KR102486858B1 - NOVEL STRAIN OF Leuconostoc mesenteroides AND USE THEREOF - Google Patents
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- KR102486858B1 KR102486858B1 KR1020220061895A KR20220061895A KR102486858B1 KR 102486858 B1 KR102486858 B1 KR 102486858B1 KR 1020220061895 A KR1020220061895 A KR 1020220061895A KR 20220061895 A KR20220061895 A KR 20220061895A KR 102486858 B1 KR102486858 B1 KR 102486858B1
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- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
본 발명은 신경 세포 사멸에 대한 보호 효과를 가지는 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P) 및 이의 용도에 관한 것이다. The present invention relates to a Leuconostoc mesenteroides strain (KCCM 12614P) having a protective effect against neuronal cell death and uses thereof.
Description
본 발명은 신경 세포 사멸에 대한 보호 효과를 가지는 신규 류코노스톡 메센테로이데스 균주 및 이의 용도에 관한 것이다. The present invention relates to a novel Leuconostoc mesenteroides strain having a protective effect against neuronal cell death and uses thereof.
프로바이오틱스는 적절한 양을 섭취시, 숙주의 건강에 도움을 주는 것으로 정의되고 있다. 상업용 프로바이오틱스로 Lactobacillus, Leuconostoc, Pediococcus, Bifidobacterium 등의 균주들이 주로 사용되고 있다. 이들 균주들은 항산화능, 항염증능, 콜레스테롤 저하능, 항당뇨 효능들이 알려져 있으며, 이러한 기능성은 균주마다 차이가 있는 것으로 보고된다. Probiotics are defined as those that, when ingested in adequate amounts, benefit the health of the host. Strains such as Lactobacillus , Leuconostoc , Pediococcus, and Bifidobacterium are mainly used as commercial probiotics. These strains are known to have antioxidant, anti-inflammatory, cholesterol-lowering, and anti-diabetic effects, and it is reported that these functionalities differ from strain to strain.
장 환경은 뇌에 영향을 끼치는 것으로 보고되고 있으며, 이들은 양방향 시그널을 나타내는 것으로 보고되고 있다. 신경, 호르몬, 면역 조절 등에 의해, 초조함, 우울증, 치매와 같은 뇌 건강 등에 영향을 끼치는 것으로 알려져 있다. The intestinal environment is reported to affect the brain, and they are reported to exhibit bidirectional signals. It is known to affect brain health such as nervousness, depression, and dementia by regulating nerves, hormones, and immunity.
종래 연구에 따르면, 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주가 발효에 사용된 예는 보고된바 있으나, 이의 신경 세포 사멸에 대한 보호 효과를 직접적으로 확인한 예는 보고된바 없다. According to prior studies, Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) Examples of strains used for fermentation have been reported, but no examples directly confirming their protective effect against neuronal cell death have been reported.
본 발명은 신경 세포 사멸에 대한 보호 효과를 가지는 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P) 등을 제공하고자 한다. The present invention is to provide a Leuconostoc mesenteroides strain (KCCM 12614P) and the like having a protective effect against neuronal cell death.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다. However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
본 발명은 신경 세포 사멸에 대한 보호 효과를 가지는 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 제공한다.The present invention provides a Leuconostoc mesenteroides strain (KCCM 12614P) having a protective effect against neuronal cell death.
상기 신경 세포 사멸은 산화적 스트레스에 의해 유도될 수 있다. The neuronal cell death can be induced by oxidative stress.
상기 신경 세포 사멸에 대한 보호 효과는 상기 균주 및 장세포 간 반응을 통해 생산된 대사산물의 작용으로 인한 것일 수 있다. The protective effect against neuronal cell death may be due to the action of metabolites produced through the reaction between the strain and enterocytes.
상기 균주는 하기 ⅰ) 또는 ⅱ)의 특징을 추가로 가질 수 있다: The strain may further have the following characteristics i) or ii):
ⅰ) 내산성, 내담즙성 및 장부착능;i) acid resistance, bile resistance and intestinal adhesion;
ⅱ) α-글루코시다아제 및 β-글루코시다아제 생산능. ii) α-glucosidase and β-glucosidase production ability.
상기 균주는 서열번호 1의 16S rRNA 염기서열을 포함할 수 있다. The strain may include the 16S rRNA nucleotide sequence of SEQ ID NO: 1.
본 발명의 일 구현예로, 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 유효성분으로 포함하는 퇴행성 신경질환 예방 또는 치료용 약학 조성물을 제공한다. In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a Leuconostoc mesenteroides strain (KCCM 12614P) as an active ingredient.
상기 퇴행성 신경질환은 알츠하이머 질환, 파킨슨 질환, 루게릭 질환, 헌팅턴 질환, 근위축성 측석 경화증, 다발성 경화증, 면역계이상 뇌기능 부전, 진행성 신경퇴행질환, 대사성 뇌질환, 니만-픽병, 뇌 허혈 및 뇌출혈로 인한 치매로 이루어진 군으로부터 선택된 하나 이상일 수 있다.The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system dysfunction, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, cerebral ischemia and cerebral hemorrhage. It may be one or more selected from the group consisting of dementia.
본 발명의 다른 구현예로, 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 유효성분으로 포함하는 퇴행성 신경질환 예방 또는 개선용 건강기능식품을 제공한다.In another embodiment of the present invention, Leuconostoc mesenteroides Des ( Leuconostoc mesenteroides ) Provides a health functional food for preventing or improving neurodegenerative diseases comprising a strain (KCCM 12614P) as an active ingredient.
본 발명의 또 다른 구현예로, 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 유효성분으로 포함하는 프로바이오틱 조성물을 제공한다.In another embodiment of the present invention, a probiotic composition comprising a Leuconostoc mesenteroides strain (KCCM 12614P) as an active ingredient is provided.
본 발명의 또 다른 구현예로, 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 포함하는 발효용 스타터를 제공한다. In another embodiment of the present invention, a starter for fermentation comprising a Leuconostoc mesenteroides strain (KCCM 12614P) is provided.
본 발명의 또 다른 구현예로, 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 포함하는 발효식품용 종균 조성물을 제공한다.In another embodiment of the present invention, Leuconostoc mesenteroides Des ( Leuconostoc mesenteroides ) Provides a seed composition for fermented food containing a strain (KCCM 12614P).
본 발명의 또 다른 구현예로, 상기 발효용 스타터 또는 상기 발효식품용 종균 조성물을 첨가하여 제조된 발효식품을 제공한다. In another embodiment of the present invention, a fermented food prepared by adding the starter for fermentation or the seed composition for fermented food is provided.
본 발명에 따른 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)는 안정적이고 안전한 프로바이오틱 특성(특히, 우수한 내담즙성 및 장부착능)을 가지면서, 신경 세포 사멸(특히, 산화적 스트레스에 의해 유도된 신경 세포 사멸)에 대한 보호 효과를 가지는바, 프로바이오틱 조성물 뿐만 아니라, 퇴행성 신경질환(특히, 알츠하이머 질환) 예방, 개선 또는 치료용 조성물로 활용될 수 있다. 더욱이, 상기 균주의 특성상 발효용 스타터 또는 발효식품용 종균 조성물로도 유용하게 활용될 수 있다. Leuconostoc mesenteroides strain (KCCM 12614P) according to the present invention has stable and safe probiotic properties (in particular, excellent bile resistance and intestinal adhesion), while neuronal cell death (in particular, oxidation) Neuronal cell death induced by enemy stress), it can be used as a probiotic composition as well as a composition for preventing, improving or treating neurodegenerative diseases (in particular, Alzheimer's disease). Moreover, due to the nature of the strain, it can be usefully used as a starter for fermentation or a seed composition for fermented foods.
도 1은 신규 Leuconostoc mesenteroides H40 균주(KCCM 12614P)의 계통도를 보여주는 그림이다.
도 2는 실시예 1 및 비교예 1-2에서 분리된 균주의 장부착능을 나타낸 그래프이다.
도 3은 실시예 1 및 비교예 1-2에서 분리된 균주 유래 CM의 인간 유래 신경 세포의 보호 효과를 나타낸 그래프이다((A) H2O2, (B) NaAsO2).
도 4는 실시예 1에서 분리된 균주 적용 요거트 유래 CM의 인간 유래 신경 세포의 보호 효과를 나타낸 사진(×100) 및 그래프이다((A) H2O2, (B) NaAsO2, (C) H2O2, (D) NaAsO2).
도 5는 실시예 1 및 비교예 1-2에서 분리된 균주 사균체의 인간 유래 대장암 세포의 BDNF 발현량을 나타낸 그래프이다.
도 6은 실시예 1에서 분리된 균주 적용 요거트의 인간 유래 대장암 세포의 BDNF 발현량을 나타낸 그래프이다.
도 7은 실시예 1 및 비교예 1-2에서 분리된 균주 유래 CM의 인간 유래 신경 세포의 Bax/Bcl-2 발현량 비율을 나타낸 그래프이다((A) H2O2, (B) NaAsO2).
도 8은 실시예 1에서 분리된 균주 적용 요거트 유래 CM의 인간 유래 신경 세포의 Bax/Bcl-2 발현량 비율 및 BDNF 발현량을 나타낸 그래프이다((A) H2O2, (B) NaAsO2).1 is a novel Leuconostoc mesenteroides H40 It is a figure showing the phylogeny of the strain (KCCM 12614P).
Figure 2 is a graph showing the intestinal attachment ability of the strains isolated in Example 1 and Comparative Example 1-2.
Figure 3 is a graph showing the protective effect of human-derived nerve cells of CM derived from strains isolated in Example 1 and Comparative Examples 1-2 ((A) H 2 O 2 , (B) NaAsO 2 ).
4 is a photograph (×100) and a graph showing the protective effect of yogurt-derived CM applied to the strain isolated in Example 1 on human-derived nerve cells ((A) H 2 O 2 , (B) NaAsO 2 , (C) H 2 O 2 , (D) NaAsO 2 ).
Figure 5 is a graph showing the BDNF expression level of human-derived colorectal cancer cells of dead cells of the strain isolated in Example 1 and Comparative Example 1-2.
6 is a graph showing the amount of BDNF expression in human-derived colorectal cancer cells of yogurt applied with the strain isolated in Example 1.
7 is a graph showing the Bax/Bcl-2 expression level ratio of human-derived neural cells in CM derived from strains isolated in Example 1 and Comparative Examples 1-2 ((A) H 2 O 2 , (B) NaAsO 2 ).
