젤) 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains. Or it can be prepared by introducing into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, or gel (eg, hydrogel), and may additionally include a dispersant or stabilizer. .
본 발명의 다른 양태는 락토바실러스 사케이 배양액 유래 세포외소포체를 포함하는 골 질환 개선용 식품 조성물이다.Another aspect of the present invention is a food composition for improving bone disease comprising an extracellular vesicle derived from a Lactobacillus Sake culture medium.
본 발명에 있어서 락토바실러스 사케이는 수탁번호 KCTC13816BP로 기탁된 락토바실러스 사케이 CVL-001 균주인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the Lactobacillus saccharis may be the Lactobacillus sacillus CVL-001 strain deposited under the accession number KCTC13816BP, but is not limited thereto.
본 발명에 있어서 골 질환은 골다공증, 뼈전이암, 골연화증, 구루병, 섬유성 골염, 무형성 골질환, 대사성 골질환 및 치주질환으로 이루어진 군으로부터 선택되는 것일 수 있고, 예를 들어, 골다공증인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the bone disease may be selected from the group consisting of osteoporosis, bone metastasis, osteomalacia, rickets, fibrous osteitis, aplastic bone disease, metabolic bone disease, and periodontal disease, for example, it may be osteoporosis. , but is not limited thereto.
본 발명에 있어서 배양액은 락토바실러스 사케이 CVL-001 균주를 배양하고, 균주를 제거한 후 수득된 배양 상층액, 이의 농축물, 이의 분획물 또는 이의 동결건조물인 것일 수 있다.In the present invention, the culture medium may be a culture supernatant obtained after culturing the Lactobacillus Sakei CVL-001 strain and removing the strain, a concentrate thereof, a fraction thereof, or a freeze-dried product thereof.
본 발명에 있어서 배양액은 락토바실러스 사케이 CVL-001 균주를 12시간 내지 30시간, 12시간 내지 24시간, 18시간 내지 30시간 또는 18시간 내지 24시간, 예를 들어, 22시간 내지 26시간 동안 배양하여 수득한 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culture medium is the Lactobacillus Sake CVL-001 strain for 12 hours to 30 hours, 12 hours to 24 hours, 18 hours to 30 hours or 18 hours to 24 hours, for example, culturing for 22 hours to 26 hours. It may be obtained by doing, but is not limited thereto.
본 발명에 있어서 배양액은 pH 6.5 내지 8.5, 6.5 내지 8.0, 6.5 내지 7.5, 7.0 내지 8.5 또는 7.0 내지 8.0, 예를 들어, pH 7.0 내지 7.5인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the culture medium may have a pH of 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5 or 7.0 to 8.0, for example, pH 7.0 to 7.5, but is not limited thereto.
본 발명의 식품 조성물을 식품 첨가물로 사용할 경우, 상기 식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 식품 조성물은 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가될 수 있다.When the food composition of the present invention is used as a food additive, the 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. In general, in the production of food or beverage, the food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 초콜 릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 식품을 모두 포함한다.There is no particular limitation on the type of the food. Examples of foods to which the above substances 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, drinks , alcoholic beverages and vitamin complexes, and includes all foods in a conventional sense.
상기 음료는 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. As the above-mentioned natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. . The ratio of the natural carbohydrate may be appropriately determined by the selection of those skilled in the art.
상기 외에 본 발명의 식품 조성물은 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, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 식품 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다.In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.
본 발명은 락토바실러스 사케이(Lactobacillus sakei) 배양액 유래 세포외소포체(Extracellular Vesicle; EV)를 포함하는 골 질환 예방, 치료 또는 개선용 조성물에 관한 것으로서, 락토바실러스 사케이 CVL-001 균주의 배양액으로부터 분리된 세포외소포체가 파골세포 분화 억제 효과를 나타내므로, 이를 효과적으로 골 질환의 예방, 치료 또는 개선에 이용할 수 있다.The present invention relates to a composition for preventing, treating, or improving bone diseases, including Lactobacillus sakei culture-derived extracellular vesicles (EV), and is isolated from the culture solution of Lactobacillus sakei CVL-001 strain. Since the endoplasmic reticulum exhibits an inhibitory effect on osteoclast differentiation, it can be effectively used for prevention, treatment or improvement of bone diseases.
도 1은 본 발명의 실시예에 따라 락토바실러스 사케이(Lactobacillus sakei) 배양액으로부터 분리된 세포외소포체(Extracellular Vesicle; EV)의 NTA(nanoparticle tracking analysis) 결과 그래프이다.
도 2는 본 발명의 실시예에 따라 락토바실러스 사케이 배양액으로부터 분리된 세포외소포체의 투과전자현미경(transmission electron microscope; TEM) 사진이다.
