KR20190075039A - Lactobacillus plantarum KCC-24 and composition comprising the same - Google Patents
Lactobacillus plantarum KCC-24 and composition comprising the same Download PDFInfo
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- KR20190075039A KR20190075039A KR1020190074420A KR20190074420A KR20190075039A KR 20190075039 A KR20190075039 A KR 20190075039A KR 1020190074420 A KR1020190074420 A KR 1020190074420A KR 20190074420 A KR20190074420 A KR 20190074420A KR 20190075039 A KR20190075039 A KR 20190075039A
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- lactobacillus plantarum
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- plantarum
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Abstract
Description
본 발명은 신규한 락토바실러스 플란타룸 균주 및 이를 포함하는 조성물에 관한 것으로, 더욱 상세하게는 항곰팡이 활성이 우수한 신규한 락토바실러스 플란타룸 균주 및 이를 이용한 품질이 개선된 사일리지에 관한 것이다. The present invention relates to a novel Lactobacillus plantarum strain and a composition comprising the same, and more particularly to a novel Lactobacillus plantarum strain having excellent antifungal activity and a silage with improved quality using the same.
식품 부패를 예방하기 위해 식품, 약학, 화학 산업에 젖산균에 대한 필요성이 증가하고 있다. 젖산의 산업적 생산은 화학적 합성 또는 미생물 발효에 의해 생산된다. 화학적으로 생산된 젖산은 항상 라세미 혼합물이다. 반면 생물학적으로 생산된 젖산은 순수하며, 박테리아 균주를 생산하는 젖산을 사용하여 수득할 수 있다. 오늘날 생균제 미생물의 섭취가 건강에 유익한 점에 대한 관심이 급증하면서 전 세계적으로 젖산균의 소비가 증가하고 있다. 생균제는 충분한 양으로 투여될 때, 개체에 건강상의 이익을 제공하는, 살아있는 미생물로 정의된다. 이는 병원성 균과 경쟁, 해로운 균의 사멸, 면역 시스템을 가속화하여, 생리학적 기능을 향상시키는 데 중요한 역할을 한다(Guarner, F., Perdigon, G., Coerthier, G., Salminen, S., Azcarate peril, M. A., and Altermann, E.: Genomic features of lactic acid bacteria effecting bioprocessing and health, FEMS. Microbial. Heal., 29, 393-409 (2005).In order to prevent food corruption, there is an increasing need for lactic acid bacteria in the food, pharmaceutical, and chemical industries. Industrial production of lactic acid is produced by chemical synthesis or microbial fermentation. Chemically produced lactic acid is always a racemic mixture. Biologically produced lactic acid, on the other hand, is pure and can be obtained using lactic acid to produce bacterial strains. Today, the consumption of lactic acid bacteria is increasing worldwide as the interest in the health benefits of the procurement of probiotic microorganisms increases rapidly. Probiotics are defined as living microorganisms that, when administered in sufficient amounts, provide a health benefit to an individual. It plays an important role in enhancing the physiological function by accelerating the immune system and the competition with pathogenic bacteria, the destruction of harmful bacteria, and the immune system (Guarner, F., Perdigon, G., Coerthier, G., Salminen, S., Azcarate Peril, MA, and Altermann, E .: Genomic features of lactic acid bacteria effecting bioprocessing and health, FEMS Microbiol. Heal., 29, 393-409 (2005).
조사료는 일시에 다량 생산되므로 연중 안정적인 급여를 위해서는 저장을 해야 하는데, 수분을 제거한 저장이 건초인 반면 수분이 있는 상태에서 저장을 하는 것이 사일리지(silage)이다. 최근 들어 사일리지의 품질 향상을 위한 첨가제의 필요성에 대한 인식이 확산되면서 국내산 사일리지용 첨가제를 개발하기 위한 연구가 이루어지고 있다.Since the forage is produced in large quantities at a time, it is necessary to store it for stable salary throughout the year. The silage is the storage of moisture while the moisture is removed from the hay. In recent years, as the awareness of necessity of additives for improving the quality of silage has spread, studies for developing additive for domestic silage have been conducted.
