KR101239806B1 - The new Lactobacillus plantarum HY7712 stimulate immunity - Google Patents
The new Lactobacillus plantarum HY7712 stimulate immunity Download PDFInfo
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- KR101239806B1 KR101239806B1 KR1020110030183A KR20110030183A KR101239806B1 KR 101239806 B1 KR101239806 B1 KR 101239806B1 KR 1020110030183 A KR1020110030183 A KR 1020110030183A KR 20110030183 A KR20110030183 A KR 20110030183A KR 101239806 B1 KR101239806 B1 KR 101239806B1
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- lactobacillus plantarum
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Abstract
본 발명은 면역증강 및 항산화 활성을 가지는 신규한 락토바실러스 플란타룸 HY7712 및 이를 유효성분으로 함유하는 제품에 관한 것으로서, 본 발명에 따른 락토바실러스 플란타룸 HY7712는 대식세포의 TNF-α의 생산을 유도함으로써 면역증강활성이 매우 뛰어날 뿐만 아니라, 항산화 활성 및 야채를 발효능을 가지고 있어 면역증강용 약학조성물, 건강기능식품, 기능성음료, 발효제품 등 다양하게 이용될 수 있다.
과제번호: 810002-03-1-SB410
부처: 농림수산식품기술기획평가원
과제명: 국내농산물을 이용한 기능성 발효음료 개발The present invention relates to a novel Lactobacillus plantarum HY7712 having an immuno-enhancing and antioxidant activity and a product containing the same as an active ingredient, the Lactobacillus plantarum HY7712 according to the present invention is for the production of TNF-α of macrophages By inducing not only very excellent immune enhancing activity, but also has an antioxidant activity and fermentation ability of vegetables can be used in a variety of pharmaceutical composition, immune functional foods, functional drinks, fermentation products for immune enhancement.
Assignment number: 810002-03-1-SB410
Ministry of Agriculture, Forestry and Fisheries Food Technology Planning and Evaluation
Title: Development of functional fermented beverages using domestic agricultural products
Description
본 발명은 면역증강 및 항산화 활성을 가지는 신규한 락토바실러스 플란타룸 HY7712 및 이를 유효성분으로 함유하는 제품에 관한으로서, 보다 상세하게는 사람의 대식세포에서 면역지표인 TNF-α의 생산을 유도함으로써 면역증강 활성을 가짐은 물론 항산화 활성 및 야채 발효능을 가지는 신규한 락토바실러스 플란타룸 HY7712 및 이를 유효성분으로 함유하는 약학 조성물, 건강기능식품, 기능성음료, 발효제품에 관한 것이다.
The present invention relates to a novel Lactobacillus plantarum HY7712 having immuno-enhancing and antioxidant activity and a product containing the same as an active ingredient, more specifically by inducing the production of TNF-α, an immunomarker in human macrophages. The present invention relates to a novel Lactobacillus plantarum HY7712 having an immune enhancing activity as well as an antioxidant activity and a vegetable fermenting ability, and a pharmaceutical composition, a health food, a functional beverage, and a fermentation product containing the same as an active ingredient.
김치와 같은 전통 발효식품에 풍부하게 존재하는 유산균은 인체의 소화계에 공생하면서 섬유질 및 복합 단백질을 분해하여 중요한 영양성분으로 만드는 역할을 담당하며, 장내 pH를 산성으로 유지시켜 대장균이나 크로스트리디움(Chlostridum sp.)과 같은 유해균의 번식을 억제하고 설사와 변비를 개선할 뿐만 아니라, 비타민 합성, 혈중 콜레스테롤 저하 등의 역할을 한다. 특히 유산균은 장의 점막과 상피세포에 강하게 결합할 수 있는 특성이 있어 정장작용에 많은 도움을 준다. 또한, 유산균은 대식세포의 증식을 촉진하여 대식세포(macrophage)의 장내 유해 세균에 대한 인지능력, 살균능력을 강화시키고, 면역관련 물질의 분비를 촉진하여 면역 증강효과를 나타내는 것으로 알려져 있다(Gabriela peridgon et al. J of food Protection 53: 404-411, 1990: Katsumasa sato et al., Microbiol Immunol., 32(7): 689-698, 1998). Lactobacillus, abundant in traditional fermented foods such as kimchi, plays a role in decomposing fibrous and complex proteins as important nutrients while living in the digestive system of the human body, and keeps the intestinal pH acidic so that E. coli or Crosstridium ( Chlostridum) In addition to suppressing the reproduction of harmful bacteria such as sp.) and improving diarrhea and constipation, it also plays a role in vitamin synthesis and lowering blood cholesterol. In particular, lactic acid bacteria have a strong ability to bind to the mucous membrane and epithelial cells of the intestine help a lot of work. In addition, lactic acid bacteria are known to promote the proliferation of macrophages to enhance the cognitive and bactericidal capacity of macrophage intestinal harmful bacteria, and to promote the secretion of immune-related substances (Gabriela peridgon). et al. J of food Protection 53: 404-411, 1990: Katsumasa sato et al., Microbiol Immunol., 32 (7): 689-698, 1998).
또한, 유산균은 면역 조절 이상과 관련된 알레르기 및 아토피 질환에도 효과가 있어 유아에 투여 시 아토피 발생이 줄어들고(Kalliomki et al., Lancet 357:1076-1079, 2001) 아토피 습진부위 및 정도가 감소한다는 보고가 있다(Rsenfeldt et al., Dermatologic and ocular diseases 111: 389-395, 2003).
In addition, lactobacillus is also effective in allergic and atopic diseases related to immunomodulatory dysfunction, resulting in decreased atopic incidence when administered to infants (Kalliomki et al., Lancet 357: 1076-1079, 2001) and decreased atopic eczema sites and severity. (Rsenfeldt et al., Dermatologic and ocular diseases 111: 389-395, 2003).
특히, 락토바실러스 플란타룸은 락토바실러스 속 유산균으로 주로 김치, 피클과 같은 침채류, 토마토, 우유, 치즈, 버터 등에서 분리되며 자연계에서 가장 분포가 넓은 유산균 중 하나로서, 김치가 발효가 많이 되어 신맛이 날 때 주로 생장하는 균으로 내산성과 내담즙성이 강하며, 푸자리움(Fusarium), 슈도모나스(Pseudomonas)에 대한 항균활성을 가지고, 항산화 및 항염증 효과를 가지고 있어 매우 유용한 균주이다.
In particular, Lactobacillus plantarum is a lactic acid bacterium of the genus Lactobacillus, mainly separated from kimchi, pickles such as pickles, tomatoes, milk, cheese, and butter, and is one of the most widely distributed lactic acid bacteria in nature. It is a very useful strain because it has a strong acid resistance and bile resistance, has antibacterial activity against Fusarium and Pseudomonas, and has antioxidant and anti-inflammatory effects.
한편, 대한민국 특허공개 제2010-0063503호(발명의 명칭: 신규한 락토바실러스 플란타룸 및 이를 포함하는 조성물)에서는 항체성 면역인 Th2 과잉반응으로 인한 Th1/Th2 불균형을 조절하는 효과가 우수한 락토바실러스 플란타룸(Lactobacillus plantarum) CJLP136이 공개되어 있으나, 이는 과민성 면역반응 조절에 관한 활성으로 일반적인 면역증강 활성과는 다르다.
On the other hand, Korean Patent Publication No. 2010-0063503 (name of the invention: a novel Lactobacillus plantarum and a composition comprising the same) Lactobacillus is excellent in controlling the Th1 / Th2 imbalance due to Th2 overreaction, which is an antibody immunity Planta room ( Lactobacillus plantarum ) CJLP136 has been published, but this activity differs from the general immunopotentiating activity for the control of overactive immune response.
이에 본 발명자들은 락토바실러스 플란타룸 중 사람의 대식세포에서 TNF-α의 생산을 유도하여 대식세포를 활성화시키는 면역증강 활성을 가지는 락토바실러스 플란타룸을 선발함으로써 본 발명을 완성하였다.
Therefore, the present inventors completed the present invention by selecting Lactobacillus plantarum having immuno-enhancing activity of activating macrophages by inducing TNF-α production in human macrophages in Lactobacillus plantarum.
본 발명은 사람의 대식세포에서 면역지표인 TNF-α의 생산을 유도함으로써 면역증강 활성을 가짐은 물론 항산화 활성 및 야채 발효능을 가지는 신규한 락토바실러스 플란타룸 HY7712 및 이를 유효성분으로 함유하는 제품을 제공하는 것을 목적으로 한다.
The present invention is a novel Lactobacillus plantarum HY7712 and a product containing the same as an active ingredient, which has immune enhancing activity as well as antioxidant activity and vegetable fermentation ability by inducing the production of TNF-α, an immune marker in human macrophages The purpose is to provide.
상기의 목적을 달성하기 위하여, 본 발명은 사람의 대식세포에서 면역지표인 TNF-α의 생산을 유도함으로써 면역증강 활성을 가짐은 물론 항산화 활성 및 야채 발효능을 가지는 신규한 락토바실러스 플란타룸 HY7712 및 이를 유효성분으로 함유하는 제품을 제공하는 것을 특징으로 한다.