도 3은 본 발명의 실시예에 따라 락토바실러스 사케이 배양액으로부터 세포외소포체의 농도별 처리에 따른 파골세포 분화 억제 효과를 나타내는 사진이다.
도 4는 본 발명의 실시예에 따라 락토바실러스 사케이 배양액으로부터 분리된 세포외소포체의 처리에 따른 파골세포 분화 관련 유전자의 발현량 측정 그래프이다.1 is a graph showing the results of NTA (nanoparticle tracking analysis) of extracellular vesicles (EV) isolated from Lactobacillus sakei culture according to an embodiment of the present invention.
FIG. 2 is a transmission electron microscope (TEM) photograph of an extracellular vesicle isolated from a Lactobacillus saccharis culture medium according to an embodiment of the present invention.
Figure 3 is a photograph showing the osteoclast differentiation inhibitory effect according to the treatment of each concentration of the extracellular vesicles from the Lactobacillus Sakei culture medium according to an embodiment of the present invention.
Figure 4 is a graph showing the expression level of osteoclast differentiation-related genes according to the treatment of the extracellular vesicles isolated from the Lactobacillus sacki culture medium according to an embodiment of the present invention.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량)%, 고체/액체는 (중량/부피)%, 그리고 액체/액체는 (부피/부피)%이다.Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% solid/solid, (weight/volume)%, and (weight/volume)% for solid/solid, and Liquid/liquid is (vol/vol) %.
실시예 1: 락토바실러스 사케이 배양액 유래 세포외소포체 분리Example 1: Isolation of extracellular vesicles derived from Lactobacillus Sake culture medium
세포에 처리하기 위한 배양액을 제조하기 위하여 alpha-MEM(Alpha Modification of Eagle's Minimum Essential Media) 배지에 수탁번호 KCTC13816BP로 기탁된 락토바실러스 사케이(Lactobacillus sakei) CVL-001 균주를 24h 동안 배양하고 원심분리 후 상층액을 분리하여 pH 7.4로 맞추었다. 이로부터 세포외소포체(extracellular vesicle; EV)를 분리하였다.To prepare a culture solution for treatment with cells, the Lactobacillus sakei CVL-001 strain deposited with the accession number KCTC13816BP in alpha-MEM (Alpha Modification of Eagle's Minimum Essential Media) medium was cultured for 24 h and centrifuged. The supernatant was separated and adjusted to pH 7.4. From this, an extracellular vesicle (EV) was isolated.
구체적으로, 배양된 락토바실러스 사케이 CVL-001의 배양액은 하기 단계에 따라 순차적으로 여과되었다:Specifically, the culture solution of the cultured Lactobacillus Sakei CVL-001 was sequentially filtered according to the following steps:
1) 락토바실러스 사케이 CVL-001의 배양액을 2000 g, 10분 동안 원심분리하여 상등액을 수득함으로써 배양액으로부터 균주를 분리 및 제거하였다.1) The strain was separated and removed from the culture solution by centrifuging the culture solution of Lactobacillus Sakei CVL-001 at 2000 g for 10 minutes to obtain a supernatant.
2) 상기 1)에서 원심분리된 배양액을 0.2 ㎛ 바틀 탑 필터(Bottle Top Filter)를 통해 여과함으로써 배양액의 순도를 증가시켰다.2) The purity of the culture solution was increased by filtering the culture solution centrifuged in 1) through a 0.2 μm bottle top filter.
3) 접선유동여과(tangential flow filtration; TFF) 방법을 수행하여 배양액을 10배로 농축하였다.3) A tangential flow filtration (TFF) method was performed to concentrate the
4) 농축된 배양액을 0.22 ㎛ 실린지 필터를 통해 여과하였다.4) The concentrated culture solution was filtered through a 0.22 μm syringe filter.
5) 여과한 배양액을 4℃에서 150,000 g, 3시간 동안 초고속원심분리하여 상등액을 제거하고 남은 세포외소포체 펠렛(pellet)을 수득하였다.5) The supernatant was removed by ultra-high speed centrifugation of the filtered culture solution at 150,000 g at 4° C. for 3 hours, and the remaining extracellular vesicle pellets were obtained.
최종적으로 수득한 세포외소포체를 완충용액(phosphate buffered saline; PBS)에 재현탁시켜 준비하였다.Finally, the obtained extracellular vesicles were prepared by resuspending in a buffer solution (phosphate buffered saline; PBS).