그러나, 사일리지는 가열공정을 거치지 않기 때문에 곰팡이 등과 같은 미생물들은 적당한 환경(온도, 영양 및 pH 조건)이 구비되면 번식이 용이하여 사일리지의 보관에 어려움을 겪고 있다. 또한, 사일리지 제조기술 부족과 유통, 보관 시 부주의로 곰팡이 등이 발생하면서 가축의 기호성이 감소되고 섭취량이 줄어드는 문제가 발생한다. 또한, 호기조건에 있는 사일리지는 영양적으로도 커다란 손실을 유발한다. However, since the silage is not subjected to the heating process, the microorganisms such as fungi are difficult to store silage because they are easily breeded when the environment (temperature, nutrition and pH conditions) is provided. In addition, lack of silage manufacturing technology and inadvertence of fungi during distribution and storage lead to decrease of palatability and decrease of intake of livestock. In addition, silage in aerobic conditions causes nutritionally large losses.
따라서, 사일리지에 발생하는 곰팡이를 억제함으로써 발효 효율 및 사료 저장성을 증진시킬 뿐만 아니라 사료의 영양성, 기호성 및 소화율도 향상시킬 수 있는 첨가제의 개발이 필요하다. 또한 병원성 균을 사멸시키며, 면역 시스템을 가속화하여, 생리학적 기능을 향상시킬 수 있는 생균제의 개발이 필요하다.Therefore, it is necessary to develop an additive capable of improving the nutritional, palatability and digestibility of the feed as well as enhancing the fermentation efficiency and feed retention by inhibiting molds generated in the silage. It is also necessary to develop a probiotic agent that can kill pathogenic bacteria, accelerate the immune system, and improve physiological function.
본 발명의 목적은 상기와 같은 기술적 과제를 해결하기 위하여, 신규 락토바실러스 플란타룸(Lactobacillus plantarum) KCC-24 (KACC91962P) 균주를 제공하는 것이다.DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel Lactobacillus plantarum KCC-24 (KACC91962P) strain in order to solve the above technical problem.
또한 본 발명의 목적은 상기 균주를 포함하는 항균제 조성물, 항산화용 조성물, 사일리지 제조를 위한 미생물 첨가제, 사일리지 제조방법, 또는 사료 첨가제 등을 제공하는 것이다.It is another object of the present invention to provide an antimicrobial composition, an antioxidant composition, a microbial additive for producing silage, a silage production method, or a feed additive, etc., containing the strain.
상기 기술적 과제를 달성하기 위하여, 본 발명은 신규 락토바실러스 플란타룸(Lactobacillus plantarum) KCC-24 (KACC91962P) 균주를 제공한다. In order to accomplish the above object, the present invention provides a novel Lactobacillus plantarum KCC-24 (KACC91962P) strain.
상기 락토바실러스 플란타룸 KCC-24 균주는 2014년 06월 13일자로 농업생명공학연구원(KACC)에 기탁하였으며, 수탁번호 KACC91962P를 부여받았다.The Lactobacillus planta KCC-24 strain was deposited with the Institute of Agricultural Biotechnology (KACC) on June 13, 2014 and received the accession number KACC91962P.
곰팡이 등은 발효가 불량한 사일리지에서 다량 발견되고 발효가 양호하게 된 사일리지에서는 육안으로 판별하기 어려울 정도이기 때문에 사일리지 곰팡이 억제기능이 우수한 젖산균을 선발 및 분리하여 사일리지 보존성을 높이고자 하였다.Mildew were found in silage with poor fermentation and it was difficult to distinguish them by naked eyes in the silage where fermentation became good. Thus, lactic acid bacteria having excellent silage mold inhibiting ability were selected and separated to improve silage preservation.
본 발명의 발명자들은 락토바실러스 플란타룸 균주를 식품으로부터 분리하는 단계; 상기 분리한 락토바실러스 플란타룸 균주의 효소 활성능을 검정하는 단계; 상기 분리한 신규 미생물인 락토바실러스 플란타룸 균주의 동정 단계; 및 상기 분리된 락토바실러스 플란타룸 균주를 이용하여 프로바이오틱 특성을 검정하는 단계를 거쳐, 항균 활성, 항산화 활성이 우수한 신규 미생물인 락토바실러스 플란타룸 (Lactobacillus plantarum) KCC-24 (KACC91962P) 균주를 얻었다.The inventors of the present invention have found that a step of isolating a Lactobacillus plantarum strain from a foodstuff; Assaying the enzymatic activity of the isolated Lactobacillus plantarum strain; Identifying the isolated new microorganism, Lactobacillus plantarum strain; And Lactobacillus plantarum KCC-24 (KACC91962P) strain, which is a novel microorganism having excellent antimicrobial activity and antioxidative activity, through a step of assaying the probiotic characteristics using the isolated Lactobacillus plantarum strain .