8 is a graph showing the Bax/Bcl-2 expression level ratio and BDNF expression level of human-derived neural cells of the strain-applied yogurt-derived CM isolated in Example 1 ((A) H 2 O 2 , (B) NaAsO 2 ).
본 발명자들은 프로바이오틱 특성을 가지는 Leuconostoc mesenteroides 균주 중에서도, 특히, 내담즙성 및 장부착능이 우수한 Leuconostoc mesenteroides H40 균주를 분리 및 동정하고, 이를 이용한 발효식품으로서 요거트를 제조하여, 신경 세포 사멸에 대한 보호 효과를 평가함으로써, 본 발명을 완성하였다. Among the Leuconostoc mesenteroides strains having probiotic properties, the present inventors, in particular, Leuconostoc mesenteroides H40, which has excellent bile resistance and intestinal adhesion, The present invention was completed by isolating and identifying the strain, preparing yogurt as a fermented food using the strain, and evaluating the protective effect against neuronal cell death.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 신경 세포 사멸에 대한 보호 효과를 가지는 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 제공한다. The present invention provides a Leuconostoc mesenteroides strain (KCCM 12614P) having a protective effect against neuronal cell death.
상기 균주(KCCM 12614P)는 류코노스톡 메센테로이데스의 아종으로 볼 수 있다. 종래 알려진 류코노스톡 메센테로이데스는 젖산균으로 김치의 발효 초기에 주로 생육하는데, 이는 김치의 발효에 종균으로서 사용될 수도 있다. 상기 균주(KCCM 12614P)는 류코노스톡 메센테로이데스 중에서도, 특히, 내담즙성 및 장부착능이 우수하고, 신경 세포 사멸에 대한 보호 효과가 우수한 특징을 가진다. The strain (KCCM 12614P) can be seen as a subspecies of Leuconostoc mesenteroides. Conventionally known Leuconostoc mesenteroides is a lactic acid bacterium that grows mainly in the early stage of fermentation of kimchi, which may be used as a starter for fermentation of kimchi. The strain (KCCM 12614P) has excellent characteristics among Leuconostoc mesenteroides, particularly, excellent bile resistance and intestinal adhesion, and excellent protective effect against neuronal cell death.
상기 균주(KCCM 12614P)의 계통도는 도 1에 도시한 바와 같으며, 배추김치로부터 분리된 것일 수 있다. 상기 분리된 균주를 동정하기 위해 16S rRNA 서열을 분석한 결과, 상기 균주(KCCM 12614P)는 서열번호 1의 16S rRNA 염기서열을 가지는 것으로 확인되며, 2019년 1O월 25일자로 한국미생물보존센터에 기탁하여 수탁번호 KCCM 12614P를 부여받았다.The phylogeny of the strain (KCCM 12614P) is as shown in FIG. 1, and may be isolated from cabbage kimchi. As a result of analyzing the 16S rRNA sequence to identify the isolated strain, the strain (KCCM 12614P) was confirmed to have the 16S rRNA sequence of SEQ ID NO: 1, and was deposited with the Korea Microorganism Conservation Center on October 25, 2019 and received the accession number KCCM 12614P.
먼저, 상기 균주(KCCM 12614P)는 신경 세포 사멸에 대한 보호 효과를 가지는 것을 특징으로 한다. First, the strain (KCCM 12614P) is characterized by having a protective effect against neuronal cell death.
구체적으로, 상기 신경세포 사멸에 대한 보호 효과는 상기 균주(KCCM 12614P)의 작용으로 인한 것일 수도 있다. 이러한 효과는 상기 균주(KCCM 12614P)를 장세포에 처리했을 때, BDNF 발현양이 증가하는 것을 통해 확인될 수 있다. 이때, BDNF의 낮은 분비는 인간의 기억과 해마의 기능에 영향을 미칠 수 있으며, 세포 사멸이 BDNF 분비에 영향을 미칠 수 있다. Specifically, the protective effect against neuronal cell death may be due to the action of the strain (KCCM 12614P). This effect can be confirmed through the increase in the amount of BDNF expression when the strain (KCCM 12614P) is treated with intestinal cells. At this time, low secretion of BDNF may affect human memory and function of the hippocampus, and cell death may affect BDNF secretion.
그밖에, 상기 신경세포 사멸에 대한 보호 효과는 상기 균주(KCCM 12614P) 및 장세포 간 반응을 통해 생산된 대사산물의 작용으로 인한 것일 수 있다. 상기 균주(KCCM 12614P)는 장세포와 반응을 하여 대사산물을 생산하는데, 이와 같이 생산된 대사산물은 신경 세포에 대해 세포 독성을 유발하지 않으면서, 산화적 스트레스(H2O2 및 NaAsO2)에 대한 세포 보호 효과를 가질 수 있다. 이러한 효과는 이와 같이 생산된 대사산물을 신경 세포에 처리했을 때, 신경 세포에 대해 세포 독성을 유발하지 않으면서, 산화적 스트레스(H2O2 및 NaAsO2)에 의해 증가된 Bax/Bcl-2 발현양 비율을 크게 감소시키는 것을 통해 확인될 수 있다. 이때, 세포사멸과 관련된 Bax/Bcl-2 발현양 비율은 신경세포의 사멸을 제어하는 인자로 알려져 있다. In addition, the protective effect against neuronal cell death may be due to the action of metabolites produced through the reaction between the strain (KCCM 12614P) and enterocytes. The strain (KCCM 12614P) reacts with enterocytes to produce metabolites, and the metabolites produced in this way do not induce cytotoxicity to nerve cells and are resistant to oxidative stress (H 2 O 2 and NaAsO 2 ). may have a cytoprotective effect on This effect is due to the increased Bax/Bcl-2 by oxidative stress (H 2 O 2 and NaAsO 2 ) without causing cytotoxicity to neurons when the metabolites produced in this way are treated with neurons. It can be confirmed by greatly reducing the expression amount ratio. At this time, the Bax/Bcl-2 expression ratio related to apoptosis is known as a factor controlling neuronal cell death.
그밖에, 상기 균주(KCCM 12614P)는 하기 ⅰ) 또는 ⅱ)의 특징을 추가로 가질 수 있다: In addition, the strain (KCCM 12614P) may further have the following characteristics of i) or ii):
ⅰ) 내산성, 내담즙성 및 장부착능;i) acid resistance, bile resistance and intestinal adhesion;
ⅱ) α-글루코시다아제 및 β-글루코시다아제 생산능. ii) α-glucosidase and β-glucosidase production ability.
구체적으로, ⅰ) 내산성, 내담즙성 및 장부착능과 관련하여, 상기 균주(KCCM 12614P)의 내산성은 pH 2.5 조건에서 3시간 반응시, 균주의 생존율이 85 % 이상일 수 있고, 상기 균주(KCCM 12614P)의 내답즙성은 0.3% oxgall 조건에서 24시간 반응시, 균주의 생존율이 90% 이상(바람직하게, 100% 이상)일 수 있다. 특히, 상기 균주(KCCM 12614P)의 장부착능은 상업용 프로바이오틱스로 알려진 Lactobacillus rhamnosus LGG 균주, Lactobacillus fermentum 200060 균주 등에 비해, 우수한 것을 특징으로 한다. Specifically, i) in relation to acid resistance, bile resistance and intestinal adhesion, the acid resistance of the strain (KCCM 12614P) may have a survival rate of 85% or more when reacted for 3 hours under pH 2.5 conditions, and the strain (KCCM 12614P) 12614P) may have a survival rate of 90% or more (preferably, 100% or more) when reacted for 24 hours under 0.3% oxgall conditions. In particular, the intestinal attachment ability of the strain (KCCM 12614P) is characterized by superiority compared to Lactobacillus rhamnosus LGG strain, Lactobacillus fermentum 200060 strain, etc. known as commercial probiotics.
ⅱ) α-글루코시다아제 및 β-글루코시다아제 생산능과 관련하여, 상기 균주(KCCM 12614P)는 α-글루코시다아제 및 β-글루코시다아제 생산능을 가짐으로써, 유용 생리활성 물질의 생성을 유도할 수 있는 이점을 가진다. 아울러, 상기 균주(KCCM 12614P)는 암 유발 효소로 분류되는 β-글루쿠로니다아제(β-Glucuronidase)를 생산하지 않음으로써, 인체 안전성을 구현할 수 있다. ii) Regarding the ability to produce α-glucosidase and β-glucosidase, the strain (KCCM 12614P) has the ability to produce α-glucosidase and β-glucosidase, thereby producing useful physiologically active substances It has the advantage of being able to induce. In addition, the strain (KCCM 12614P) does not produce β-glucuronidase, which is classified as a cancer-inducing enzyme, so that human safety can be realized.
또한, 본 발명은 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 유효성분으로 포함하는 퇴행성 신경질환 예방 또는 치료용 약학 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a Leuconostoc mesenteroides strain (KCCM 12614P) as an active ingredient.
상기 균주(KCCM 12614P)에 대해서는 전술한 바와 같으며, 상기 생균체, 사균체 또는 배양여액으로 제조될 수 있다.The strain (KCCM 12614P) is as described above, and may be prepared from the live cells, dead cells or culture filtrate.
상기 균주(KCCM 12614P)의 용량은 1×106 CFU/mL 내지 1×1010 CFU/mL인 것이 바람직하나, 이에 한정되지 않는다. 이때, 상기 균주(KCCM 12614P)의 용량이 상기 범위 미만인 경우, 신경 세포 사멸에 대한 보호 효과를 발휘하기 어려운 문제점이 있고, 상기 균주(KCCM 12614P)의 용량이 상기 범위를 초과하는 경우, 세포독성을 포함한 독성의 우려사항이 있을 수 있다. The capacity of the strain (KCCM 12614P) is preferably 1×10 6 CFU/mL to 1×10 10 CFU/mL, but is not limited thereto. At this time, when the dose of the strain (KCCM 12614P) is less than the above range, there is a problem in that it is difficult to exert a protective effect against neuronal cell death, and when the dose of the strain (KCCM 12614P) exceeds the above range, cytotoxicity There may be toxicity concerns, including
본 명세서 내 "퇴행성 신경질환"이라 함은 신경 세포가 퇴행하는 병태 전부를 포함하는 의미로, 산화적 스트레스에 의해 유도된 것일 수 있다. 구체적으로, 알츠하이머 질환, 파킨슨 질환, 루게릭 질환, 헌팅턴 질환, 근위축성 측석 경화증, 다발성 경화증, 면역계이상 뇌기능 부전, 진행성 신경퇴행질환, 대사성 뇌질환, 니만-픽병, 뇌 허혈 및 뇌출혈로 인한 치매로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있고, 알츠하이머 질환을 포함하는 것이 바람직하나, 이에 한정되지 않는다. The term "degenerative neurological disease" in the present specification is meant to include all conditions in which nerve cells degenerate, and may be induced by oxidative stress. Specifically, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system dysfunction, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, dementia caused by cerebral ischemia and cerebral hemorrhage. It may include one or more selected from the group consisting of, preferably one including Alzheimer's disease, but is not limited thereto.