In order to achieve the above object, the present invention induces the production of TNF-α, an immune marker in human macrophages, has a novel Lactobacillus plantarum HY7712 that has an immunostimulatory activity as well as an antioxidant activity and vegetable fermentation ability. And it is characterized by providing a product containing this as an active ingredient.
이하, 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명에 따른 락토바실러스 플란타룸 HY7712는 김치로부터 분리 및 동정된 락토바실러스 플란타룸의 신규한 균주로서 미생물 동정 및 분류를 위한 16S rRNA 염기서열 분석결과, 락토바실러스 플란타룸 KW30(Lactobacillus plantrum KW30)과 가장 높은 상동성을 나타내어 락토바실러스 플란타룸과 가장 높은 분자계통학적 유연관계를 보여 상기 미생물을 락토바실러스 플란타룸으로 동정하고 락토바실러스 플란타룸HY7712로 명명하였으며, 생명공학연구원 유전자은행에 2011년 2월 23일자로 기탁하였다(기탁번호 KCTC 11874BP). 락토바실러스 플란타룸 HY7712의 16S rRNA 유전자의 염기서열은 본 명세서에 첨부된 염기서열 목록 SEQ No. 1과 같다.
Lactobacillus plantarum HY7712 according to the present invention is a novel strain of Lactobacillus plantarum isolated and identified from kimchi 16S rRNA sequence analysis for microorganism identification and classification, Lactobacillus plantarum KW30 ( Lactobacillus plantrum KW30 ), Which showed the highest homology with Lactobacillus plantarum and the highest molecular systemic relationship, the microorganism was identified as Lactobacillus plantarum and named Lactobacillus plantarum HY7712. Deposited 23 February 2011 (Accession No. KCTC 11874BP). The nucleotide sequence of the 16S rRNA gene of Lactobacillus plantarum HY7712 is shown in SEQ ID NO. Same as 1.
본 발명의 락토바실러스 플란타룸 HY7712는 그람 양성균이며 호기적인 조건과 혐기적인 조건에서 모두 성장이 가능한 통성 혐기성(facultive anaerobe)이며 포자를 형성하지 않고 운동성이 없으며 세포의 형태는 간균이다. 락토바실러스 플란타룸 HY7712의 보다 구체적인 생리학적 특성은 당해 기술 분야의 통상의 방법에 따라 분석한 결과 표 1과 같은 것으로 나타났다.
The Lactobacillus plantarum HY7712 of the present invention is a gram-positive bacterium, a facultive anaerobe capable of growing in both aerobic and anaerobic conditions, does not form spores, has no mobility, and has a cell form. The more specific physiological characteristics of Lactobacillus plantarum HY7712 were analyzed according to the conventional methods in the art, as shown in Table 1.
+: 양성반응, -: 음성반응 +: Positive,-: negative
본 발명의 락토바실러스 플란타룸 HY7712는 장기간 안정적으로 보존하기 위해서는 물에 글리세롤 성분을 일정량 혼합하여 만든 보관용액에 균체를 풀어 -70℃에서 보관하거나 멸균된 10% 탈지유에 현탁하여 동결건조하는 것이 바람직하다.
In order to stably preserve Lactobacillus plantarum HY7712 of the present invention, it is preferable to dissolve cells in a storage solution made by mixing a certain amount of glycerol components in water, store them at -70 ° C, or suspend them in sterilized 10% skim milk to freeze-dry them. Do.
TNF-α는 면역제어와 염증반응에 핵심적 조절자 역할을 담당하는 사이토카인(cytokine)이다. TNF-α는 세균의 세포막에 있는 내독소인 LPS(lipopolysaccharide)에 의해 활성화된 림프구에 의해 만들어지는데, 많은 종류의 사이토카인 분비를 조절하는 면역반응에 있어서 가장 초기에 일차적인 매개체로 작용한다. 또한 T 세포와 B 세포의 활성화에 공동자극제(co-stimulator)로 작용하기도 한다. 따라서 TNF-α는 염증반응에서 백혈구들이 혈관 내피세포에 부착하는 것을 촉진하며 염증세포들의 미생물 살해능력을 증가시키며, 단핵 식균세포(mononuclear phagocytes)에 작용하여 여러 가지 염증반응에 관여하는 사이토카인을 생산하게 만들어 염증반응을 촉진한다. TNF-α는 또한 종양세포에 작용하여 세포자연사를 유도하기도 한다.TNF-α is a cytokine that plays a key regulator in immune control and inflammatory responses. TNF-α is produced by lymphocytes activated by LPS (lipopolysaccharide), an endotoxin in bacterial cell membranes, and is the earliest primary mediator of immune responses that regulate many types of cytokine secretion. It also acts as a co-stimulator for the activation of T cells and B cells. Thus, TNF-α promotes the adhesion of leukocytes to vascular endothelial cells in inflammatory reactions, increases the ability of inflammatory cells to kill microorganisms, and acts on mononuclear phagocytes to produce cytokines involved in various inflammatory responses To stimulate the inflammatory response. TNF-α also acts on tumor cells to induce cellular apoptosis.
대식세포는 면역반응에 관계하는 주 세포로서 면역세포의 작용을 조절하는 사이토카인을 분비하여 면역반응 조절, 자연면역에서 미생물, 항원, 죽은 조직 등을 식균 작용으로 파괴, 체액성 면역 반응에서 작용세포(effector cell)로서의 활동, 지연성 과민 반응(delayed type hypersensivity)에서 항원 제거, TNF-α 분비를 통한 종양세포의 파괴, 획득면역반응에서 항원의 조작과 제시(antigen processing and presentation), 염증반응에 관여하는 물질이나 사이토카인을 분비하여 염증반응을 조절, 섬유아세포와 혈관내피세포의 성장인자를 생산하여 손상된 조직의 치유 등 다양한 기능을 한다.Macrophages are the main cells involved in the immune response and secrete cytokines that regulate the function of the immune cells. activity as an effector cell, antigen removal in delayed type hypersensivity, destruction of tumor cells via TNF-α secretion, manipulation and presentation of antigens in acquired immune responses, and inflammatory responses. It functions by controlling secretory substances and cytokines to control inflammatory reactions, producing growth factors of fibroblasts and vascular endothelial cells, and healing damaged tissues.
본 발명에서는 사람의 대식세포에 김치에서 분리한 락토바실러스 플란타룸 종을 처리하고 이에 TNF-α가 특히 높게 생산되는 락토바실러스 플란타룸 HY7712를 선발하였다.
In the present invention, Lactobacillus plantarum HY7712, in which human macrophages were treated with Lactobacillus plantarum species isolated from kimchi and TNF-α was produced particularly high, was selected.
본 발명의 면역증강 활성을 가지는 락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 면역증강용 약학 조성물은 제제 시에 통상적으로 이용되는 것으로, 락토스, 덱스트로스, 수크로스, 젤라틴 등의 부형제들과 함께 이용될 수 있으며 상기의 성분 외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.Immunity-enhancing pharmaceutical compositions containing Lactobacillus plantarum HY7712 having an immuno-enhancing activity of the present invention as an active ingredient are commonly used in the preparation, together with excipients such as lactose, dextrose, sucrose, gelatin and the like. It may be used and may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
본 발명의 락토바실러스 플란타룸 HY7712는 경구투여 및 기타 투여방법으로 복용하였을 시에도 다른 유익균에는 영향을 미치지 않으므로 안전하다. 본 발명의 면역증강 활성을 가지는 락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 면역증강용 약학 조성물은 건조 또는 액체상태의 제제 형태일 수 있고,락토바실러스 플란타룸 HY7712를 단일 또는 다른 미생물과 복합으로 혼합하여 이용할 수 있다.
Lactobacillus plantarum HY7712 of the present invention is safe because it does not affect other beneficial bacteria even when taken by oral administration and other administration methods. Immunostimulating pharmaceutical composition containing the Lactobacillus plantarum HY7712 having an immunostimulating activity of the present invention as an active ingredient may be in the form of a dry or liquid formulation, Lactobacillus plantarum HY7712 combined with a single or other microorganisms It can be mixed and used.
또한, 본 발명의 락토바실러스 플란타룸 HY7712는 항산화 활성이 매우 뛰어나다. 우리의 몸은 수없이 많은 산화적 손상 속에 노출되어 있는데 유해산소, 활성산소라고 하는 것이 자연발생적으로 생겨난다. 이 활성산소는 세포의 구성성분인 지질, 단백질, 당, DNA 등과 반응하여 파괴작용을 함으로써 노화는 물론, 암, 뇌질환, 심장질환 등의 각종 질병을 일으키는 원인이 된다. 활성산소를 막아주는 물질을 항산화제(antioxidants)라고 하는데 인체에 자연적으로 존재하는 항산화제는 물론, 비타민 E, C, 베타카로틴 등 외부에서 제공해주는 항산화제도 있다. 항산화 효과를 가지는 유산균은 비피도 박테리움 인판티스(Bifidobacterium infantis), 비피도 박테리움 롱검(Bifidobacterium longum), 락토바실러스 퍼멘텀(lactobacillus fermentum), 락토코커스 락티스(lactococcus lactis) ssp. 락티스(lactis) 등이 알려져 있으며, 본 발명의 신규한 락토바실러스 플란타룸 HY7712 역시 항산화 활성을 가지고 있다.