본 발명의 실시예들에 따르면, 본 발명에 따른 락토바실러스 플란타룸 (Lactobacillus plantarum) KCC-24 (KACC91962P) 균주는 부패 미생물의 활성을 저하시키고 생육을 억제시켜 항균 활성을 나타낼 수 있다.According to the embodiments of the present invention, the Lactobacillus plantarum KCC-24 (KACC91962P) strain according to the present invention may exhibit antimicrobial activity by inhibiting the activity of the spoilage microorganism and suppressing growth thereof.
본 발명에 있어서, 상기 부패 미생물은 아스퍼질러스 푸미가테스(Aspergillus Fumigates), 페니실리움 크리소게눔(Penicillium Chrysogenum), 페니실리움 로케포티(Penicillium Roqueforti), 보트리티스 엘립티카(botrytis elliptica), 및 후자리움 옥시스포럼(Fusarium Oxysporum)으로 이루어진 군에서 선택된 어느 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the spoilage microorganism may be selected from the group consisting of Aspergillus fumigates , Penicillium chrysogenum , Penicillium roqueforti , botrytis elliptica , , And Fusarium oxysporum . However, the present invention is not limited thereto.
상기와 같은 우수한 항균 효과로 인해 상기 균주는 항균용 조성물 제조에 이용할 수 있으며, 특히, 우수한 프로바이오틱 효과로 인해서 다양한 항균용 조성물을 포함하는 식품, 의약품, 화장품, 건강식품 등의 제조에 이용될 수 있다. Due to the excellent antibacterial effect as described above, the strain can be used in the production of antimicrobial compositions. In particular, the strains can be used for the production of foods, medicines, cosmetics, health foods and the like containing various antibacterial compositions due to their excellent probiotic effect .
본 발명의 항균용 조성물이 포함되는 식품, 의약품, 화장품, 건강식품 등의 제조는 업계에서 일반적으로 실시하는 방법을 통하여 제조될 수 있다.The manufacture of foods, medicines, cosmetics, health foods and the like containing the antimicrobial composition of the present invention can be manufactured by a method generally used in the industry.
본 발명의 발명자들은 본 발명의 신규 균주가 독성이 없으면서, 항균 활성이 뛰어나, 우수한 프로바이오틱 제제로 이용될 수 있다는 것을 실험을 통해서 밝혀내었다.The inventors of the present invention have found through experimentation that the novel strain of the present invention is excellent in antimicrobial activity without being toxic, and can be used as an excellent probiotic preparation.
본 발명의 균주는 바람직하게는 상기 우수한 항균 활성으로 인해 사일리지 제조시 저장성을 증진시킬 수 있고, 영양성분의 파괴를 예방할 수 있다.The strain of the present invention is preferably capable of enhancing storability during silage production due to the excellent antimicrobial activity and preventing destruction of nutritional ingredients.
또한, 부패 미생물등과 같은 원하지 않는 미생물에 의한 사일리지 풍미 감소 등을 예방하여, 가축의 기호성을 증진시킬 수 있는 추가 효과까지 얻을 수 있다. In addition, it is possible to prevent the decrease of the silage flavor caused by unwanted microorganisms such as spoilage microorganisms and the like, and to further enhance the palatability of the livestock.
본 발명의 또 다른 실시예들에 따르면, 본 발명의 신규 균주는 우수한 항산화 활성을 가지며, 본 발명의 균주를 포함하는 항산화용 조성물을 제공할 수 있다.According to still another embodiment of the present invention, the novel strain of the present invention has an excellent antioxidative activity and can provide a composition for antioxidation containing the strain of the present invention.