실시예 2: 김치로부터 분리된 락토바실러스 사케이 배양액 유래 세포외소포체 확인 Example 2: Identification of extracellular vesicles derived from Lactobacillus Sakei culture medium isolated from kimchi
분리한 유산균 유래 세포외소포체는 NTA(nanoparticle tracking analysis)와 투과전자현미경(transmission electron microscope; TEM)으로 크기와 형태를 분석하였다.The size and shape of the isolated lactic acid bacteria-derived extracellular vesicles were analyzed by NTA (nanoparticle tracking analysis) and transmission electron microscope (TEM).
도 1 및 2에서 확인할 수 있듯이, 세포외소포체는 대체로 구형을 이루고 직경이 약 150 nm 인 것을 확인하였다. 측정된 직경의 범위는 10 내지 300 nm인 것으로 나타났고, 평균 직경은 150 nm인 것으로 분석되었다.As can be seen in FIGS. 1 and 2 , it was confirmed that the extracellular vesicles were generally spherical and had a diameter of about 150 nm. The measured diameter was found to be in the range of 10 to 300 nm, and the average diameter was analyzed to be 150 nm.
실시예 3: 김치로부터 분리된 락토바실러스 사케이 배양액 유래 세포외소포체의 파골세포 분화에 대한 억제 평가 (TRAP staining) 확인Example 3: Confirmation of inhibition evaluation (TRAP staining) for osteoclast differentiation of extracellular vesicles derived from Lactobacillus Sake culture isolated from kimchi
마우스 대식세포를 12 웰 플레이트(well plate)에 2 x 105/well의 밀도로 24h 동안 배양하였으며 10% 소태아혈청(fatal bovine serum; FBS), 1% 포스파티딜세린(phosphatidylserine; PS), 25 ng/ml의 대식세포집락자극인자(macrophage colony stimulating factor; M-CSF)가 첨가된 배지로 교체해준 후 세포외소포체 0.5, 5.0 및 50.0 x 108 particles/ml을 2h 동안 처리하였다. 이후 100 ng/ml의 RANKL(Receptor activator of nuclear factor kappa-Βligand)을 처리하여 24h 동안 반응시켰고, 위와 같은 방법으로 4일 동안 분화시켰다.Mouse macrophages were cultured for 24 h at a density of 2 x 10 5 /well in a 12-well plate, 10% fetal bovine serum (FBS), 1% phosphatidylserine (PS), 25 ng After replacing the medium with /ml macrophage colony stimulating factor (M-CSF) added, 0.5, 5.0 and 50.0 x 10 8 particles/ml of extracellular vesicles were treated for 2h. Then, 100 ng/ml of RANKL (Receptor activator of nuclear factor kappa-Βligand) was treated and reacted for 24 h, and differentiated for 4 days in the same manner as above.
대식세포에 RANKL을 처리하면 RANK에 결합하며 타타르산염 내성 산성 포스타페이즈(tartrate resistance acid phosphatase; TRAP) 양성 세포로 분화하게 된다. 이후 파골세포의 세포 화학적 표지효소인 TRAP에 발색성 기질을 첨가하여 핵을 염색하였고 핵이 3개 이상으로 다핵화된 세포를 관찰하고 이미지화하였다.When macrophages are treated with RANKL, they bind to RANK and differentiate into tartrate resistance acid phosphatase (TRAP)-positive cells. After that, the nucleus was stained by adding a chromogenic substrate to TRAP, a cytochemical marker enzyme of osteoclasts, and cells with three or more nuclei were observed and imaged.
표 1 및 도 3에서 확인할 수 있듯이, 대식세포에 RANKL을 처리했을 때 파골세포의 분화가 증가하였으며 세포외소포체 처리에 의해서 파골세포의 수가 농도의존적으로 현저히 감소된 것을 확인하였다. 이것으로 보아 락토바실러스 사케이 배양액 유래 세포외소포체가 대식세포로부터 파골세포로의 분화를 억제시킴을 확인할 수 있었다.As can be seen in Table 1 and Figure 3, when macrophages were treated with RANKL, the differentiation of osteoclasts was increased, and it was confirmed that the number of osteoclasts was significantly reduced in a concentration-dependent manner by treatment with the extracellular vesicles. From this, it was confirmed that the extracellular vesicles derived from the Lactobacillus sacillus culture medium inhibited the differentiation of macrophages into osteoclasts.