In addition, the Lactobacillus plantarum HY7712 of the present invention is very excellent in antioxidant activity. Our bodies are exposed to numerous oxidative damages, and free radicals and free radicals occur naturally. This reactive oxygen reacts with lipids, proteins, sugars, and DNA, which are the constituents of the cell, and causes destruction, thereby causing various diseases such as aging, cancer, brain disease, and heart disease. Antioxidants are substances that block the active oxygen, which is an antioxidant that provides naturally occurring antioxidants in the body as well as vitamin E, C, and beta carotene. Lactic acid bacteria having antioxidant effects include Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus fermentum, Lactococcus lactis ssp. Lactis and the like are known, and the novel Lactobacillus plantarum HY7712 of the present invention also has antioxidant activity.
또한, 본 발명의 락토바실러스 플란타룸 HY7712는 발효 야채주스를 제조하는 데에도 적합하다. 야채발효는 야채 고유의 맛과 향을 증진시키고 항산화 효과를 증진시키는 데에 도움이 된다. 풍미 이외에도 야채발효를 통해 소화력을 높이고 영양성분을 증가시키는 효과도 얻을 수 있으며 비타민 C의 경우 발효야채에서 보존이 더욱 잘된다는 연구 결과가 있다(Kalantzopolos G. 1997. Anaerobe. 3: 185-190. Nout MJR, Motarjemi Y. 1996. Food Control. 8: 553-559). 이에 따라 본 발명의 신규한 락토바실러스 플란타룸 HY7712는 야채주스를 발효시켜 풍미와 영양성분이 증진됨은 물론, 항산화 효과와 면역증강 효과를 가지는 야채주스를 제공할 수 있다.
In addition, the Lactobacillus plantarum HY7712 of the present invention is also suitable for preparing fermented vegetable juice. Vegetable fermentation helps to increase the inherent taste and aroma of vegetables and to increase the antioxidant effect. In addition to flavors, the fermentation of vegetables can increase digestion and increase nutrients. Vitamin C is also better preserved in fermented vegetables (Kalantzopolos G. 1997. Anaerobe. 3: 185-190. Nout MJR, Motarjemi Y. 1996. Food Control. 8: 553-559). Accordingly, the novel Lactobacillus plantarum HY7712 of the present invention can ferment vegetable juice to enhance flavor and nutrients, as well as provide vegetable juice having antioxidant and immune enhancing effects.
이상에서와 같이, 본 발명의 락토바실러스 플란타룸 HY7712는 면역증강 활성, 황산화 활성 및 야채 발효능이 뛰어나, 이를 유효성분으로 함유하는 약학 조성물, 건강기능식품, 기능성음료, 발효제품 등으로 이용될 수 있다.
As described above, the Lactobacillus plantarum HY7712 of the present invention is excellent in immunopotentiation activity, sulfate activity and vegetable fermentation ability, it is used as a pharmaceutical composition, health functional food, functional beverage, fermentation products, etc. Can be.
도 1은 김치로부터 분리된 락토바실러스 플란타룸 균주들 중에 락토바실러스 플란타룸 HY7712가 가장 높은 면역증강율을 보임을 나타낸 그래프이다.
a: KM22-1, b: KM2-1, c: KM20-3, d: KM19-6, e: KM18-3, f: KM16-3, g: KM14-7, h: KM12-3 i: KM1-2, j: K7-9 k: K7-1, l:K6-3, m: K5-8, n: K5-5, o: K3-2 p: K20-15, q: K12-7, r: K12-2
도 2는 락토바실러스 플란타룸 HY7712의 농도별 면역지표인 TNF-α의 생산유도를 나타낸 그래프이다.
도 3은 락토바실러스 플란타룸 균종의 DPPH 라디칼 소거능을 측정한 결과를 나타낸 그래프이다.
도 4는 락토바실러스 플란타룸 균종의 환원활성을 측정한 결과를 나타낸 그래프이다.
도 5는 락토바실러스 플란타룸 균종의 활성산소 흡수력을 측정한 결과를 나타낸 그래프이다.
도 6은 락토바실러스 플란타룸 HY7712의 야채주스에서의 생장 곡선을 나타낸 그래프이다.FIG. 1 is a graph showing the highest immunopotentiation rate of Lactobacillus plantarum HY7712 among Lactobacillus plantarum strains isolated from kimchi.
a: KM22-1, b: KM2-1, c: KM20-3, d: KM19-6, e: KM18-3, f: KM16-3, g: KM14-7, h: KM12-3 i: KM1 -2, j: K7-9 k: K7-1, l: K6-3, m: K5-8, n: K5-5, o: K3-2 p: K20-15, q: K12-7, r K12-2
Figure 2 is a graph showing the production induction of TNF-α, the immune markers by concentration of Lactobacillus plantarum HY7712.
Figure 3 is a graph showing the results of measuring the DPPH radical scavenging ability of the Lactobacillus plantarum species.
Figure 4 is a graph showing the results of measuring the reducing activity of the Lactobacillus plantarum species.
5 is a graph showing the results of measuring the active oxygen absorption capacity of the Lactobacillus plantarum species.
Figure 6 is a graph showing the growth curve in vegetable juice of Lactobacillus plantarum HY7712.
이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 다음의 실시예는 본 발명의 범위를 한정하는 것은 아니며, 본 발명의 기술적 사상의 범위 내에서 당업자에 의한 통상적인 변화가 가능하다.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are not intended to limit the scope of the present invention, and ordinary changes by those skilled in the art are possible within the scope of the technical idea of the present invention.
<실시예 1>≪ Example 1 >
김치로부터 락토바실러스 플란타룸 HY7712의 분리 및 동정Isolation and Identification of Lactobacillus Planta Room HY7712 from Kimchi
본 발명에 따른 락토바실러스 플란타룸은 각 지역에서 수집한 김치 국물에서 분리되었다. 김치 국물을 십진희석하여 1.5% 한천(agar)이 포함된 MRS 고체배지에 도말하여 37℃에서 48시간 배양한 후 콜로니(colony)를 백금이(loop)로 취하여 새로운 MRS 고체배지에 접종하고 순수분리 하였다. 순수분리된 콜로니를 MRS 액체배지에 접종하여 37℃에서 24시간 배양하였다. 실험에 이용한 락토바실러스 플란타룸은 총 53종으로 대부분 서로 다른 김치에서 분리하였다. Lactobacillus plantarum according to the present invention was isolated from kimchi broth collected in each region. Kimchi broth was diluted with dilution and smeared on MRS solid medium containing 1.5% agar and incubated for 48 hours at 37 ° C. Colonies were taken as platinum loops and inoculated in new MRS solid medium and purified. It was. Purely isolated colonies were inoculated in MRS liquid medium and incubated at 37 ° C. for 24 hours. Lactobacillus plantarum used in the experiment was a total of 53 species, mostly separated from different kimchi.
이후, 분리한 균주의 형태 및 생리학적 특성을 API 50CH 및 API 50CHL 키트를 사용하여 결정하였다. 상기 표 1은 HY7712 균주의 API 키트 결과로서, HY7712 균주는 락토바실러스 플란타룸의 당이용성과 동일하였다. 또한, 유산균 동정 및 분류를 위한 16S rRNA 유전자 염기서열을 분석하였으며, 그 결과 확인된 HY7712 균주의 16S rRNA 유전자 염기서열은 서열목록에 기재하였다(SEQ No. 1). 16S rRNA 유전자 염기서열 분석결과 HY7712 균주는 락토바실러스 플란타룸 표준균주와 가장 높은 상동성을 나타내어 락토바실러스 플란타룸(Lactobacillus plantarum)으로 동정하고, 락토바실러스 플란타룸 HY7712로 명명하였으며, 생명공학연구원 유전자 은행에 2011년 2월 23자로 기탁하였다(기탁번호: KCTC 11874BP).
The morphology and physiological properties of the isolated strains were then determined using API 50CH and API 50CHL kits. Table 1 shows the results of the API kit of the HY7712 strain, the HY7712 strain was the same as the sugar availability of Lactobacillus plantarum. In addition, 16S rRNA gene sequences for lactic acid bacteria identification and classification were analyzed, and the resulting 16S rRNA gene sequences were identified in the sequence list (SEQ No. 1). As a result of 16S rRNA gene sequencing analysis, HY7712 strain showed the highest homology with Lactobacillus plantarum standard strain and was identified as Lactobacillus plantarum and named as Lactobacillus plantarum HY7712. Deposited 23 February 2011 to the Gene Bank (Accession Number: KCTC 11874BP).