또한, 본 발명은 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 유효성분으로 포함하는 퇴행성 신경질환 예방 또는 개선용 건강기능식품을 제공한다. In addition, the present invention provides a health functional food for preventing or improving neurodegenerative diseases comprising a Leuconostoc mesenteroides strain (KCCM 12614P) as an active ingredient.
상기 균주(KCCM 12614P)에 대해서는 전술한 바와 같으며, 상기 생균체, 사균체 또는 배양여액으로 제조될 수 있다.The strain (KCCM 12614P) is as described above, and may be prepared from the live cells, dead cells or culture filtrate.
상기 균주(KCCM 12614P)의 용량은 1×106 CFU/mL 내지 1×1010 CFU/mL인 것이 바람직하나, 이에 한정되지 않는다. 이때, 상기 균주(KCCM 12614P)의 용량이 상기 범위 미만인 경우, 신경 세포 사멸에 대한 보호 효과를 발휘하기 어려운 문제점이 있고, 상기 균주(KCCM 12614P)의 용량이 상기 범위를 초과하는 경우, 세포독성을 포함한 독성의 우려사항이 있을 수 있다. The capacity of the strain (KCCM 12614P) is preferably 1×10 6 CFU/mL to 1×10 10 CFU/mL, but is not limited thereto. At this time, when the dose of the strain (KCCM 12614P) is less than the above range, there is a problem in that it is difficult to exert a protective effect against neuronal cell death, and when the dose of the strain (KCCM 12614P) exceeds the above range, cytotoxicity There may be toxicity concerns, including
또한, 본 발명은 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 유효성분으로 포함하는 프로바이오틱 조성물을 제공한다. In addition, the present invention provides a probiotic composition comprising a Leuconostoc mesenteroides strain (KCCM 12614P) as an active ingredient.
상기 균주(KCCM 12614P)에 대해서는 전술한 바와 같으며, 상기 생균체, 사균체 또는 배양여액으로 제조될 수 있다. The strain (KCCM 12614P) is as described above, and may be prepared from the live cells, dead cells or culture filtrate.
또한, 본 발명은 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)를 포함하는 발효용 스타터 또는 발효식품용 종균 조성물을 제공한다. In addition, the present invention provides a starter for fermentation or a seed composition for fermented food containing a Leuconostoc mesenteroides strain (KCCM 12614P).
상기 균주(KCCM 12614P)에 대해서는 전술한 바와 같으며, 상기 생균체, 사균체 또는 배양여액으로 제조될 수 있다. The strain (KCCM 12614P) is as described above, and may be prepared from the live cells, dead cells or culture filtrate.
그밖에, 상기 발효용 스타터 또는 상기 발효식품용 종균 조성물을 첨가하여 제조한 발효식품을 제공한다. In addition, a fermented food prepared by adding the starter for fermentation or the seed composition for fermented food is provided.
본 발명에 따른 발효식품은 요거트, 김치, 치즈 등 다양할 수 있는데, 상기 발효식품 역시 신경 세포 사멸(특히, 산화적 스트레스에 의해 유도된 신경 세포 사멸)에 대한 보호 효과를 가질 수 있고, 따라서, 퇴행성 신경질환(특히, 알츠하이머 질환) 예방, 개선 또는 치료에 효과적이다. The fermented food according to the present invention may be various, such as yogurt, kimchi, and cheese, and the fermented food may also have a protective effect against neuronal cell death (in particular, neuronal cell death induced by oxidative stress), and thus, It is effective in preventing, improving or treating neurodegenerative diseases (particularly, Alzheimer's disease).
구체적으로, 상기 신경세포 사멸에 대한 보호 효과는, 상기 발효식품을 장세포에 처리했을 때, BDNF 발현양이 증가하는 것을 통해 확인될 수 있다. Specifically, the protective effect against neuronal cell death can be confirmed through an increase in the amount of BDNF expression when the enterocytes are treated with the fermented food.
그밖에, 상기 신경세포 사멸에 대한 보호 효과는 상기 발효식품 및 장세포 간 반응을 통해 생산된 대사산물의 작용으로 인한 것일 수 있다. 상기 발효식품은 장세포와 반응을 하여 대사산물을 생산하는데, 이와 같이 생산된 대사산물은 신경 세포에 대해 세포 독성을 유발하지 않으면서, 산화적 스트레스(H2O2 및 NaAsO2)에 대한 세포 보호 효과를 가질 수 있다. 이러한 효과는 이와 같이 생산된 대사산물을 신경 세포에 처리했을 때, 신경 세포에 대해 세포 독성을 유발하지 않으면서, 산화적 스트레스(H2O2 및 NaAsO2)에 의해 증가된 Bax/Bcl-2 발현양 비율을 크게 감소시키고, 산화적 스트레스(H2O2 및 NaAsO2)에 의해 감소된 BDNF 발현양을 증가시키는 것을 통해 확인될 수 있다.In addition, the protective effect against neuronal cell death may be due to the action of metabolites produced through the reaction between the fermented food and enterocytes. The fermented food reacts with enterocytes to produce metabolites, and the metabolites produced in this way do not induce cytotoxicity to nerve cells and are effective against oxidative stress (H 2 O 2 and NaAsO 2 ). may have a protective effect. This effect is due to the increased Bax/Bcl-2 by oxidative stress (H 2 O 2 and NaAsO 2 ) without causing cytotoxicity to neurons when the metabolites produced in this way are treated with neurons. It can be confirmed by significantly reducing the expression amount ratio and increasing the BDNF expression amount reduced by oxidative stress (H 2 O 2 and NaAsO 2 ).
본 발명에 따른 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구제 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화되어 사용할 수 있고, 제형화를 위하여 약학 조성물의 제조에 통상적으로 사용되는 적절한 담체, 부형제 또는 희석제를 포함할 수 있다.The pharmaceutical composition according to the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. And, for formulation, suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions may be included.
상기 담체 또는, 부형제 또는 희석제로는 락토즈, 덱스트로즈, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리게이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다.Examples of the carrier or excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undecided various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
제제화할 경우에는 보통 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조할 수 있다.When formulated, it can be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants.
경구 투여를 위한 고형제제는 상기 균주(KCCM 12614P)에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘 보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 제조할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용할 수 있다.A solid preparation for oral administration may be prepared by mixing the strain (KCCM 12614P) with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.
경구를 위한 액상 제제로는 현탁액, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용하는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 포함할 수 있다.Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. .
비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등을 사용할 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(Tween) 61, 카카오지, 라우린지, 글리세롤젤라틴 등을 사용할 수 있다.Formulations for parenteral administration include sterilized aqueous solutions, water-insoluble agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base material for suppositories, witepsol, macrogol, Tween 61, cacao butter, laurin paper, glycerol gelatin, and the like can be used.
본 발명에 따른 약학 조성물의 바람직한 투여량은 환자의 상태, 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 바람직한 효과를 위해서는 1일 0.0001 내지 2,000 mg/kg으로, 바람직하게는 0.001 내지 2,000 mg/kg으로 투여할 수 있다. 투여는 하루에 한 번 투여할 수도 있고, 수회 나누어서 투여할 수도 있다. 다만, 상기 투여량에 의해서 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, body weight, disease severity, drug type, administration route and period, but can be appropriately selected by those skilled in the art. However, for desirable effects, it may be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. Administration may be administered once a day or divided into several times. However, the scope of the present invention is not limited by the dosage.
본 발명에 따른 약학 조성물은 쥐, 생쥐, 가축, 인간 등의 포유 동물에 다양한 경로로 투여할 수 있다. 투여의 모든 방식은 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관내(intracerebroventricular) 주사에 의해서 투여할 수 있다.The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
본 발명에 따른 건강기능식품 조성물에 있어서, 상기 균주(KCCM 12614P)를 건강기능식품의 첨가물로 사용하는 경우 이를 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효 성분의 혼합양은 예방, 건강 또는 치료 등의 각 사용 목적에 따라 적합하게 결정할 수 있다.In the health functional food composition according to the present invention, when the strain (KCCM 12614P) is used as an additive for health functional food, it can be added as it is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. can The mixing amount of the active ingredient can be appropriately determined according to each purpose of use, such as prevention, health, or treatment.
건강기능식품의 제형은 산제, 과립제, 환, 정제, 캡슐제의 형태뿐만 아니라 일반 식품 또는 음료의 형태 어느 것이나 가능하다. The formulation of the health functional food may be in the form of a powder, granule, pill, tablet, or capsule, as well as a general food or beverage form.
상기 식품의 종류에는 특별히 제한은 없고, 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸콜렛, 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 포함할 수 있다.The type of food is not particularly limited, and examples of food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products including ice cream. , various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc., and may include all foods in a conventional sense.
일반적으로, 식품 또는 음료의 제조시에 상기 균주(KCCM 12614P)는 원료 100 중량부에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가할 수 있다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.In general, when preparing food or beverage, the strain (KCCM 12614P) may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range.
본 발명에 따른 건강기능식품 중 음료는 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜일 수 있다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명에 따른 음료 100 mL당 약 0.01 ~ 0.04 g, 바람직하게는 약 0.02 ~ 0.03 g일 수 있다.Among the health functional foods according to the present invention, beverages may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.
상기 외에 본 발명에 따른 건강기능식품 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, 탄산음료에 사용되는 탄산화제를 함유할 수 있다. 그 밖에 본 발명에 따른 건강기능식품 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 제한되지 않으나 본 발명에 따른 건강기능식품 조성물 100 중량부 대비 0.01 ~ 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the health functional food composition according to the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, It may contain glycerin, alcohol, and carbonation agents used in carbonated beverages. In addition, the health functional food composition according to the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not limited, but is generally selected from the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition according to the present invention.