일반적으로 활성산소는 단백질의 SH기와 반응해서 효소의 활성을 잃게 하거나 가교결합의 형성, DNA, RNA, 효소 및 세포막을 구성하고 있는 불포화 지방산을 산화시켜 과산화지질을 형성하게 하는데, 과산화지질은 생체에 매우 유독하여 생체의 기능을 저하시켜서 결국에는 혈액순환장애, 피로, 심장병, 간장장애, 발암 등과 같은 질병의 원인이 되고 궁극적으로 노화의 원인이 된다.In general, reactive oxygen species react with SH groups of proteins to cause loss of activity of enzymes, oxidation of unsaturated fatty acids constituting crosslinks, DNA, RNA, enzymes and cell membranes to form lipid peroxides. It is very toxic and lowers the function of the living body and eventually causes diseases such as blood circulation disorder, fatigue, heart disease, hepatic disorder, carcinogen, etc., and ultimately causes aging.
본 발명의 발명자들은 본 발명의 락토바실러스 플란타룸 (Lactobacillus plantarum) KCC-24 (KACC91962P) 균주가 항균 효과뿐만 아니라, 상기 활성산소의 억제 효과, 즉 항산화 효과가 우수함을 실험을 통해 확인하였다. The inventors of the present invention have confirmed through experiments that the Lactobacillus plantarum KCC-24 (KACC91962P) strain of the present invention has an antimicrobial effect as well as an excellent antioxidant effect.
상기와 같은 우수한 항산화 효과로 인해 상기 균주는 항산화용 조성물 제조에 이용할 수 있으며, 특히, 우수한 프로바이오틱 효과로 인해서 다양한 항산화용 조성물을 포함하는 식품, 의약품, 화장품, 건강식품 등의 제조에 이용될 수 있다. Due to the excellent antioxidative effect as described above, the strain can be used for the production of antioxidant compositions, and in particular, it can be used for the production of foods, medicines, cosmetics, health foods and the like containing various antioxidant compositions due to their excellent probiotic effect .
발명의 항산화용 조성물이 포함되는 식품, 의약품, 화장품, 건강식품 등의 제조는 업계에서 일반적으로 실시하는 방법을 통하여 제조될 수 있다.The manufacture of foods, medicines, cosmetics, health foods and the like containing the antioxidant composition of the present invention can be produced by a method generally used in the industry.
본 발명의 발명자들은 본 발명의 신규 균주가 독성이 없으면서, 항산화 활성이 뛰어나, 우수한 프로바이오틱 제제로 이용될 수 있다는 것을 실험을 통해서 밝혀내었다.The inventors of the present invention have found through experimentation that the novel strain of the present invention is excellent in antioxidative activity while being not toxic and can be used as an excellent probiotic preparation.
본 발명은 상기 락토바실러스 플란타룸 (Lactobacillus plantarum) KCC-24 (KACC91962P) 균주를 포함하는 식물체 사일리지 발효용 미생물 첨가제를 또한 제공한다. The present invention also provides a microorganism additive for plant silage fermentation comprising the strain Lactobacillus plantarum KCC-24 (KACC91962P).
상기 식물체 사일리지 발효용 미생물 첨가제를 첨가하는 대상이 되는 식물체는, 예를 들어 볏짚, 라이그라스(ryegrass), 총체보리, 총체벼, 호밀 또는 옥수수일 수 있으나 이에 제한되는 것은 아니며, 가축의 사료로 이용될 수 있는 식물체라면 모두 이용될 수 있다. The plant to which the microorganism additive for plant silage fermentation is added may be, for example, rice straw, ryegrass, whole barley, whole rice, rye or corn, but not limited to, Any plant that can be used can be used.
상기 식물체 사일리지 발효용 미생물 첨가제는 락토바실러스 플란타룸 (Lactobacillus plantarum) KCC-24 (KACC91962P) 균주를 포함하는 배양원액, 또는 상기 배양원액의 희석액의 형태로 첨가할 수 있다.The microorganism additive for plant silage fermentation may be added in the form of a culture stock solution containing Lactobacillus plantarum KCC-24 (KACC91962P) or a dilution solution of the culture stock solution.
또한 본 발명은 식물체에 락토바실러스 플란타룸(Lactobacillus plantarum) KCC-24 (KACC91962P) 균주를 첨가하는 것을 특징으로 하는 식물체 사일리지 제조방법을 제공한다. The present invention also provides a method for producing plant silage, which comprises adding a strain of Lactobacillus plantarum KCC-24 (KACC91962P) to a plant.