<실시예 2><Example 2>
락토바실러스 플란타룸 HY7712를 포함한 동결건조분말 제조Preparation of lyophilized powders, including Lactobacillus plantarum HY7712
상기 실시예 1의 락토바실러스 플란타룸 HY7712를 엠알에스 배지(MRS broth)에 접종하고 37℃ 배양기에서 24시간 동안 정치배양 하였다. 이것을 5000rpm에서 20분간 원심분리하여 상층액을 제거하고, 식염수로 균체를 세척한 후 다시 5000 rpm에서 20분간 원심분리하여 상층액을 제거하였다. 수거한 균체는 보존제인 탈지분유 분말과 1:1로 섞어 동결건조하여 분말을 제조하였다.Lactobacillus plantarum HY7712 of Example 1 was inoculated in MRS broth, and cultured in a 37 ° C. incubator for 24 hours. The supernatant was removed by centrifugation at 5000 rpm for 20 minutes, the cells were washed with saline, and then centrifuged at 5000 rpm for 20 minutes to remove the supernatant. The collected cells were lyophilized by mixing 1: 1 with skim milk powder as a preservative to prepare a powder.
한편, 본 발명의 락토바실러스 플란타룸 HY7712는 상기와 같이 동결건조된 분말 형태 또는 배양물 형태로 제공될 수 있다.
On the other hand, Lactobacillus plantarum HY7712 of the present invention may be provided in the form of lyophilized powder or culture as described above.
<실시예 3><Example 3>
락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 약학적 조성물의 제조Preparation of a pharmaceutical composition containing Lactobacillus plantarum HY7712 as an active ingredient
액제의 제조Preparation of liquid
상기 실시예 2의 락토바실러스 플란타룸 HY7712를 포함한 동결건조분말 100㎎,Lyophilized powder 100mg, including Lactobacillus plantarum HY7712 of Example 2,
이성화당 10g, 만니톨 5g을 통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 정제수를 가하여 전체 100㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조하였다.
10 g isomerized sugar and 5 g of mannitol were dissolved in purified water according to the conventional method for preparing a liquid solution, and lemon flavor was added thereto, and then purified water was added to adjust the total amount to 100 ml. It was.
캡슐제의 제조Preparation of capsules
상기 실시예 2의 락토바실러스 플란타룸 HY7712를 포함한 동결건조분말 100㎎에 옥수수 전분 100㎎, 유당 100㎎, 스테아린산 마그네슘 2㎎을 완전히 혼합한 후 통상의 캡슐제의 제조방법에 따라 경질 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.
100 mg of lyophilized powder including Lactobacillus plantarum HY7712 of Example 2 was thoroughly mixed with 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate, and then hard gelatine capsules were prepared according to a conventional capsule preparation method. The capsule was prepared by filling.
<실시예 4> <Example 4>
락토바실러스 플란타룸 HY7712을 유효성분으로 함유하는 발효유의 제조Preparation of fermented milk containing Lactobacillus plantarum HY7712 as an active ingredient
본 발명의 면역증강 및 항산화 활성을 갖는 락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 발효유를 제조하는 방법은 다음과 같다. Method for producing a fermented milk containing Lactobacillus plantarum HY7712 having an immune enhancing and antioxidant activity of the present invention as an active ingredient is as follows.
먼저, 유산균 배양액은 원유 95.36중량%와 탈지분유(또는 혼합분유) 4.6중량%를 교반하여 15℃에서의 비중은 1.0473~1.0475, 적정산도는 0.200~0.220%, pH는 6.65~6.70, 20℃에서의 브릭스(Brixo)는 16.3~16.5%정도가 되도록 혼합하였다. 혼합 후에 이를 UHT 열처리(135℃에서 2초간 살균)하고 40℃로 냉각한 뒤, 스트렙토코커스 써모필러스균과 유당분해효소(Valley laboratory, USA)를 각기 0.02중량%씩 첨가하고 6시간 동안 배양하여 BCP 배지에서의 총 유산균수가 1.0×109 cfu/㎖ 이상, 적정산도가 0.89~0.91%, pH는 4.55~4.65가 되도록 하여 제조하였다.The lactic acid bacteria culture solution was prepared by stirring 95.36 wt% of crude oil and 4.6 wt% of skim milk powder (or mixed powdered milk) and measuring specific gravity at 15 ° C from 1.0473 to 1.0475, a titratable acidity of 0.200 to 0.220%, pH of 6.65 to 6.70, of Brix (Brix o) were mixed such that the degree of 16.3 ~ 16.5%. After mixing, it was UHT heat-treated (sterilized at 135 ° C for 2 seconds), cooled to 40 ° C, and then Streptococcus thermophilus and Lactobacillus (Valley laboratory, USA) were added at 0.02% by weight, respectively, and incubated for 6 hours, followed by BCP. Total lactic acid bacteria in the medium was 1.0 × 10 9 cfu / ㎖ or more, the titratable acidity was 0.89 ~ 0.91%, pH was prepared to be 4.55 ~ 4.65.
그런 다음, 혼합과즙시럽은 액상과당 13중량%, 백설탕 5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.Then, mixed juice syrup, liquid fructose, 13 wt.%, Sugar 5 wt.%, Mixed juice concentrate 56Brix o 10.9% by weight of pectin, 1.0% by weight, fresh fruit mix Essence 0.1% and purified water 70% by weight at 30 ~ 35 ℃ Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled.
그런 다음, 상기 유산균배양액 69.5중량%와 실시예 2의 락토바실러스 플란타룸 HY7712를 포함한 동결건조분말 0.1중량% 및 상기 혼합과즙시럽 30.4중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각하여 락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 발효유를 제조하였다.
Then, 69.5% by weight of the lactic acid bacteria culture medium, 0.1% by weight of lyophilized powder including Lactobacillus plantarum HY7712 of Example 2, and 30.4% by weight of the mixed fruit juice syrup were homogenized at 150 bar and then cooled to 10 ° C. or less. Fermented milk containing Lactobacillus plantarum HY7712 as an active ingredient was prepared.
<실시예 5> <Example 5>
락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 야채주스의 제조Preparation of vegetable juice containing Lactobacillus plantarum HY7712 as an active ingredient
본 발명의 면역증강 및 항산화 활성을 갖는 락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 야채주스를 제조하는 방법은 다음과 같다.Method for producing a vegetable juice containing Lactobacillus plantarum HY7712 having an immune enhancing and antioxidant activity of the present invention as an active ingredient is as follows.
고구마, 단호박 등의 혼합야채즙 1.7중량%, 당근즙 52중량%, 토마토즙 42중량%, 시금치즙 1중량%, 양상추즙 2중량%, 셀러리즙 1중량%, 무수구연산 0.2중량%, 상기 실시예 2의 락토바실러스 플란타룸 HY7712를 포함한 동결건조분말 0.1중량%를 배합기에서 균일하게 혼합한 후 살균기를 이용하여 85~97℃에서 살균하여 락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 야채주스를 제조하였다.
1.7% by weight of mixed vegetable juices such as sweet potatoes and sweet pumpkin, 52% by weight carrot juice, 42% by weight tomato juice, 1% by weight spinach juice, 2% by weight lettuce juice, 1% by weight celery juice, 0.2% by weight citric anhydride, Vegetable juice containing Lactobacillus plantarum HY7712 as an active ingredient by uniformly mixing 0.1% by weight of the lyophilized powder containing Lactobacillus plantarum HY7712 of Example 2 in a blender and then sterilizing at 85 ~ 97 ℃ using a sterilizer. Was prepared.
<실시예 6> <Example 6>
락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 기능성 음료의 제조Preparation of functional beverage containing Lactobacillus plantarum HY7712 as an active ingredient
본 발명의 면역증강 및 항산화 활성을 갖는 락토바실러스 플란타룸 HY7712을 유효성분으로 함유하는 기능성 음료를 제조하는 방법은 다음과 같다.Method for producing a functional beverage containing Lactobacillus plantarum HY7712 having an immune enhancing and antioxidant activity of the present invention as an active ingredient is as follows.
먼저, 혼합과즙시럽은 액상과당 13중량%, 백설탕 2.5중량%, 갈색설탕 2.5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.First, a mixed juice syrup was 13% by weight of liquid fructose, white sugar, 2.5 wt%, brown sugar 2.5% by weight, mixed juice concentrate 56Brix o 10.9% by weight of pectin, 1.0% by weight, fresh 0.1% by weight fruit mix essence and purified water 70% The mixture was stirred at 30 to 35 ° C, mixed, and heat-treated by UHT (sterilized at 135 ° C for 2 seconds) and cooled.
그리고, 상기의 방법으로 제조된 혼합과즙시럽 30.4중량%와 상기 실시예 2의 락토바실러스 플란타룸 HY7712를 포함한 동결건조분말 0.1중량% 및 나머지 정제수 69.5중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 이를 유리병, 패트병 등 소포장 용기에 포장하여 락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 기능성 음료를 제조하였다.
Then, the mixture was prepared in the above method and mixed with 30.4% by weight of the juice syrup, 0.1% by weight of the lyophilized powder including the Lactobacillus plantarum HY7712 of Example 2 and 69.5% by weight of the remaining purified water were homogenized at 150 bar and then 10 ° C. After cooling to below, it was packaged in a small packaging container such as a glass bottle or a plastic bottle to prepare a functional beverage containing Lactobacillus plantarum HY7712 as an active ingredient.