전술한 바와 같이, 본 발명에 따른 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주(KCCM 12614P)는 안정적이고 안전한 프로바이오틱 특성(특히, 우수한 내담즙성 및 장부착능)을 가지면서, 신경 세포 사멸(특히, 산화적 스트레스에 의해 유도된 신경 세포 사멸)에 대한 보호 효과를 가지는 바, 프로바이오틱 조성물 뿐만 아니라, 퇴행성 신경질환(특히, 알츠하이머 질환) 예방, 개선 또는 치료용 조성물로 활용될 수 있다. 더욱이, 상기 균주의 특성상 발효용 스타터 또는 발효식품용 종균 조성물로도 유용하게 활용될 수 있다. As described above, the Leuconostoc mesenteroides strain (KCCM 12614P) according to the present invention has stable and safe probiotic properties (particularly, excellent bile resistance and intestinal adhesion), while Since it has a protective effect against death (particularly, neuronal cell death induced by oxidative stress), it can be used not only as a probiotic composition, but also as a composition for preventing, improving or treating neurodegenerative diseases (particularly, Alzheimer's disease). there is. Moreover, due to the nature of the strain, it can be usefully used as a starter for fermentation or a seed composition for fermented foods.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.
<실시예><Example>
실시예 1: Example 1: Leuconostoc mesenteroides Leuconostoc mesenteroides H40 균주 분리 및 동정H40 strain isolation and identification
신규 균주를 분리하기 위해, 집에서 담근 배추김치 국물을 샘플링하여 시료로 사용하였다. 배추김치 국물을 10배 희석법을 사용하여 MRS 한천 배지에 도말하고 여러 차례 획선 도말하고 배양한 후 콜로니 형태를 관찰하여 신규 균주를 분리하였다. 분리된 신규 균주의 동정을 위해 Bionics(Seoul, Korea)에 16S rRNA 서열분석을 의뢰하였다. 구체적으로, 16S rRNA 서열분석을 수행하였고, 16S rRNA 유전자를 27F와 1492R 프라이머(primer)를 이용하여 PCR을 진행하였다. 염기서열을 분석하여 나온 결과를 토대로 BLAST 프로그램을 이용하여 Genbank의 데이터베이스에 등록된 다른 표준균주와 상동성을 비교하였다. 신규 균주는 Leuconostoc mesenteroides H40 균주로 확인되었고, 유사성은 99%로 나타났다. 또한, Mega 7 program을 이용하여 상동성을 분석하고 계통도를 얻었다(도 1). 이때, Leuconostoc mesenteroides H40 균주는 2019년 1O월 25일자로 한국미생물보존센터에 기탁하여 수탁번호 KCCM 12614P를 부여받았다.In order to isolate the new strain, the cabbage kimchi broth made at home was sampled and used as a sample. The cabbage kimchi broth was smeared on MRS agar medium using a 10-fold dilution method, streaked and cultured several times, and then colony morphology was observed to isolate new strains. 16S rRNA sequencing was requested to Bionics (Seoul, Korea) to identify the isolated new strain. Specifically, 16S rRNA sequencing was performed, and 16S rRNA gene was subjected to PCR using 27F and 1492R primers. Based on the results of the sequencing analysis, homology was compared with other standard strains registered in the Genbank database using the BLAST program. The new strain was identified as the Leuconostoc mesenteroides H40 strain, and the similarity was 99%. In addition, homology was analyzed using the Mega 7 program and a phylogeny was obtained (FIG. 1). At this time, the Leuconostoc mesenteroides H40 strain was deposited with the Korea Microorganism Conservation Center on October 25, 2019 and was given accession number KCCM 12614P.
이후, Leuconostoc mesenteroides H40 균주를 lactobacilli MRS broth에서 37℃, 12시간 동안 배양하여 초기 균수를 1×109 CFU/mL로 조절한 후 원심분리(5,000×g, 10분)하여 침전물(균체)과 상층액(배양여액)으로 분리하였고. 침전물을 동결건조시킨 후 생균체를 제조하였고, 여기에 추가적인 열처리(121℃, 15 분)를 통하여 균주 사균체를 제조하였다. 그다음, 균주 사균체를 0.1% 펩톤수로 3번 세척 및 재현탁하여 실험에 사용하였다.Thereafter, the Leuconostoc mesenteroides H40 strain was cultured in lactobacilli MRS broth at 37 ° C for 12 hours to adjust the initial number of bacteria to 1 × 10 9 CFU / mL, and then centrifuged (5,000 × g, 10 minutes) to precipitate (cells) and upper layer It was isolated as a liquid (culture filtrate). After freeze-drying the precipitate, live cells were prepared, and strain-killed cells were prepared through additional heat treatment (121 ° C., 15 minutes). Then, the dead cells of the strain were washed and resuspended three times with 0.1% peptone water and used in the experiment.
한편, 시유(서울우유社)를 90℃에서 10분 동안 열처리하여 파스퇴르화한 후, 40℃까지 식혔다. 여기에, ABT-B 스타터(Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium longum 및 Streptococcus thermophilus)(삼익유가공社)와 균주 생균체를 동등한 균수로 투입하여 혼합하였고, pH가 4.5가 될 때까지 배양하여 균주 적용 요거트를 제조하였다. 이화학적 평가는 우유분석기(Milko scan minor, Foss, Hillerod, Denmark), pH 미터기 및 산도 AOAC(1999)를 통해 측정하였다.On the other hand, seed milk (Seoul Milk Co.) was pasteurized by heat treatment at 90°C for 10 minutes, and then cooled to 40°C. Here, ABT-B starters ( Lactobacillus acidophilus , Lactobacillus bulgaricus , Bifidobacterium longum , and Streptococcus thermophilus ) (Samik Yu Co., Ltd.) and strain probiotics were added in equal numbers and mixed, and cultured until the pH reached 4.5 to apply the strain. was manufactured. Physicochemical evaluation was performed using a milk analyzer (Milko scan minor, Foss, Hillerod, Denmark), pH meter, and acidity AOAC (1999).
비교예 1: Comparative Example 1: Lactobacillus rhamnosusLactobacillus rhamnosus LGG 균주 준비 LGG strain preparation
상업용 프로바이오틱스로 알려진 Lactobacillus rhamnosus LGG 균주를 준비하였다. 배양 조건은 Leuconostoc mesenteroides H40 균주와 동일하게 하였다. Lactobacillus rhamnosus LGG strain known as commercial probiotics was prepared. Culture conditions were the same as for Leuconostoc mesenteroides H40 strain.
비교예 2: Comparative Example 2: Lactobacillus fermentumLactobacillus fermentum 200060 균주 준비 200060 strain preparation
상업용 프로바이오틱스로 알려진 Lactobacillus fermentum 200060 균주를 준비하였다. 배양 조건은 Leuconostoc mesenteroides H40 균주와 동일하게 하였다. Lactobacillus fermentum 200060 strain known as commercial probiotics was prepared. Culture conditions were the same as for Leuconostoc mesenteroides H40 strain.
실험예 1: 균주의 생리적 특성 평가Experimental Example 1: Evaluation of physiological characteristics of strains
실시예 1 및 비교예 1-2에서 분리된 균주의 생리적 특성으로서, 내산성, 담즙성 및 장부착능을 평가하였다. As physiological characteristics of the strains isolated in Example 1 and Comparative Example 1-2, acid resistance, bile property and intestinal adhesion were evaluated.
(1) 내산성 평가(1) Acid resistance evaluation
실시예 1 및 비교예 1-2에서 분리된 균주를 24시간 배양하였다. pH 2.5로 조절한 배양액 실험관에 pepsin 3 mg/mL를 첨가하고, 미리 배양된 실시예 1 및 비교예 1-2에서 분리된 균주를 각 실험관에 1 mL씩 접종하였다. 0시간을 대조군으로 사용하였고, 0시간 및 3시간 후의 총 균수를 확인하여 내산성을 평가하였고, 그 결과는 하기 표 1에 나타내었다.The strains isolated in Example 1 and Comparative Example 1-2 were cultured for 24 hours. 3 mg/mL of pepsin was added to the culture solution test tube adjusted to pH 2.5, and each test tube was inoculated with 1 mL of the pre-cultured strains isolated from Example 1 and Comparative Examples 1-2. 0 hour was used as a control group, and acid resistance was evaluated by checking the total number of bacteria after 0 hour and 3 hours, and the results are shown in Table 1 below.
(2) 내담즙성 평가(2) evaluation of bile resistance
실시예 1 및 비교예 1-2에서 분리된 균주를 24시간 배양하였다. 배양액 실험관에 Oxgall 3.0 mg/mL를 첨가하고, 미리 배양된 실시예 1 및 비교예 1-2에서 분리된 균주를 각 실험관에 1 mL씩 접종하였다. 0시간을 대조군으로 사용하였고, 0 시간 및 24시간 후의 총 균수를 확인하여 내담즙성을 평가하였고, 그 결과는 하기 표 1에 나타내었다. The strains isolated in Example 1 and Comparative Example 1-2 were cultured for 24 hours. 3.0 mg/mL of Oxgall was added to the culture test tube, and 1 mL of the strains isolated from the pre-cultured Example 1 and Comparative Examples 1-2 were inoculated into each test tube. 0 hour was used as a control group, and biliary resistance was evaluated by checking the total number of bacteria after 0 hour and 24 hours, and the results are shown in Table 1 below.
표 1에 나타난 바와 같이, 내산성 평가 결과, 실시예 1에서 분리된 균주는 초기 균수와 비교하여 균수가 다소 감소하였지만, 우수한 내산성을 가지는 것으로 확인된다. 한편, 내담즙성 평가 결과, 실시예 1에서 분리된 균주는 초기 균수와 비교하여 균수가 약 0.09 Log CFU/mL 증가하여 우수한 내담즙성을 가지는 것으로 확인된다. 반면, 비교예 1에서 분리된 균주는 초기 균수와 비교하여 균수가 약 0.03 Log CFU/mL만 증가하였고, 비교예 2에서 분리된 균주는 초기 균수와 비교하여 균수가 약 0.85 Log CFU/mL 감소하였다. As shown in Table 1, as a result of acid resistance evaluation, the strain isolated in Example 1 was confirmed to have excellent acid resistance, although the number of bacteria slightly decreased compared to the initial number of bacteria. On the other hand, as a result of evaluation of bile resistance, the strain isolated in Example 1 was found to have excellent chole resistance as the number of bacteria increased by about 0.09 Log CFU/mL compared to the initial number of bacteria. On the other hand, the strain isolated in Comparative Example 1 increased the number of bacteria by only about 0.03 Log CFU / mL compared to the initial number of bacteria, and the strain isolated in Comparative Example 2 increased the number of bacteria by about 0.85 Log CFU / mL compared to the initial number of bacteria. .