실시예 4: 김치로부터 분리된 락토바실러스 사케이 배양액 유래 세포외소포체의 파골세포 분화에 대한 억제 평가 (Real-time PCR) 확인Example 4: Confirmation of inhibition evaluation (Real-time PCR) for osteoclast differentiation of extracellular vesicles derived from Lactobacillus Sake culture isolated from kimchi
마우스 대식세포를 12 웰 플레이트에 2 x 105/well의 밀도로 24h 동안 배양하였으며 10% FBS, 1% PS, 25 ng/ml의 M-CSF가 첨가된 배지로 교체해준 후 세포외소포체 0.5, 5.0 및 50.0 x 108 particles/ml을 2h 동안 처리하였다. 이후 100 ng/ml의 RANKL을 처리하여 24h 동안 반응시켰고, 위와 같은 방법으로 4일 동안 분화시켰다.Mouse macrophages were cultured in a 12-well plate at a density of 2 x 10 5 /well for 24 h and replaced with a medium supplemented with 10% FBS, 1% PS, and 25 ng/ml M-CSF, and then 0.5, 5.0 and 50.0 x 10 8 particles/ml were treated for 2 h. Thereafter, 100 ng/ml of RANKL was treated, reacted for 24 h, and differentiated for 4 days in the same manner as above.
분화시킨 후 TRIzol 용액을 이용하여 세포내 RNA를 분리하고, RNA 정량값을 토대로 RT 프리믹스(premix)를 이용하여 cDNA를 합성하였다. 합성된 cDNA는 프라이머(primer)를 이용하여 Real time PCR을 통해 증폭시켰다. 사용된 프라이머 Mouse TRAP, DC-STAMP, Cathepsin K는 하기 표 2로 나타내었으며, 대조군(Control) 유전자인 Actin과 비교하여 상대적 양을 비교하였다.After differentiation, intracellular RNA was isolated using TRIzol solution, and cDNA was synthesized using RT premix based on the RNA quantitative value. The synthesized cDNA was amplified through real-time PCR using a primer. The used primers Mouse TRAP, DC-STAMP, and Cathepsin K are shown in Table 2 below, and their relative amounts were compared with Actin, a control gene.
일반적으로 대식세포에 RANKL을 처리하면 RANK에 결합하여 TRAP 양성인 다핵화된 파골세포로 분화된다. 또한 파골세포로 분화하는데 중요한 전사 인자인 NFATc1을 활성화시키고 파골세포 형성(osteoclastogenesis)에 관여하는 TRAP, Cathepsine K, DC-STAMP의 발현을 증가시킨다.In general, when macrophages are treated with RANKL, they bind to RANK and differentiate into TRAP-positive multinucleated osteoclasts. It also activates NFATc1, a transcription factor important for differentiation into osteoclasts, and increases the expression of TRAP, Cathepsine K, and DC-STAMP involved in osteoclastogenesis.
Real-time PCR 분석 결과는 하기 표 3에 나타내었다.Real-time PCR analysis results are shown in Table 3 below.
표 3 및 도 4에서 확인할 수 있듯이, RANKL 처리에 의하여 증가된 TRAP, DC-STAMP, Cathepsin K의 발현이 락토바실러스 사케이 배양액 유래 세포외소포체에 의해 감소하였다. 이것으로 보아 락토바실러스 사케이 배양액 유래 세포외소포체가 대식세포로부터 파골세포로의 분화 시 증가되는 유전자 발현을 억제시킴으로써 파골세포 분화를 억제시킴을 확인할 수 있었다.As can be seen in Table 3 and Figure 4, the expression of TRAP, DC-STAMP, and Cathepsin K increased by RANKL treatment was decreased by the Lactobacillus sacchar culture-derived extracellular vesicles. From this, it was confirmed that the Lactobacillus sacillus-derived extracellular vesicles inhibited osteoclast differentiation by suppressing the gene expression that is increased during differentiation from macrophages to osteoclasts.
<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for preventing, treating or improving bone diseases containing extracellular vesicles derived from Lactobacillus sakei CVL001 strain culture medium <130> PN200341 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TRAP F primer <400> 1 ctggagtgca cgatgccagc gaca 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TRAP R primer <400> 2 tccgtgctcg gcgatggacc aga 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DC-STAMP F primer <400> 3 ccaaggagtc gtccatgatt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DC-STAMP R primer <400> 4 ggctgctttg atcgtttctc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cathepsin K F primer <400> 5 ggccaactca agaagaaaac 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cathepsin K R primer <400> 6 gtgcttgctt cccttctgg 19 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for preventing, treating or improving bone diseases containing extracellular vesicles derived from Lactobacillus sakei CVL001 strain culture medium <130> PN200341 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TRAP F primer <400> 1 ctggagtgca cgatgccagc gaca 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TRAP R primer <400> 2 tccgtgctcg gcgatggacc aga 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DC-STAMP F primer <400> 3 ccaaggagtc gtccatgatt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DC-STAMP R primer <400> 4 ggctgctttg atcgtttctc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cathepsin K F primer <400> 5 ggccaactca agaagaaaac 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cathepsin K R primer <400> 6 gtgcttgctt cccttctgg 19
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