<실시예 7> ≪ Example 7 >
락토바실러스 플란타룸 HY7712를 유효성분으로 함유하는 건강기능식품의 제조Preparation of dietary supplement containing Lactobacillus plantarum HY7712 as an active ingredient
상기 실시예 2의 락토바실러스 플란타룸 HY7712를 포함한 동결건조분말 0.1중량%에 영양보조성분(비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드) 및 올리고당을 상기의 실시예 2의 락토바실러스 플란타룸 HY7712를 포함한 동결건조분말 100중량부에 대하여 10중량부가 되도록 첨가하여 고속회전 혼합기에서 혼합하였다. 상기 혼합물에 멸균 정제수 10중량부를 첨가, 혼합하고 직경 1~2mm의 과립상으로 성형하였다. 상기 성형된 과립은 40~50℃의 진공건조기에서 건조시킨 후 12~14메쉬(mesh)를 통과시켜 균일하게 과립을 제조하였다. 상기와 같이 제조된 과립은 적당량씩 압출 성형되어 정제 또는 분말로 되거나 경질캡슐에 충전되어 경질캡슐제품으로 제조하였다.
In 0.1% by weight of the lyophilized powder containing Lactobacillus plantarum HY7712 of Example 2, nutritional supplements (vitamins B1, B2, B5, B6, E and acetate esters, nicotinic acid amide) and oligosaccharides of Example 2 To 100 parts by weight of the lyophilized powder, including Lactobacillus plantarum HY7712 was added to 10 parts by weight and mixed in a high speed rotary mixer. 10 parts by weight of sterile purified water was added to the mixture, mixed, and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum dryer at 40 to 50 ° C., and then passed through 12 to 14 meshes to prepare granules uniformly. The granules prepared as described above were extruded by appropriate amounts into tablets or powders or filled into hard capsules to produce hard capsule products.
<시험예 1>≪ Test Example 1 >
락토바실러스 플란타룸 HY7712의 TNF-α생산에 대한 영향 조사Investigation of the effects of Lactobacillus plantarum HY7712 on TNF-α production
TNF-α는 면역제어와 염증반응에 핵심적 조절자 역할을 담당하는 사이토카인이다. 따라서 본 발명의 김치분리균인 락토바실러스 플란타룸 균종들이 TNF-α의 생산에 어떠한 영향을 미치는지 조사하였다.
TNF-α is a cytokine that plays a key regulator of immune regulation and inflammatory response. Therefore, the effects of the Lactobacillus plantarum strains of the kimchi isolate of the present invention on the production of TNF-α were investigated.
<1-1> 대식세포의 배양 <1-1> Macrophage Culture
본 시험에 사용된 동물 세포주로는 인간유래 단핵백혈구(monocyte) 세포주인 THP-1 세포주를 한국세포주은행(cellbank.snu.ac.kr)에서 분양받아 사용하였다. 일반적인 세포의 유지를 위해서 85% RPMI 1640 배지(Gibco#22400)와 15%의 FBS(Gibco#16000)가 함유된 배지에서 세포를 배양하였으며, 1:4의 비율로 주 1회 계대 배양을 수행하였다.
As the animal cell line used in this test, THP-1 cell line, a human-derived mononuclear leukocyte (monocyte) cell line, was used by the Korea Cell Line Bank (cellbank.snu.ac.kr). To maintain normal cells, cells were cultured in a medium containing 85% RPMI 1640 medium (Gibco # 22400) and 15% FBS (Gibco # 16000), and passaged once a week at a ratio of 1: 4. .
<1-2> 유산균 시료의 제조 <1-2> Preparation of lactic acid bacteria sample
MRS 액체배지에 배양된 각 지역에서 수집한 김치 국물에서 분리된 락토바실러스 플란타룸 배양액을 취하여 원심분리 후 균체를 PBS로 3회 세척하였다. 세척된 락토바실러스 플란타룸 균체를 100℃에서 15분간 중탕으로 끓임으로서 사멸시킨 후 실험에 이용하였다.
The Lactobacillus plantarum culture medium separated from the kimchi broth collected in each region incubated in MRS liquid medium was taken, and the cells were washed three times with PBS after centrifugation. The washed Lactobacillus plantarum cells were killed by boiling in a hot bath at 100 ° C. for 15 minutes and then used in the experiment.
<1-3> 락토바실러스 플란타룸 처리에 의한 THP-1 세포에서의 TNF-α 발현량 측정 <1-3> Measurement of TNF-α Expression in THP-1 Cells by Lactobacillus Plantarum Treatment
THP-1 세포를 1:4의 비율로 96 well plate에 계대배양 한 후 48시간 동안 배양하였다. 각 well당 105 CFU의 사멸된 락토바실러스 플란타룸 균종을 처리한 후 18시간동안 추가 배양하였다. 각 well에서 조심스럽게 배지를 수거 후 원심분리하여 유산균체를 제거한 상등액을 취하고 TNF-α ELISA kit(BD#550610)을 사용하여 배지에 존재하는 TNF-α의 양을 정량하였다.THP-1 cells were passaged in 96 well plates at a ratio of 1: 4 and incubated for 48 hours. Each well was treated with 10 5 CFU of killed Lactobacillus plantarum strains and further incubated for 18 hours. After carefully collecting the medium from each well, the supernatant from which lactic acid cells were removed by centrifugation was taken, and the amount of TNF-α present in the medium was quantified using a TNF-α ELISA kit (BD # 550610).
간단하게 실험방법을 기술하면, ELISA plate의 각 well 당 50㎕의 ELISA diluent를 첨가하여 코팅(coating)된 항체(antibody)를 활성화 시킨 후, 여기에 샘플 또는 standard 100㎕를 첨가하고 플레이트(plate)를 sealing한 후 2시간 동안 상온에서 반응시켰다. 300㎕의 Wash buffer로 5회 세척 후, anti-TNF-α 항체 희석액을 100㎕ 첨가 한 후 플레이트를 sealing한 후 1시간 동안 반응시켰다. 300㎕의 Wash buffer로 7회 세척 후, TMB 발색 시약을 100㎕ 첨가하여 상온에서 30분간 발색 반응을 유도한 후 50㎕의 Stop solution을 첨가하고 450㎚에서 흡광도를 측정하였다. 모든 실험은 3반복을 수행하여 각 시료 당 평균과 표준 편차를 계산하였다.If the test method is described simply by adding 50 μl of the ELISA diluent to each well of the ELISA plate to activate the coated antibody, add 100 μl of the sample or standard thereto, And then reacted at room temperature for 2 hours. After washing 5 times with 300 μl of wash buffer, 100 μl of anti-TNF-α antibody dilution was added, and the plate was sealed and reacted for 1 hour. After washing 7 times with 300 μl of wash buffer, 100 μl of TMB coloring reagent was added, and a color reaction was induced at room temperature for 30 minutes. Then, 50 μl of stop solution was added and absorbance was measured at 450 nm. All experiments were performed with three iterations to calculate the mean and standard deviation per sample.
그 결과를 도 1에 나타내었다.
The results are shown in FIG.
도 1에서 확인할 수 있는 바와 같이, 각 지역에서 수집한 김치 국물에서 분리된 락토바실러스 플란타룸 균주들 중 본 발명의 락토바실러스 플란타룸 HY7712만이 특히 높은 수준으로 TNF-α를 유도하여 THP-1세포에서 면역반응을 증가시킴을 확인할 수 있었다.
As can be seen in Figure 1, of the Lactobacillus plantarum strains isolated from the Kimchi broth collected in each region only Lactobacillus plantarum HY7712 of the present invention induce TNF-α to a particularly high level THP-1 Increased immune response in the cell was confirmed.
<1-4> 락토바실러스 플란타룸 HY7712의 농도별 처리 <1-4> Concentration Treatment of Lactobacillus Planta Room HY7712
위의 방법과 마찬가지로 THP-1세포에 락토바실러스 플란타룸 HY7712를 농도별로 처리하여 TNF-α의 유도 정도를 비교하였다. In the same manner as above, THP-1 cells were treated with Lactobacillus plantarum HY7712 at different concentrations to compare the induction of TNF-α.
무처리군은 세포에 아무 처리도 하지 않은 상태에서의 TNF-α의 양을 측정하였고, LPS 처리군은 세포에 LPS를 100 ng/mL 농도로 처리한 상태에서의 TNF-α의 양을 측정하였고, HY7712(저농도)는 HY7712 105 CFU/well, HY7712(중농도)는 HY7712 106 CFU/well, HY7712(고농도)는 HY7712 107 CFU/well로 처리한 상태에서 TNF-α의 양을 측정하였다.The untreated group measured the amount of TNF-α in the cells without any treatment, and the LPS treated group measured the amount of TNF-α in the cells treated with LPS at a concentration of 100 ng / mL. , HY7712 (low concentration) was measured with
그 결과를 도 2에 나타내었다.
The results are shown in FIG.
도 2에서 확인할 수 있는 바와 같이, HY7712(저농도)는 TNF-α의 발현 유도를 LPS 처리군과 유사하게 일으키며, 농도의존적으로 TNF-α의 발현량을 증가시킴을 알 수 있는데, 이를 통하여 본 발명의 락토바실러스 플란타룸 HY7712는 농도별로 THP-1 세포에서 면역반응을 증가시켜 면역지표의 생산 유도가 락토바실러스 플란타룸 HY7712 때문임을 확인할 수 있었다.