(3) 장부착능 평가(3) Evaluation of adhesion ability
실시예 1 및 비교예 1-2에서 분리된 균주를 샘플로 준비하여, HT-29 cells (human colon adenocarcinoma cell line, KCLB 30038)를 1×105 cell/mL 농도로 1 mL씩 넣은 후 24시간 배양하였다. 준비된 샘플을 100 μL(1×108 CFU/mL)씩 넣은 후 2 시간 동안 배양한 후, 부착된 cells를 1% Triton × 100 용액을 이용하여 떼어준 후 부착 균수를 확인하여 장부착능을 하기와 같이 평가하였고, 그 결과는 하기 도 2에 나타내었다: The strains isolated in Example 1 and Comparative Examples 1-2 were prepared as samples, and HT-29 cells (human colon adenocarcinoma cell line, KCLB 30038) were added at a concentration of 1 × 10 5 cell / mL by 1 mL, followed by 24 hours cultured. After adding 100 μL (1×10 8 CFU/mL) of the prepared sample and incubating for 2 hours, the adhered cells are detached using 1% Triton × 100 solution, and the number of adherent bacteria is checked to determine the adherent ability. It was evaluated as, and the results are shown in Figure 2 below:
장부착능 = (부착 균수(Log CFU/mL)/접종 균수(Log CFU/mL)) × 100.Intestinal adhesion capacity = (number of adherent bacteria (Log CFU/mL)/number of inoculated bacteria (Log CFU/mL)) × 100.
도 2에 나타난 바와 같이, 장부착능 평가 결과, 실시예 1에서 분리된 균주의 장부착능은 약 2.86 ± 0.16%로 아주 뛰어난 것으로 확인된다. 반면, 비교예 1-2에서 분리된 균주의 장부착능은 각각 약 2.34 ± 0.26% 및 약 1.18 ± 0.08 정도로 미미한 수준에 불과하였다. As shown in FIG. 2, as a result of the evaluation of intestinal adhesion, the intestinal adhesion of the strain isolated in Example 1 was about 2.86 ± 0.16%, which was confirmed to be very excellent. On the other hand, the intestinal adhesion ability of the strains isolated in Comparative Example 1-2 was only insignificant, about 2.34 ± 0.26% and about 1.18 ± 0.08, respectively.
즉, 실시예 1에서 분리된 균주는 생리적 특성이 모두 우수하므로, 인체에 유익한 유산균으로 볼 수 있다. That is, since the strain isolated in Example 1 has excellent physiological characteristics, it can be regarded as a lactic acid bacteria beneficial to the human body.
실험예 2: 균주의 효소 생산능 평가Experimental Example 2: Evaluation of enzyme production ability of strains
실시예 1 및 비교예 1-2에서 분리된 균주의 효소 생산능을 평가하였다.The enzyme production ability of the strains isolated in Example 1 and Comparative Example 1-2 was evaluated.
구체적으로, 실시예 1 및 비교예 1-2에서 분리된 균주를 1×106 CFU/mL로 조절한 후, API ZYM kit를 이용하여 효소 생산능을 확인하였고, 그 결과는 하기 표 2에 나타내었다. Specifically, after adjusting the strains isolated in Example 1 and Comparative Examples 1-2 to 1×10 6 CFU/mL, the enzyme production ability was confirmed using the API ZYM kit, and the results are shown in Table 2 below. was
* 0, 1, 2, 3, 4 및 5는 효소 생산능을 의미하는 것으로, 0은 0 nmol; 1은 5 nmol; 2는 10 nmol; 3은 20 nmol; 4는 30 nmol; 및 5는 40 nmol 이상을 의미한다. * 0, 1, 2, 3, 4 and 5 denote enzyme production capacity, 0 is 0 nmol; 1 is 5 nmol; 2 is 10 nmol; 3 is 20 nmol; 4 is 30 nmol; and 5 means 40 nmol or more.
상기 표 2에 나타난 바와 같이, 실시예 1에서 분리된 균주는 암 유발 효소인 β-글루쿠로니다아제(β-Glucuronidase)를 생산하지 않음으로써, 생체 안전성이 확보된 것으로 확인된다. 또한, 유용 생리활성 물질의 생성을 유도하는 α-글루코시다아제(α-Glucosidase) 및 β-글루코시다아제(β-Glucosidase) 생산능이 뛰어나 유용성을 가지는 것으로 확인된다. As shown in Table 2, the strain isolated in Example 1 does not produce β-glucuronidase, which is a cancer-inducing enzyme, so it is confirmed that biosafety is secured. In addition, it is confirmed to have usefulness because of its excellent ability to produce α-glucosidase and β-glucosidase, which induce the production of useful physiologically active substances.
실험예 3: 균주 유래 대사산물 또는 균주 적용 요거트 유래 대사산물의 신경 세포 사멸 보호 평가Experimental Example 3: Neuronal cell death protection evaluation of strain-derived metabolites or strain-applied yogurt-derived metabolites
먼저, 인간 유래 대장암 세포인 HT-29 세포주를 6-well plate에 접종하여 (5×105 cells/mL) 단일층을 형성할 때까지 배양한 후 균주 사균체 또는 균주 적용 요거트를 200 μL (1×109 CFU/mL) 접종하여 24시간 동안 배양하였다. 이후 상등액을 수거하여 13,000×g, 4℃에서 5분 동안 원심분리 후 0.45 μM 필터를 사용해 필터링하여 균주 유래 대사산물(conditioned medium; CM)을 제조하였다. First, human-derived colorectal cancer cells, HT-29 cell line, were inoculated into a 6-well plate (5×10 5 cells/mL), cultured until a monolayer was formed, and then strain-killed cells or strain-applied yogurt were added to 200 μL ( 1×10 9 CFU/mL) and cultured for 24 hours. Thereafter, the supernatant was collected, centrifuged at 13,000 × g at 4° C. for 5 minutes, and filtered using a 0.45 μM filter to prepare a strain-derived metabolite (conditioned medium; CM).
균주 유래 CM 또는 균주 적용 요거트 유래 CM의 인간 유래 신경 세포인 SH-SY5Y 세포주에 대한 세포 독성 유발 여부와 산화적 스트레스인 H2O2 및 NaAsO2에 대한 세포 보호 효과를 측정하기 위해 MTT(methylthizol-2-yl-2,5-diphenyl tetrazoliumbromide) 어세이를 실시하였다. 96 well-plate에 DMEM에 현탁한 SH-SY5Y 세포주를 0.1 mL 넣어 24시간 동안 배양하였다. 균주 유래 CM 또는 균주 적용 요거트 유래 CM을 세포독성 여부를 측정하기 위해서는 균주 유래 CM 또는 균주 적용 요거트 유래 CM을 24시간 동안 처리하였고, H2O2 및 NaAsO2에 대한 세포 보호 효과를 측정하기 위해서는 800 μL의 균주 유래 CM 또는 균주 적용 요거트 유래 CM를 4시간 동안 미리 처리한 다음 H2O2 (50 μM) 및 NaAsO2 (10 μM)을 처리하여 20시간 배양하였다. 이후 배지를 제거한 후 well에 MTT(5 mg/mL)을 20 μL 첨가하여 1시간 동안 배양하면서 환원반응을 유도한 후 배양액을 제거하고 0.1 mL의 DMSO(Dimethyl sulfoxide) 용액을 첨가하여 15분간 반응시켜 생성된 결정을 녹여 주었다. 이후 흡광도를 540 ㎚ 측정하였으며 세포 생존률에 관한 식은 다음과 같고, 그 결과는 도 3 및 도 4에 각각 나타내었다: MTT ( methylthizol- 2-yl-2,5-diphenyl tetrazoliumbromide) assay was performed. 0.1 mL of the SH-SY5Y cell line suspended in DMEM was placed in a 96 well-plate and cultured for 24 hours. In order to measure the cytotoxicity of strain-derived CM or strain-applied yogurt-derived CM, the strain-derived CM or strain-applied yogurt-derived CM was treated for 24 hours, and to measure the cytoprotective effect against H 2 O 2 and NaAsO 2 , 800 μL of strain-derived CM or strain-applied yogurt-derived CM was previously treated for 4 hours, and then treated with H 2 O 2 (50 μM) and NaAsO 2 (10 μM), followed by incubation for 20 hours. After removing the medium, 20 μL of MTT (5 mg/mL) was added to the well and incubated for 1 hour to induce a reduction reaction. The crystals formed were melted. Then, absorbance was measured at 540 nm, and the expression for cell viability was as follows, and the results were shown in FIGS. 3 and 4, respectively:
도 3에 나타난 바와 같이, 대조군의 세포 생존률을 100%로 설정했을 때 H2O2를 처리한 세포의 세포 생존률은 약 44.12%로 나타났으며, 순서대로 비교예 1-2 및 실시예 1에서 분리된 균주 유래 CM을 처리하고 세포 독성을 유발했을 때 세포 생존률은 각각 약 55.93%, 약 45.19% 및 약 66.32%인 것으로 확인된다(p<0.05). 또한, NaAsO2를 처리한 세포의 세포 생존률은 약 56.67%로 나타났으며, 순서대로 비교예 1-2 및 실시예 1에서 분리된 균주 유래 CM을 처리하고 세포 독성을 유발했을 때 세포 생존률은 각각 약 56.00%, 약 63.28% 및 약 76.36%인 것으로 확인된다(p<0.05). 따라서, 실시예 1에서 분리된 균주는 다른 상업용 프로바이오틱스에 비해, 신경 세포 사멸에 대한 보호 효과가 우수하다고 볼 수 있다. As shown in FIG. 3, when the cell viability of the control group was set to 100%, the cell viability of cells treated with H 2 O 2 was about 44.12%, and in Comparative Examples 1-2 and Example 1 in order. When the CM derived from the isolated strain was treated and cytotoxicity was induced, the cell viability was confirmed to be about 55.93%, about 45.19%, and about 66.32%, respectively (p<0.05). In addition, the cell viability of the cells treated with NaAsO 2 was about 56.67%, and when the CM derived from the strains isolated from Comparative Examples 1-2 and Example 1 were sequentially treated and cytotoxicity was induced, the cell viability was about 56.00%, about 63.28% and about 76.36% (p<0.05). Therefore, it can be seen that the strain isolated in Example 1 has an excellent protective effect against neuronal cell death compared to other commercial probiotics.
도 4에 나타난 바와 같이, 대조군의 세포 생존률을 100%로 설정했을 때 H2O2를 처리한 세포의 세포 생존률은 약 67.44%로 나타났으며, 실시예 1에서 분리된 균주 적용 요거트 유래 CM을 처리하고 세포 독성을 유발했을 때 세포 생존률은 약 137.98%인 것으로 확인된다(p<0.05). 한편, 실시예 1에서 분리된 균주가 포함되지 않은 요거트(대조군 요거트) 유래 CM을 처리하고 세포 독성을 유발했을 때 세포 생존율은 약 86.82%에 불과하였다. 또한, NaAsO2를 처리한 세포의 세포 생존률은 약 35.5%로 나타났으며, 실시예 1에서 분리된 균주 적용 요거트 유래 CM을 처리하고 세포 독성을 유발했을 때 세포 생존률은 각각 약 109.5%인 것으로 확인된다(p<0.05). 한편, 실시예 1에서 분리된 균주가 포함되지 않은 요거트(대조군 요거트) 유래 CM을 처리하고 세포 독성을 유발했을 때 세포 생존율은 약 34.5%에 불과하였다. 따라서, 실시예 1에서 분리된 균주 적용 요거트 유래 CM은 대조군에 비해, 신경 세포 사멸에 대한 보호 효과가 우수하다고 볼 수 있다. As shown in Figure 4, when the cell viability of the control group was set to 100%, the cell viability of the cells treated with H 2 O 2 was about 67.44%, and the yogurt-derived CM applied to the strain isolated in Example 1 When treated and cytotoxic, the cell viability was found to be about 137.98% (p<0.05). On the other hand, when the CM derived from yogurt (control yogurt) not containing the strain isolated in Example 1 was treated and cytotoxicity was induced, the cell viability was only about 86.82%. In addition, the cell viability of the cells treated with NaAsO 2 was about 35.5%, and when the yogurt-derived CM applied to the strain isolated in Example 1 was treated and cytotoxicity was induced, the cell viability was confirmed to be about 109.5%, respectively. (p<0.05). On the other hand, when the CM derived from yogurt (control yogurt) not containing the strain isolated in Example 1 was treated and cytotoxicity was induced, the cell viability was only about 34.5%. Therefore, it can be seen that the strain-applied yogurt-derived CM isolated in Example 1 has an excellent protective effect against neuronal cell death compared to the control group.
실험예 4: 균주 사균체 또는 균주 적용 요거트의 인간 유래 대장암 세포에 대한 유전자 발현 조사Experimental Example 4: Investigation of gene expression in human-derived colorectal cancer cells of strain-killed cells or strain-applied yogurt
균주 사균체 또는 균주 적용 요거트를 인간 유래 대장암 세포에 처리했을 때 신경 성장 촉진인자인 BDNF(brain derived neurotrophic factor) 증감 여부를 real time PCR으로 측정하였다. 인간 유래 대장암 세포인 HT-29 세포주(1Х106 cells/well)를 6-well plate에 접종하여 단층이 형성될 때까지 배양하였다. 이후, 800 μL의 균주 사균체 또는 균주 적용 요거트를 4시간 동안 처리하였다. 유전자 발현을 RNA 프라이머(표 3) 및 real-time PCR을 이용하여 확인하였고, 그 결과는 도 5 및 6에 각각 나타내었다. The increase or decrease of BDNF (brain derived neurotrophic factor), a nerve growth promoter, was measured by real-time PCR when human-derived colorectal cancer cells were treated with dead strain cells or strain-applied yogurt. HT-29 cell line (1Х10 6 cells/well), a human-derived colon cancer cell, was inoculated into a 6-well plate and cultured until a monolayer was formed. Thereafter, 800 μL of dead cells of the strain or strain-applied yogurt was treated for 4 hours. Gene expression was confirmed using RNA primers (Table 3) and real-time PCR, and the results are shown in FIGS. 5 and 6, respectively.
RNA 순도는 Multiscan GO (Thermo Fisher Scientific, Waltham, MA, USA)를 이용하였고, cDNA로 Revertaid first strand cDNA synthesis kit(Thermo Fisher Scientific)를 이용하여 전환하였다. Semi-quantitative real-time PCR은 SYBR Green PCR Master Mix를 이용하였고, PCR 조건은 총 20 μL 볼륨으로, 95℃ for 2 min as initial denaturation; 95℃ for 5 s as denaturation (40 cycle); 60℃ for 15 s as annealing and extension으로 하였다. RNA purity was measured using Multiscan GO (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA was converted using the Revertaid first strand cDNA synthesis kit (Thermo Fisher Scientific). Semi-quantitative real-time PCR was performed using SYBR Green PCR Master Mix, PCR conditions were 20 μL in total volume, 95 ° C for 2 min as initial denaturation; 95℃ for 5 s as denaturation (40 cycles); 60 ℃ for 15 s as annealing and extension.
normalizing internal standardGlyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
normalizing internal standard
도 5에 나타난 바와 같이, 인간 유래 대장암 세포의 BDNF 발현량을 살펴본 결과, HT-29 세포주에 실시예 1에서 분리된 균주 사균체를 처리했을 경우 대조군에 비해 약 1.94배 증가한 BDNF 발현량을 보였는바, 실시예 1에서 분리된 균주 사균체는 장세포와 반응하여 뛰어난 뇌 유래 신경영양인자인 BDNF 생성능을 보이는 것으로 확인된다. 반면, HT-29 세포주에 비교예 2에서 분리된 균주 사균체를 처리한 경우 대조군에 비해 약 1.24배 증가한 BDNF 발현량을 보인 것에 불과하였다.As shown in FIG. 5, as a result of examining the BDNF expression level of human-derived colon cancer cells, when the HT-29 cell line was treated with the dead cells of the strain isolated in Example 1, the BDNF expression level was increased by about 1.94 times compared to the control group. Bar, it was confirmed that the dead cells of the strain isolated in Example 1 reacted with enterocytes to show the ability to produce BDNF, a brain-derived neurotrophic factor. On the other hand, when the HT-29 cell line was treated with dead cells of the strain isolated in Comparative Example 2, the BDNF expression level increased by about 1.24 times compared to the control group.
도 6에 나타난 바와 같이, 인간 유래 대장암 세포의 BDNF 발현량을 살펴본 결과, HT-29 세포주에 실시예 1에서 분리된 균주 적용 요거트를 처리했을 경우 대조군에 비해 약 1.6배 증가한 BDNF 발현량을 보였는바, 실시예 1에서 분리된 균주 적용 요거트는 장세포와 반응하여 뛰어난 뇌 유래 신경영양인자인 BDNF 생성능을 보이는 것으로 확인된다. 반면, HT-29 세포주에 실시예 1에서 분리된 균주가 포함되지 않은 요거트(대조군 요거트)를 처리한 경우 대조군에 비해 약 1.19배 증가한 BDNF 발현량을 보인 것에 불과하였다.As shown in FIG. 6, as a result of examining the BDNF expression level of human-derived colon cancer cells, when the HT-29 cell line was treated with yogurt applied to the strain isolated in Example 1, the BDNF expression level was increased by about 1.6 times compared to the control group. Bar, it is confirmed that the strain-applied yogurt isolated in Example 1 reacts with enterocytes to show the ability to produce BDNF, a brain-derived neurotrophic factor. On the other hand, when the HT-29 cell line was treated with yogurt (control yogurt) not containing the strain isolated in Example 1, the BDNF expression level increased by about 1.19 times compared to the control group.
실험예 5: 균주 유래 대사산물 또는 균주 적용 요거트 유래 대사산물의 인간 유래 신경 세포에 대한 유전자 발현 조사Experimental Example 5: Investigation of gene expression of strain-derived metabolites or strain-applied yogurt-derived metabolites in human-derived nerve cells
균주 유래 대사산물 또는 균주 적용 요거트 유래 대사산물을 인간 유래 신경 세포에 처리했을 때 세포사멸에 관련된 인자인 Bax(Bcl-2 associated protein X) 및 Bcl-2(B-cell lymphoma 2) 증감 여부와, 신경 성장 촉진인자인 BDNF(brain derived neurotrophic factor) 증감 여부를 real time PCR으로 측정하였다. 인간 유래 신경 세포인 SH-SY5Y 세포주(1Х106 cells/well)를 6-well plate에 접종하여 단층이 형성될 때까지 배양하였다. 이후, 800 μL의 균주 유래 CM 또는 균주 적용 요거트 유래 CM을 4시간 동안 처리한 후, 산화적 스트레스를 유도하기 위해 200 μL의 H2O2 (50 μM) 또는 NaAsO2 (10 μM)을 20 시간 동안 처리하였다. 유전자 발현을 RNA 프라이머(표 4) 및 real-time PCR을 이용하여 확인하였고, 그 결과는 도 7 및 도 8에 나타내었다. When strain-derived metabolites or strain-applied yogurt-derived metabolites were treated with human-derived nerve cells, whether or not Bax (Bcl-2 associated protein X) and Bcl-2 (B-cell lymphoma 2), which are factors related to apoptosis, were increased or decreased, The increase or decrease of BDNF (brain derived neurotrophic factor), a nerve growth promoting factor, was measured by real time PCR. SH-SY5Y cell line (1Х10 6 cells/well), a human-derived neuronal cell, was inoculated into a 6-well plate and cultured until a monolayer was formed. Thereafter, 800 μL of strain-derived CM or strain-applied yogurt-derived CM was treated for 4 hours, and then 200 μL of H 2 O 2 (50 μM) or NaAsO 2 (10 μM) was added for 20 hours to induce oxidative stress. treated during. Gene expression was confirmed using RNA primers (Table 4) and real-time PCR, and the results are shown in FIGS. 7 and 8 .