As can be seen in Figure 2, HY7712 (low concentration) causes the expression of TNF-α similar to the LPS treatment group, it can be seen that increases the expression of TNF-α concentration-dependently, through the present invention Lactobacillus plantarum HY7712 increased the immune response in THP-1 cells by concentration, it was confirmed that the production of immune markers is due to Lactobacillus plantarum HY7712.
<시험예 2>≪ Test Example 2 &
락토바실러스 플란타룸 HY7712의 항산화 활성에 대한 조사Investigation of Antioxidant Activity of Lactobacillus Planta Room HY7712
본 발명의 락토바실러스 플란타룸 HY7712 균주가 다른 락토바실러스 플란타룸 균주와 다른 활성을 갖는지 알아보기 위하여 균주의 항산화 활성을 조사하였다.
To determine whether the Lactobacillus plantarum HY7712 strain of the present invention has different activity from other Lactobacillus plantarum strains, the antioxidant activity of the strain was investigated.
<2-1> 락토바실러스 플란타룸 HY7712의 DPPH 라디칼 소거활성 측정 <2-1> DPPH radical scavenging activity of Lactobacillus plantarum HY7712
(1)본 발명의 락토바실러스 플란타룸 HY7712 균주를 MRS 액체배지(10㎖)에 접종하여 37℃에서 20시간 배양하였다. 이 배양액을 3,000rpm, 4℃에서 10분간 원심분리하여 균체를 얻은 후 PBS 완충용액으로 3회 세척한 다음, PBS 완충용액으로 부피를 10㎖로 조절하여 intact cell로서 시험에 사용하였다.(1) The Lactobacillus plantarum HY7712 strain of the present invention was inoculated in MRS liquid medium (10 ml) and incubated at 37 ° C. for 20 hours. The culture solution was centrifuged at 3,000 rpm, 4 ° C. for 10 minutes to obtain cells, and then washed three times with PBS buffer. The volume was adjusted to 10 ml with PBS buffer and used as an intact cell for testing.
DPPH 라디칼 소거능(%)은 Lin과 Chang의 방법(Lin M-Y와 Chang F-J. 2000. Antioxidative Effect of Intestinal Bacteria Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilus ATCC 4356. Dig. Dis. Sci. 45:1617-1622)을 이용하여 다음과 같이 측정하였다.The DPPH radical scavenging activity (%) was measured by the method of Lin and Chang (Lin MY and Chang FJ 2000. Antioxidative Effect of Intestinal Bacteria Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilus ATCC 4356. Dig. Dis. Sci. The following measurements were made.
상기 Intact cell 800㎕과 DPPH 용액(0.2mM) 1㎖을 혼합하여 30분간 반응시킨 다음 5,000rpm에서 원심분리한 후 상등액의 흡광도를 517nm에서 측정하였다. DPPH 라디칼 소거능(%)은 아래와 같은 식으로 계산하였다.
800 μl of the Intact cell and 1 ml of a DPPH solution (0.2 mM) were mixed and reacted for 30 minutes. After centrifugation at 5,000 rpm, the absorbance of the supernatant was measured at 517 nm. The DPPH radical scavenging activity (%) was calculated by the following equation.
DPPH 라디칼 소거능(%) = 100 X (1-유산균의 흡광도/blank의 흡광도)
DPPH radical scavenging activity (%) = 100 X (absorbance of lactic acid bacteria / absorbance of blank)
(2)락토바실러스 플란타룸(Lactobacillus plantarum) KM22-1, 락토바실러스 플란타룸 KM16-3, 락토바실러스 플란타룸 K7-9, 락토바실러스 플란타룸 K5-8, 락토바실러스 플란타룸 K3-2, 락토바실러스 플란타룸 K20-15, 락토바실러스 플란타룸 K12-7, 그리고 락토바실러스 플란타룸 K12-2 각각에 대하여도 상기 (1)의 본 발명의 락토바실러스 플란타룸 HY7712의 DPPH 라디칼 소거능 측정방법과 동일한 방법으로 DPPH 라디칼 소거능을 측정하였다.(2) Lactobacillus plantarum KM22-1, Lactobacillus plantarum KM16-3, Lactobacillus plantarum K7-9, Lactobacillus plantarum K5-8, Lactobacillus plantarum K3- 2, DPPH radicals of Lactobacillus plantarum HY7712 of the present invention of (1) also for Lactobacillus plantarum K20-15, Lactobacillus plantarum K12-7, and Lactobacillus plantarum K12-2, respectively. DPPH radical scavenging ability was measured in the same manner as the scavenging activity measuring method.
그 결과를 도 3에 나타내었다.The results are shown in Fig.
도 3의 가로축에는 락토바실러스 플란타룸(Lactobacillus plantarum) KM22-1, 락토바실러스 플란타룸 KM16-3, 락토바실러스 플란타룸 K7-9, 락토바실러스 플란타룸 K5-8, 락토바실러스 플란타룸 K3-2, 락토바실러스 플란타룸 K20-15, 락토바실러스 플란타룸 K12-7, 락토바실러스 플란타룸 K12-2, 락토바실러스 플란타룸 HY7712를 각각 'KM22-1', 'KM16-3', 'K7-9', 'K5-8', 'K3-2', 'K20-15', 'K12-7', 'K12-2', 'HY7712'로 표시하였다.
3, Lactobacillus plantarum KM22-1, Lactobacillus plantarum KM16-3, Lactobacillus plantarum K7-9, Lactobacillus plantarum K5-8, Lactobacillus plantarum K3-2, Lactobacillus plantarum K20-15, Lactobacillus plantarum K12-7, Lactobacillus plantarum K12-2, and Lactobacillus plantarum HY7712 are 'KM22-1' and 'KM16-3', respectively. , 'K7-9', 'K5-8', 'K3-2', 'K20-15', 'K12-7', 'K12-2' and 'HY7712'.
도 3에서 확인할 수 있는 바와 같이, 본 발명의 락토바실러스 플란타룸 HY7712의 DPPH 라디칼 소거능이 약 37% 이상으로 다른 유산균들과 비교하여 가장 높게 나타나 항산화 활상이 가장 높음을 알 수 있었다.
As can be seen in Figure 3, the lactobacillus plantarum HY7712 DPPH radical scavenging ability of the present invention was about 37% or more, compared to other lactic acid bacteria showed the highest antioxidant activity.
<2-2> 락토바실러스 플란타룸 HY7712의 환원활성 측정 <2-2> Reduction Activity of Lactobacillus Planta Room HY7712
(1)본 발명의 락토바실러스 플란타룸 HY7712의 환원활성(reducing activity)을 Lin과 Yen의 방법(Lin M-Y과 Yen CL.1999. Antioxidative ability of lactic acid acteria. J. Agric. Food Chem. 47:1460-1466)을 이용하여 측정하였다. 본 발명의 락토바실러스 플란타룸 HY7712 균주를 MRS 액체배지(10㎖)에 접종하여 37℃에서 20시간 배양하였다. 이 배양액을 3,000rpm, 4℃에서 10분간 원심분리하여 균체를 얻은 후 PBS 완충용액으로 3회 세척한 다음, PBS 완충용액으로 부피를 10㎖로 조절하여 intact cell로 사용하였다.(1) Reducing activity of Lactobacillus plantarum HY7712 of the present invention by Lin and Yen method (Lin MY and Yen CL. 1999. Antioxidative ability of lactic acid acteria.J. Agric.Food Chem. 47: 1460-1466). The Lactobacillus plantarum HY7712 strain of the present invention was inoculated in MRS liquid medium (10 ml) and incubated at 37 ° C. for 20 hours. The culture solution was centrifuged at 3,000 rpm, 4 ° C. for 10 minutes to obtain cells, and then washed three times with PBS buffer. The volume was adjusted to 10 ml with PBS buffer and used as an intact cell.
한편, Intracellular cell free extract는 다음과 같이 준비하였다.Meanwhile, Intracellular cell free extract was prepared as follows.
원심분리 튜브에 상기 intact cell 1.5㎖ 및 glass bead(212~300㎛) 200mg을 넣은 다음, FastPrep(MP Bio, USA)을 이용하여 균체를 파쇄하고, 7,800 x g에서 10분간 원심분리한 후 상등액을 취하여 intracellular cell free extract로 사용하였다.Put 1.5ml of the intact cell and 200mg of glass bead (212 ~ 300㎛) into the centrifuge tube, crush the cells using FastPrep (MP Bio, USA), centrifuge at 7,800 xg for 10 minutes, and then take the supernatant Intracellular cell free extract was used.
본 발명의 락토바실러스 플란타룸 HY7712의 intact cell의 환원능은 다음과 같이 측정하였다. Reducing ability of the intact cell of the Lactobacillus plantarum HY7712 of the present invention was measured as follows.