RNA 순도는 Multiscan GO (Thermo Fisher Scientific, Waltham, MA, USA)를 이용하였고, cDNA로 Revertaid first strand cDNA synthesis kit(Thermo Fisher Scientific)를 이용하여 전환하였다. Semi-quantitative real-time PCR은 SYBR Green PCR Master Mix를 이용하였고, PCR 조건은 총 20 μL 볼륨으로, 95℃ for 2 min as initial denaturation; 95℃ for 5 s as denaturation (40 cycle); 60℃ for 15 s as annealing and extension으로 하였다. RNA purity was measured using Multiscan GO (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA was converted using the Revertaid first strand cDNA synthesis kit (Thermo Fisher Scientific). Semi-quantitative real-time PCR was performed using SYBR Green PCR Master Mix, PCR conditions were 20 μL in total volume, 95 ° C for 2 min as initial denaturation; 95℃ for 5 s as denaturation (40 cycles); 60 ℃ for 15 s as annealing and extension.
normalizing internal standardGlyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
normalizing internal standard
도 7에 나타난 바와 같이, 산화적 스트레스가 유도된 인간 유래 신경 세포의 Bax/Bcl-2 발현량 비율을 살펴본 결과, SH-SY5Y 세포주에 H2O2만 처리했을 경우 Bax/Bcl-2 발현량 비율이 음성대조군에 비해 약 3.19배로 증가하였으나, SH-SY5Y 세포주에 H2O2 및 실시예 1에서 분리된 균주 유래 CM을 처리했을 경우 음성대조군에 비해 약 2.41배만 증가한 Bax/Bcl-2 발현량 비율을 보였다. 즉, 실시예 1에서 분리된 균주 유래 CM은 Bax/Bcl-2 발현량 비율을 크게 낮추었는 바, 실시예 1에서 분리된 균주 유래 CM은 H2O2의 세포 사멸 유도 작용을 완화하는 것으로 확인된다. 반면, SH-SY5Y 세포주에 H2O2 및 비교예 1에서 분리된 균주 유래 CM을 처리했을 경우에는 음성대조군에 비해 약 2.67배 이상 증가한 Bax/Bcl-2 발현량 비율을 보였다. 한편, SH-SY5Y 세포주에 NaAsO2 만 처리했을 경우 Bax/Bcl-2 발현량 비율이 음성대조군에 비해 약 2.57배로 증가하였으나, SH-SY5Y 세포주에 NaAsO2 및 실시예 1에서 분리된 균주 유래 CM을 처리했을 경우 음성대조군에 비해 약 1.67배만 증가한 Bax/Bcl-2 발현량 비율을 보였다. 즉, 실시예 1에서 분리된 균주 유래 CM은 Bax/Bcl-2 발현량 비율을 크게 낮추었는 바, 실시예 1에서 분리된 균주 유래 CM은 NaAsO2의 세포 사멸 유도 작용을 완화하는 것으로 확인된다. 반면, SH-SY5Y 세포주에 NaAsO2 및 비교예 1에서 분리된 균주 유래 CM을 처리했을 경우에는 오히려 음성대조군에 비해 약 2.99배 이상 증가한 Bax/Bcl-2 발현량 비율을 보였다. As shown in FIG. 7, as a result of examining the Bax/Bcl-2 expression level ratio of human-derived neurons induced by oxidative stress, Bax/Bcl-2 expression level when SH-SY5Y cell line was treated with only H 2 O 2 The ratio increased to about 3.19 times compared to the negative control, but the H 2 O 2 in the SH-SY5Y cell line And when the CM derived from the strain isolated in Example 1 was treated, the Bax/Bcl-2 expression ratio increased by only about 2.41 times compared to the negative control group. That is, since the CM derived from the strain isolated in Example 1 significantly lowered the Bax/Bcl-2 expression level ratio, the CM derived from the strain isolated in Example 1 mitigated the apoptosis-inducing action of H 2 O 2 . . On the other hand, when the SH-SY5Y cell line was treated with H 2 O 2 and CM derived from the strain isolated in Comparative Example 1, the Bax/Bcl-2 expression ratio increased by about 2.67 times or more compared to the negative control group. On the other hand, when the SH-SY5Y cell line was treated with only NaAsO 2 , the ratio of Bax/Bcl- 2 expression increased by about 2.57 times compared to the negative control group. And when the CM derived from the strain isolated in Example 1 was treated, the Bax/Bcl-2 expression ratio increased by only about 1.67 times compared to the negative control group. That is, the CM derived from the strain isolated in Example 1 significantly lowered the Bax/Bcl-2 expression ratio, and it was confirmed that the CM derived from the strain isolated in Example 1 alleviated the apoptosis-inducing action of NaAsO 2 . On the other hand, when the SH-SY5Y cell line was treated with NaAsO 2 and CM derived from the strain isolated in Comparative Example 1, the Bax/Bcl-2 expression ratio increased by about 2.99 times or more compared to the negative control group.
도 8에 나타난 바와 같이, 산화적 스트레스가 유도된 인간 유래 신경 세포의 Bax/Bcl-2 발현량 비율을 살펴본 결과, SH-SY5Y 세포주에 H2O2만 처리했을 경우 Bax/Bcl-2 발현량 비율이 음성대조군에 비해 약 2.19배로 증가하였으나, SH-SY5Y 세포주에 H2O2 및 실시예 1에서 분리된 균주 적용 요거트 유래 CM을 처리했을 경우 음성대조군에 비해 약 1.01배만 증가한 Bax/Bcl-2 발현량 비율을 보였다. BDNF 발현량은 음성대조군에 비해 약 1.12배 증가한 것으로 확인된다. 즉, 실시예 1에서 분리된 균주 유래 적용 요거트 유래 CM은 Bax/Bcl-2 발현량 비율을 음성대조군 수준으로 낮추었고, BDNF 발현량은 음성대조군 수준 이상으로 증가시켰는 바, 실시예 1에서 분리된 균주 적용 요거트 유래 CM은 H2O2의 세포 사멸 유도 작용을 완화하는 것으로 확인된다. 반면, SH-SY5Y 세포주에 H2O2 및 실시예 1에서 분리된 균주가 포함되지 않은 요거트(대조군 요거트) 유래 CM을 처리했을 경우에는 음성대조군에 비해 약 1.66배 이상 증가한 Bax/Bcl-2 발현량 비율을 보였다. As shown in FIG. 8, as a result of examining the Bax/Bcl-2 expression level ratio of human-derived neurons induced by oxidative stress, Bax/Bcl-2 expression level when the SH-SY5Y cell line was treated with only H 2 O 2 The ratio increased to about 2.19 times compared to the negative control, but the H 2 O 2 in the SH-SY5Y cell line And when the yogurt-derived CM applied with the strain isolated in Example 1 was treated, the Bax/Bcl-2 expression ratio increased by only about 1.01 times compared to the negative control group. It is confirmed that the BDNF expression level increased by about 1.12 times compared to the negative control group. That is, the yogurt-derived CM derived from the strain isolated in Example 1 lowered the Bax/Bcl-2 expression level ratio to the negative control level and increased the BDNF expression level to the negative control level or higher. It is confirmed that strain-applied yogurt-derived CM alleviates the apoptosis-inducing action of H 2 O 2 . On the other hand, when the SH-SY5Y cell line was treated with CM derived from yogurt (control yogurt) that did not contain H 2 O 2 and the strain isolated in Example 1, Bax/Bcl-2 expression increased by about 1.66 times or more compared to the negative control group. showed the volume ratio.
한편, SH-SY5Y 세포주에 NaAsO2 만 처리했을 경우 Bax/Bcl-2 발현량 비율이 음성대조군에 비해 약 1.92배로 증가하였으나, SH-SY5Y 세포주에 NaAsO2 및 실시예 1에서 분리된 균주 적용 요거트 유래 CM을 처리했을 경우 음성대조군에 비해 약 1.13배만 증가한 Bax/Bcl-2 발현량 비율을 보였다. BDNF 발현량은 음성대조군에 비해 약 1.08배 증가한 것으로 확인된다. 즉, 실시예 1에서 분리된 균주 적용 요거트 유래 CM은 Bax/Bcl-2 발현량 비율을 음성대조군 수준으로 낮추었고, BDNF 발현량은 음성대조군 수준 이상으로 증가시켰는바, 실시예 1에서 분리된 균주 적용 요거트 유래 CM은 NaAsO2의 세포 사멸 유도 작용을 완화하는 것으로 확인된다. 반면, SH-SY5Y 세포주에 NaAsO2 및 실시예 1에서 분리된 균주가 포함되지 않은 요거트(대조군 요거트) 유래 CM을 처리했을 경우에는 오히려 음성대조군에 비해 약 1.62배 이상 증가한 Bax/Bcl-2 발현량 비율을 보였다. On the other hand, when the SH-SY5Y cell line was treated with only NaAsO 2 , the Bax/Bcl-2 expression level ratio increased by about 1.92 times compared to the negative control group, but NaAsO 2 in the SH-SY5Y cell line And when the yogurt-derived CM applied with the strain isolated in Example 1 was treated, the Bax/Bcl-2 expression ratio increased by only about 1.13 times compared to the negative control group. It is confirmed that the BDNF expression level increased by about 1.08 times compared to the negative control group. That is, the strain-applied yogurt-derived CM isolated in Example 1 lowered the Bax/Bcl-2 expression level ratio to the negative control level, and increased the BDNF expression level to more than the negative control level. Application Yogurt-derived CM is confirmed to alleviate the apoptosis-inducing action of NaAsO 2 . On the other hand, when the SH-SY5Y cell line was treated with CM derived from yogurt (control yogurt) that did not contain NaAsO 2 and the strain isolated in Example 1, Bax / Bcl-2 expression increased by about 1.62 times or more compared to the negative control group ratio was shown.
하기에 본 발명의 균주를 유효성분으로 포함하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Hereinafter, formulation examples of compositions containing the strain of the present invention as an active ingredient will be described, but the present invention is not intended to limit them, but only to be specifically described.
제제예 1: 산제의 제조Formulation Example 1: Preparation of powder
Leuconostoc mesenteroides H40 균주 20 mg Leuconostoc mesenteroides H40 strain 20 mg
유당수화물 100 mg
탈크 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조하였다.A powder was prepared by mixing the above components and filling in airtight bags.
제제예 2: 정제의 제조Formulation Example 2: Preparation of tablets
Leuconostoc mesenteroides H40 균주 10 mg Leuconostoc mesenteroides H40 strain 10 mg
옥수수전분 100 mg
유당수화물 100 mg
스테아르산마그네슘 2 mg
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet manufacturing method.