상기 Intact cell 500㎕, 페리시안산칼륨(Potassium ferricyanide;1% w/v) 500㎕ 및 소디움포스페이트 완충용액(0.02M) 500㎕를 혼합한 다음 50℃에서 20분간 반응시키고 냉각시켰다. 여기에 트리클로로아세트산(Trichloroacetic acid; 10% w/v) 500㎕를 첨가하고 혼합한 다음 3,000rpm에서 10분간 원심분리하였다. 원심분리된 상등액 1.5㎖에 염화제2철(FeCl3 0.1% w/v) 0.2㎖을 첨가하고 혼합한 후 700nm에서 흡광도를 측정하였다. L-시스테인(L-cysteine)을 표준시료로 이용하여 위와 동일한 방법으로 처리하여 흡광도를 측정하여 표준곡선을 작성한 다음 이를 이용하여 본 발명의 락토바실러스 플란타룸 HY7712의 intact cell의 환원능을 equivalent cysteine(μM)로 표시하였다. 또한, 본 발명의 락토바실러스 플란타룸 HY7712의 intracellular cell free extract의 환원능도 상기 intact cell의 환원능 측정방법과 동일하게 실시하여 측정하였다.
500 μl of the Intact cell, 500 μl of potassium ferricyanide (1% w / v), and 500 μl of sodium phosphate buffer (0.02 M) were mixed, and then reacted and cooled at 50 ° C. for 20 minutes. 500 µl of trichloroacetic acid (Trichloroacetic acid; 10% w / v) was added thereto, mixed, and centrifuged at 3,000 rpm for 10 minutes. To 1.5 ml of the centrifuged supernatant, 0.2 ml of ferric chloride (FeCl 3 0.1% w / v) was added and mixed, and the absorbance was measured at 700 nm. L-cysteine (L-cysteine) as a standard sample was treated in the same way as described above to measure the absorbance to create a standard curve and then using this to reduce the ability of the intact cell of the Lactobacillus plantarum HY7712 equivalent cysteine (μM). In addition, the reducing ability of the intracellular cell free extract of Lactobacillus plantarum HY7712 of the present invention was also measured in the same manner as the measuring method of the reducing activity of the intact cell.
(2)락토바실러스 플란타룸(Lactobacillus plantarum) KM22-1, 락토바실러스 플란타룸 KM16-3, 락토바실러스 플란타룸 K7-9, 락토바실러스 플란타룸 K5-8, 락토바실러스 플란타룸 K3-2, 락토바실러스 플란타룸 K20-15, 락토바실러스 플란타룸 K12-7, 락토바실러스 플란타룸 K12-2 각각에 대하여도 상기 (1)의 본 발명의 락토바실러스 플란타룸 HY7712의 intact cell과 intracellular cell free extract의 환원능 측정방법과 동일한 방법으로 측정하여 각 균주들의 intact cell과 intracellular cell free extract의 환원능을 측정하였다.(2) Lactobacillus plantarum KM22-1, Lactobacillus plantarum KM16-3, Lactobacillus plantarum K7-9, Lactobacillus plantarum K5-8, Lactobacillus plantarum K3- 2, Lactobacillus plantarum K20-15, Lactobacillus plantarum K12-7, and Lactobacillus plantarum K12-2, respectively, the intact cell of the Lactobacillus plantarum HY7712 of the present invention (1) The reduction ability of intact cell and intracellular cell free extract of each strain was measured by the same method as that of intracellular cell free extract.
그 결과를 도 4에 나타내었다. The results are shown in FIG.
도 4의 가로축에는 락토바실러스 플란타룸(Lactobacillus plantarum) KM22-1, 락토바실러스 플란타룸 KM16-3, 락토바실러스 플란타룸 K7-9, 락토바실러스 플란타룸 K5-8, 락토바실러스 플란타룸 K3-2, 락토바실러스 플란타룸 K20-15, 락토바실러스 플란타룸 K12-7, 락토바실러스 플란타룸 K12-2, 락토바실러스 플란타룸 HY7712를 각각 'KM22-1', 'KM16-3', 'K7-9', 'K5-8', 'K3-2', 'K20-15', 'K12-7', 'K12-2', 'HY7712'로 표시하였다.
4, Lactobacillus plantarum KM22-1, Lactobacillus plantarum KM16-3, Lactobacillus plantarum K7-9, Lactobacillus plantarum K5-8, Lactobacillus plantarum K3-2, Lactobacillus plantarum K20-15, Lactobacillus plantarum K12-7, Lactobacillus plantarum K12-2, and Lactobacillus plantarum HY7712 are 'KM22-1' and 'KM16-3', respectively. , 'K7-9', 'K5-8', 'K3-2', 'K20-15', 'K12-7', 'K12-2' and 'HY7712'.
도 4에서 확인할 수 있는 바와 같이, 본 발명의 락토바실러스 플란타룸 HY7712의 intact cell과 intracellular cell free extract의 환원능이 각각 99와 85 equivalent cysteine(μM)로 다른 유산균들과 비교하여 가장 높게 나타나, 본 발명의 락토바실러스 플란타룸 HY7712는 항산화 활성이 가장 높음을 알 수 있었다.
As can be seen in Figure 4, the reducing ability of the intact cell and intracellular cell free extract of the Lactobacillus plantarum HY7712 of the present invention is 99 and 85 equivalent cysteine (μM) showed the highest compared to other lactic acid bacteria, respectively, Lactobacillus plantarum HY7712 of the invention was found to have the highest antioxidant activity.
<2-3> 락토바실러스 플란타룸 HY7712의 활성산소흡수력(ORAC) 측정 <2-3> Determination of free radicals (ORAC) of Lactobacillus plantarum HY7712
본 발명의 락토바실러스 플란타룸 HY7712의 활성산소흡수력을 Huang 등[Huang D, Ou B, Hampsch-Woodill M, Flanagan J, 그리고 Prior R. 2002. High-throughput Assay of Oxygen Radical Absorbance Capacity(ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format. J. Agric. Food Chem. 50:4437-4444]이 제시한 ORAC assay법에 따라 다음과 같이 측정하였다.The active oxygen absorption capacity of the Lactobacillus plantarum HY7712 of the present invention is determined by Huang et al., Huang D, Ou B, Hampsch-Woodill M, Flanagan J, and Prior R. 2002. High-throughput Assay of Oxygen Radical Absorbance Capacity a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format. J. Agric. Food Chem. 50: 4437-4444] according to the ORAC assay described below.
본 발명의 락토바실러스 플란타룸 HY7712를 MRS 액체배지에서 약 20시간 배양한 다음 3,000rpm에서 10분간 원심분리하여, 상층액을 버리고 cell pellet을 PBS 완충용액에 현탁하여 3,000rpm에서 원심분리하고 상층액을 버리는 과정을 3회 반복하였다. Cell pellet을 assay buffer(75 mM phosphate, pH 7.4) 1㎖에 현탁하여 ORAC 분석에 사용하였다. Fluorescein stock solution은 Sodium fluorescein을 4μM의 농도로 assay buffer에 녹여 차광한 후, 5℃에 보관하였다.The Lactobacillus plantarum HY7712 of the present invention was incubated for about 20 hours in an MRS liquid medium and then centrifuged at 3,000 rpm for 10 minutes to discard the supernatant, and the cell pellet was suspended in PBS buffer and centrifuged at 3,000 rpm. The process of discarding was repeated three times. The cell pellet was suspended in 1 ml of assay buffer (75 mM phosphate, pH 7.4) and used for ORAC analysis. Fluorescein stock solution was dissolved in Sodium Fluorescein in assay buffer at a concentration of 4μM, and then stored at 5 ° C.
양성대조군으로는 Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)를 6.25, 12.5, 25, 50, 100μM의 농도로 assay buffer에 녹여 준비하였다. 96 well, black-sided, special optics clear bottom plate의 맨 가장자리 well에 물을 300㎕씩 채워 온도 변화에 의한 실험적 오차를 줄였다. Plate의 안쪽 well에 fluorescein stock solution을 phosphate buffer로 100배 희석하여 150㎕씩 넣은 후, 시험군, 양성대조군 및 blank(phosphate buffer)를 25㎕씩 넣었다. plate를 Synergy HT Multi-Detection Microplate reader로 37℃에서 30분간 평형에 도달하게 하였다. AAPH{2,2'-Azobis(2-amidinopropane) dihydrochloride solution}을 153mM 의 농도로 assay buffer에 녹여, well당 25㎕씩 넣고, 60분간 31회 형광을 측정하였다(Excitation : 485nm, Emission : 528 nm, sensitivity : 60).As a positive control, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was dissolved in assay buffer at concentrations of 6.25, 12.5, 25, 50 and 100 μM. 96 wells, black-sided, special optics Clear bottom plate was filled with 300 μl of water at the farthest well to reduce experimental error by temperature change. The fluorescein stock solution was diluted 100 times with phosphate buffer in the inner well of the plate, and 150 μl of each dilution was added to each well. Then, 25 μl of test group, positive control group and blank (phosphate buffer) were added. plate was allowed to reach equilibrium at 37 ° C for 30 minutes with a Synergy HT Multi-Detection Microplate Reader. AAPH (2-amidinopropane) dihydrochloride solution was dissolved in assay buffer at a concentration of 153 mM, and 25 μl of the solution was added to each well. The fluorescence was measured 31 times for 60 minutes (excitation: 485 nm, emission: 528 nm , sensitivity: 60).
실험 결과를 KC4TM data reduction software를 이용하여, 다음과 같은 수식에 의해 Kinetic curve의 AUC(Area Under the Curve)를 구하고 양성대조군의 AUC와 비교하여 TE(Trolox equivalents)를 산출하였다[Saide JA와 Gilliland SE. 2005. Antioxidative activity of lactobacilli measured by oxygen radical absorbance capacity. J. Dairy Sci. 88(4):1352-1357].The experimental results using the KC4 TM data reduction software, yielding a TE (Trolox equivalents) to obtain the AUC (Area Under the Curve) of the Kinetic curve compared to the AUC of positive control group by the following formula [Saide JA and Gilliland SE. 2005. Antioxidative activity of lactobacilli measured by oxygen radical absorbance capacity. J. Dairy Sci. 88 (4): 1352-1357).
AUC는 아래와 같이 계산하였다.
AUC was calculated as follows.
AUC = 0.5+(R2/R1)+(R3/R1)+(R4/R1)+……+ 0.5(Rn/R1)
AUC = 0.5 + (R2 / R1) + (R3 / R1) + (R4 / R1) +... ... + 0.5 (Rn / R1)
R1은 반응 초기의 형광 측정값이고, Rn은 맨 마지막 형광 측정값이다. R1 is the fluorescence measurement at the beginning of the reaction and Rn is the last fluorescence measurement.
본 발명의 락토바실러스 플란타룸 HY7712의 활성산소흡수력(ORAC)은 아래와 같이 락토바실러스 플란타룸 HY7712 균수의 109 cells에 대한 TE(Trolox equivalents)로 나타내었다.The active oxygen absorption capacity (ORAC) of the Lactobacillus plantarum HY7712 of the present invention is expressed as TE (Trolox equivalents) for 10 9 cells of the Lactobacillus plantarum HY7712 bacteria.
Trolox eq./109 cells = 
Trolox eq./10 9 cells =
(2)락토바실러스 플란타룸(Lactobacillus plantarum) KM22-1, 락토바실러스 플란타룸 KM16-3, 락토바실러스 플란타룸 K7-9, 락토바실러스 플란타룸 K5-8, 락토바실러스 플란타룸 K3-2, 락토바실러스 플란타룸 K20-15, 락토바실러스 플란타룸 K12-7, 락토바실러스 플란타룸 K12-2 각각에 대하여도 상기 (1)의 본 발명의 락토바실러스 플란타룸 HY7712의 활성산소흡수력(ORAC) 측정방법과 동일하게 각 균주들의 활성산소흡수력(ORAC)을 측정하였다.(2) Lactobacillus plantarum KM22-1, Lactobacillus plantarum KM16-3, Lactobacillus plantarum K7-9, Lactobacillus plantarum K5-8, Lactobacillus plantarum K3- 2, Lactobacillus plantarum K20-15, Lactobacillus plantarum K12-7, Lactobacillus plantarum K12-2, respectively, also for the active oxygen absorption capacity of the Lactobacillus plantarum HY7712 of the present invention (1) ORAC was measured in the same manner as the (ORAC) measurement method.
그 결과를 도 5에 나타내었다.The results are shown in Fig.
도 5의 가로축에는 락토바실러스 플란타룸(Lactobacillus plantarum) KM22-1, 락토바실러스 플란타룸 KM16-3, 락토바실러스 플란타룸 K7-9, 락토바실러스 플란타룸 K5-8, 락토바실러스 플란타룸 K3-2, 락토바실러스 플란타룸 K20-15, 락토바실러스 플란타룸 K12-7, 락토바실러스 플란타룸 K12-2, 락토바실러스 플란타룸 HY7712를 각각 'KM22-1', 'KM16-3', 'K7-9', 'K5-8', 'K3-2', 'K20-15', 'K12-7', 'K12-2', 'HY7712'로 표시하였다.
5, Lactobacillus plantarum KM22-1, Lactobacillus plantarum KM16-3, Lactobacillus plantarum K7-9, Lactobacillus plantarum K5-8, Lactobacillus plantarum K3-2, Lactobacillus plantarum K20-15, Lactobacillus plantarum K12-7, Lactobacillus plantarum K12-2, and Lactobacillus plantarum HY7712 are 'KM22-1' and 'KM16-3', respectively. , 'K7-9', 'K5-8', 'K3-2', 'K20-15', 'K12-7', 'K12-2' and 'HY7712'.
도 5에서 확인할 수 있는 바와 같이, 본 발명의 락토바실러스 플란타룸 HY7712의 활성산소흡수력(ORAC)이 약 27.3 TE로 다른 유산균들과 비교하여 가장 높게 나타나, 본 발명의 락토바실러스 플란타룸 HY7712의 항산화 활성이 가장 높음을 알 수 있었다.
As can be seen in Figure 5, the active oxygen absorption capacity (ORAC) of the Lactobacillus plantarum HY7712 of the present invention is about 27.3 TE is the highest compared to other lactic acid bacteria, the Lactobacillus plantarum HY7712 of the present invention It was found that the antioxidant activity was the highest.
<시험예 3><Test Example 3>
락토바실러스 플란타룸 HY7712의 야채주스 발효능 조사Fermentation of Vegetable Juice from Lactobacillus Planta Room HY7712
상기 실시예 5의 혼합야채즙을 끓는 물에 5분간 중탕하여 살균시킨 후 식혀서 준비한 다음, 락토바실러스 플란타룸 HY7712를 106 CFU/㎖ 수준으로 접종하고 35℃에서 16~18 시간동안 배양하여 혼합야채즙 속 균주의 생장 상태를 조사하였다.The mixed vegetable juice of Example 5 was sterilized by boiling in boiling water for 5 minutes, then cooled and prepared, and then inoculated with Lactobacillus plantarum HY7712 at a level of 10 6 CFU / mL and incubated at 35 ° C. for 16-18 hours to mix. The growth state of the strain in the vegetable juice was investigated.
그 결과를 도 6에 나타내었다.
The results are shown in Fig.
도 6에서 확인할 수 있는 바와 같이, 상기 혼합야채즙의 배양물은 pH가 낮아지고 상큼한 풍미가 있었음은 물론, 균주 농도가 1X109 CFU/㎖ 수준으로 매우 높아져 본 발명의 락토바실러스 플란타룸 HY7712는 야채주스를 발효시키는 능력이 뛰어남을 알 수 있었다.
As can be seen in Figure 6, the culture of the mixed vegetable juice has a low pH and fresh flavor, as well as the strain concentration is very high to 1X10 9 CFU / ㎖ Lactobacillus plantarum HY7712 of the present invention The ability to ferment vegetable juice was found to be excellent.
<110> KOREA YAKULT CO., LTD <120> The new Lactobacillus plantarum HY7712 stimulate immunity and products containing thereof as effective component <130> P11-03 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1031 <212> DNA <213> Lactobacillus plantarum <400> 1 atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa agccttcggc 60 tcaaccgaag aagtgcatcg gaaactggga aacttgagtg cagaagagga cagtggaact 120 ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga aggcggctgt 180 ctggtctgta actgacgctg aggctcgaaa gtatgggtag caaacaggat tagataccct 240 ggtagtccat accgtaaacg atgaatgcta agtgttggag ggtttccgcc cttcagtgct 300 gcagctaacg cattaagcat tccgcctggg gagtacggcc gcaaggctga aactcaaagg 360 aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc tacgcgaatt 420 ggagggtttc cgcccttcag tgctgcagct aacgcattaa gcattccgcc tggggagtac 480 ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 540 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatactatg caaatctaag 600 agattagacg ttcccttcgg ggacatggat acaggtggtg catggttgtc gtcagctcgt 660 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattatca gttgccagca 720 ttaagttggg cactctggtg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 780 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 840 gttgcgaact cgcgagagta agctaatctc ttaaagccat tctcagttcg gattgtaggc 900 tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga 960 atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa 1020 gtcggtgggg t 1031 <110> KOREA YAKULT CO., LTD <120> The new Lactobacillus plantarum HY7712 stimulate immunity and products containing according as effective component <130> P11-03 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1031 <212> DNA <213> Lactobacillus plantarum <400> 1 atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa agccttcggc 60 tcaaccgaag aagtgcatcg gaaactggga aacttgagtg cagaagagga cagtggaact 120 ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga aggcggctgt 180 ctggtctgta actgacgctg aggctcgaaa gtatgggtag caaacaggat tagataccct 240 ggtagtccat accgtaaacg atgaatgcta agtgttggag ggtttccgcc cttcagtgct 300 gcagctaacg cattaagcat tccgcctggg gagtacggcc gcaaggctga aactcaaagg 360 aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc tacgcgaatt 420 ggagggtttc cgcccttcag tgctgcagct aacgcattaa gcattccgcc tggggagtac 480 ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 540 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatactatg caaatctaag 600 agattagacg ttcccttcgg ggacatggat acaggtggtg catggttgtc gtcagctcgt 660 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattatca gttgccagca 720 ttaagttggg cactctggtg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 780 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 840 gttgcgaact cgcgagagta agctaatctc ttaaagccat tctcagttcg gattgtaggc 900 tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga 960 atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa 1020 gtcggtgggg t 1031
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| KR102028744B1 (en) | 2018-07-23 | 2019-10-07 | 주식회사한국야쿠르트 | Lactobacillus plantarum HY7717 strain having immune-enhancing activity, antioxidative activity and digestive fluid resistance and use thereof |
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