제제예 3: 캅셀제의 제조Formulation Example 3: Preparation of capsule formulation
Leuconostoc mesenteroides H40 균주 10 mg Leuconostoc mesenteroides H40 strain 10 mg
미결정셀룰로오스 3 mgMicrocrystalline Cellulose 3 mg
유당수화물 14.8 mgLactose monohydrate 14.8 mg
스테아르산마그네슘 0.2 mgMagnesium Stearate 0.2 mg
상기의 성분을 혼합한 후, 통상의 캅셀제의 제조방법에 따라서 젤라틴캡슐에 충전하여 캅셀제를 제조하였다.After mixing the above ingredients, capsules were prepared by filling gelatin capsules according to a conventional capsule preparation method.
제제예 4: 주사제의 제조Formulation Example 4: Preparation of injection
Leuconostoc mesenteroides H40 균주 10 mg Leuconostoc mesenteroides H40 strain 10 mg
만니톨 180 mg
주사용 멸균 증류수 2974 mgSterile Distilled Water for Injection 2974 mg
인산일수소나트륨 26 mgSodium monohydrogen phosphate 26 mg
상기의 성분을 혼합한 후, 통상의 주사제의 제조방법에 따라 1앰플당(2 mL) 상기의 성분 함량으로 제조하였다.After mixing the above components, it was prepared with the above component content per 1 ampoule (2 mL) according to a conventional method for preparing injections.
제제예 5: 액제의 제조Formulation Example 5: Preparation of liquid formulation
Leuconostoc mesenteroides H40 균주 10 mg Leuconostoc mesenteroides H40 strain 10 mg
이성화당 10 gIsomerized sugar 10 g
만니톨 5 g5 g mannitol
정제수 적량Appropriate amount of purified water
레몬향 적량Lemon flavor appropriate amount
상기의 성분을 통상의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 정제수를 가하여 전체 100 mL로 조절한 후 멸균시켜 갈색병에 충진하여 액제를 제조한다. The above components are added to and dissolved in purified water according to a conventional manufacturing method, lemon flavor is added in an appropriate amount, purified water is added to adjust the total volume to 100 mL, and then sterilized and filled in a brown bottle to prepare a liquid formulation.
제제예 6: 건강기능식품의 제조Formulation Example 6: Manufacture of health functional food
Leuconostoc mesenteroides H40 균주 10 mg Leuconostoc mesenteroides H40 strain 10 mg
비타민 혼합물 적량Appropriate amount of vitamin mixture
비타민 A 아세테이트 70 ㎍Vitamin A Acetate 70 μg
비타민 E 1.0 ㎎Vitamin E 1.0 mg
비타민 B1 0.13 ㎎Vitamin B1 0.13 mg
비타민 B2 0.15 ㎎Vitamin B2 0.15 mg
비타민 B6 0.5 ㎎Vitamin B6 0.5 mg
비타민 B12 0.2 ㎍Vitamin B12 0.2 μg
비타민 C 10 ㎎
비오틴 10 ㎍10 μg of biotin
니코틴산아미드 1.7 ㎎Nicotinamide 1.7 mg
엽산 50 ㎍
판토텐산 칼슘 0.5 ㎎Calcium Pantothenate 0.5 mg
무기질 혼합물 적량Appropriate amount of mineral mixture
황산제1철 1.75 ㎎Ferrous sulfate 1.75 mg
산화아연 0.82 ㎎Zinc Oxide 0.82 mg
탄산마그네슘 25.3 ㎎Magnesium Carbonate 25.3 mg
제1인산칼륨 15 ㎎Potassium Phosphate Monobasic 15 mg
제2인산칼슘 55 ㎎Dibasic Calcium Phosphate 55 mg
구연산칼륨 90 ㎎
탄산칼슘 100 ㎎
염화마그네슘 24.8 ㎎Magnesium Chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강기능식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강기능식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강기능식품 제조에 사용할 수 있다.Although the composition ratio of the above vitamin and mineral mixture was mixed with ingredients suitable for health functional food in a preferred embodiment, the mixing ratio may be arbitrarily modified, and the above ingredients were mixed according to the usual method for manufacturing health functional food. Then, granules are prepared and can be used in the manufacture of health functional foods according to conventional methods.
제제예 7: 건강음료의 제조Formulation Example 7: Manufacture of health drink
Leuconostoc mesenteroides H40 균주 10 mg Leuconostoc mesenteroides H40 strain 10 mg
비타민 C 15 g15 g of vitamin C
비타민 E(분말) 100 g100 g of vitamin E (powder)
젖산철 19.75 g19.75 g iron lactate
산화아연 3.5 gZinc oxide 3.5 g
니코틴산아미드 3.5 gNicotinamide 3.5 g
비타민 A 0.2 gVitamin A 0.2 g
비타민 B1 0.25 gVitamin B1 0.25 g
비타민 B2 0.3gVitamin B2 0.3g
정제수 정량Quantification of purified water
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2 L 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다.After mixing the above ingredients according to the usual health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and collected in a sterilized 2 L container, sealed and sterilized, and then refrigerated. It is used for preparing the health drink composition of the present invention.
상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
<110> Konkuk University Industrial Cooperation Corp <120> NOVEL STRAIN OF Leuconostoc mesenteroides AND USE THEREOF <130> 2020-I025 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1431 <212> RNA <213> Leuconostoc mesenteroides <400> 1 cagtcgaacg cacagcgaaa ggtgcttgca cctttcaagt gagtggcgaa cgggtgagta 60 acacgtggac aacctgcctc aaggctgggg ataacatttg gaaacagatg ctaataccga 120 ataaaactta gtgtcgcatg acaaaaagtt aaaaggcgct tcggcgtcac ctagagatgg 180 atccgcggtg cattagttag ttggtggggt aaaggcctac caagacaatg atgcatagcc 240 gagttgagag actgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300 ctgcagtagg gaatcttcca caatgggcga aagcctgatg gagcaacgcc gcgtgtgtga 360 tgaaggcttt cgggtcgtaa agcactgttg tatgggaaga acagctagaa taggaaatga 420 ttttagtttg acggtaccat accagaaagg gacggctaaa tacgtgccag cagccgcggt 480 aatacgtatg tcccgagcgt tatccggatt tattgggcgt aaagcgagcg cagacggttt 540 attaagtctg atgtgaaagc ccggagctca actccggaat ggcattggaa actggttaac 600 ttgagtgcag tagaggtaag tggaactcca tgtgtagcgg tggaatgcgt agatatatgg 660 aagaacacca gtggcgaagg cggcttactg gactgcaact gacgttgagg ctcgaaagtg 720 tgggtagcaa acaggattag ataccctggt agtccacacc gtaaacgatg aacactaggt 780 gttaggaggt ttccgcctct tagtgccgaa gctaacgcat taagtgttcc gcctggggag 840 tacgaccgca aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat 900 gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ttgaagcttt 960 tagagataga agtgttctct tcggagacaa agtgacaggt ggtgcatggt cgtcgtcagc 1020 tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatt gttagttgcc 1080 agcattcaga tgggcactct agcgagactg ccggtgacaa accggaggaa ggcggggacg 1140 acgtcagatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcgtataca 1200 acgagttgcc aacccgcgag ggtgagctaa tctcttaaag tacgtctcag ttcggattgt 1260 agtctgcaac tcgactacat gaagtcggaa tcgctagtaa tcgcggatca gcacgccgcg 1320 gtgaatacgt tcccgggtct tgtacacacc gcccgtcaca ccatgggagt ttgtaatgcc 1380 caaagccggt ggcctaacct tttaggaagg agccgtctaa gcagacagag g 1431 <110> Konkuk University Industrial Cooperation Corp. <120> NOVEL STRAIN OF Leuconostoc mesenteroides AND USE THEREOF <130> 2020-I025 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1431 <212> RNA <213> Leuconostoc mesenteroides <400> 1 cagtcgaacg cacagcgaaa ggtgcttgca cctttcaagt gagtggcgaa cgggtgagta 60 acacgtggac aacctgcctc aaggctgggg ataacatttg gaaacagatg ctaataccga 120 ataaaactta gtgtcgcatg acaaaaagtt aaaaggcgct tcggcgtcac ctagagatgg 180 atccgcggtg cattagttag ttggtggggt aaaggcctac caagacaatg atgcatagcc 240 gagttgagag actgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300 ctgcagtagg gaatcttcca caatgggcga aagcctgatg gagcaacgcc gcgtgtgtga 360 tgaaggcttt cgggtcgtaa agcactgttg tatgggaaga acagctagaa taggaaatga 420 ttttagtttg acggtaccat accagaaagg gacggctaaa tacgtgccag cagccgcggt 480 aatacgtatg tcccgagcgt tatccggatt tattgggcgt aaagcgagcg cagacggttt 540 attaagtctg atgtgaaagc ccggagctca actccggaat ggcattggaa actggttaac 600 ttgagtgcag tagaggtaag tggaactcca tggttagcgg tggaatgcgt agatatatgg 660 aagaacacca gtggcgaagg cggcttactg gactgcaact gacgttgagg ctcgaaagtg 720 tgggtagcaa acaggattag ataccctggt agtccacacc gtaaacgatg aacactaggt 780 gttaggaggt ttccgcctct tagtgccgaa gctaacgcat taagtgttcc gcctggggag 840 tacgaccgca aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat 900 gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ttgaagcttt 960 tagagataga agtgttctct tcggagacaa agtgacaggt ggtgcatggt cgtcgtcagc 1020 tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttat gttagttgcc 1080 agcattcaga tgggcactct agcgagactg ccggtgacaa accggaggaa ggcggggacg 1140 acgtcagatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcgtataca 1200 acgagttgcc aacccgcgag ggtgagctaa tctcttaaag tacgtctcag ttcggattgt 1260 agtctgcaac tcgactacat gaagtcggaa tcgctagtaa tcgcggatca gcacgccgcg 1320 gtgaatacgt tcccgggtct tgtacacacc gcccgtcaca ccatgggagt ttgtaatgcc 1380 caaagccggt ggcctaacct tttaggaagg agccgtctaa gcagacagag g 1431
Claims (2)
Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) strain (KCCM 12614P) containing as an active ingredient Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, immune system abnormal brain dysfunction, progressive neurodegenerative disease, metabolic brain disease, A pharmaceutical composition for preventing or treating neurodegenerative diseases, which is one or more selected from the group consisting of Niemann-Pick disease, dementia due to cerebral ischemia and cerebral hemorrhage.
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| Food Sci. Biotechnol. (2018) 27(1):123-129.* |
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| Leuconostoc mesenteroides strain IMAU11274 (YM2-3) 16S ribosomal RNA gene, partial sequence, Genbank: KP764082.1. |
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