본 발명에 있어서, 상기 식물체 사일리지 제조방법은 당업계에서 사용되는 일반적인 방법으로서, 예를 들어 식물체의 발효를 위한 복수의 발효 단계들을 포함하는 것을 특징으로 한다. 상기 복수의 발효 단계들은, 예를 들어 혐기성균인 초산균의 발효 단계 및 이후 젖산균의 발효 단계 등을 포함할 수 있으며, 당업자에 의해 적절한 조건으로 수행될 수 있다.In the present invention, the method for producing plant silage is a general method used in the art, for example, including a plurality of fermentation steps for fermentation of a plant. The plurality of fermentation steps may include, for example, a fermentation step of anaerobic bacteria, such as a fermentation step, followed by fermentation of lactic acid bacteria, and the like, and may be carried out by those skilled in the art under appropriate conditions.
본 발명은 또한 상기 락토바실러스 플란타룸 (Lactobacillus plantarum) KCC-24 (KACC91962P) 균주를 포함하는 사료 첨가제를 제공한다. The present invention also provides a feed additive comprising the strain Lactobacillus plantarum KCC-24 (KACC91962P).
본 발명의 사료 첨가제는 업계에서 일반적으로 이용되는 방법으로 제조될 수 있다. The feed additive of the present invention can be produced by a method commonly used in the art.
본 발명의 상기 신규 균주가 독성이 없으면서, 항균 활성이 뛰어나, 우수한 프로바이오틱 제제로 이용될 수 있고, 이러한 신규 균주는 우수한 항균 활성으로 인해 사료 첨가제 제조시 저장성을 증진시킬 수 있고, 영양성분의 파괴를 예방할 수 있다.The novel strain of the present invention is excellent in antimicrobial activity without being toxic and can be used as an excellent probiotic preparation. The novel strain has excellent antimicrobial activity and can improve storage stability in the production of feed additive, Destruction can be prevented.
본 발명에 따른 락토바실러스 플란타룸 KCC-24는 우수한 항균 활성을 가진다.Lactobacillus plantarum KCC-24 according to the present invention has excellent antibacterial activity.
또한, 본 발명에 따른 락토바실러스 플란타룸 KCC-24는 항산화 효과가 우수하다.Lactobacillus plantarum KCC-24 according to the present invention is also excellent in antioxidative effect.
본 발명에 따른 상기 균주를 이용하여 다양한 항균용 조성물 또는 항산화용 조성물을 제조할 수 있다. Various antimicrobial compositions or compositions for antioxidation can be prepared using the strain according to the present invention.
본 발명의 균주를 이용하여 제조된 식물체 사일리지는 가축의 소화율을 개선할 수 있고, 항균 효과가 우수하며, 풍미 개선으로 기호성을 향상시킬 수 있어 가축의 품질과 생산성을 높일 수 있다.The plant silage produced by using the strain of the present invention can improve the digestibility of livestock, improve antimicrobial effect, improve the taste by improving flavor, and improve the quality and productivity of livestock.
또한 상기 사일리지는 저장성이 향상되어 농가 소득 증대에 크게 기여할 수 있다.In addition, the silage can greatly contribute to the increase in the farm household income by improving the storage stability.
본 발명의 항균용 조성물 또는 항산화용 조성물은 독성이 없어 우수한 프로바이오틱 효과를 가지므로, 식품, 의약품, 화장품, 건강식품 등 다양한 형태로 활용될 수 있다. Since the antimicrobial composition or antioxidant composition of the present invention has no toxicity and has an excellent probiotic effect, it can be utilized in various forms such as foods, medicines, cosmetics, and health foods.
도 1은 식품 부패균에 대한 Lactobacillus plantarum KCC-24 균주의 항곰팡이 활성을 나타낸 사진 및 KCC-24의 A. fumigatus, P. Chrysogenum, P. roqueforti, B. elliptica, F. oxysporum에 대한 억제율을 나타내었다.
도 2는 L. plantarum KCC-24 배양물이 담즙염 내 생장하는 능력을 나타낸다.
도 3은 L. plantarum KCC-24가 탈지유 배지 상에 클리어 존을 형성하여 단백질분해 활성을 나타낸 사진이다.
도 4는 L. plantarum KCC-24의 시간의 흐름에 따른 자가 응집% 및 자일렌 및 클로로폼에서 소수성%를 나타낸다.
도 5의 a는 L. plantarum KCC-24 무세포 추출물의 DPPH 라디칼 제거 활성을 나타내고, b는 L. plantarum KCC-24의 활성에 과산화수소의 영향을 나타낸다.FIG. 1 shows photographs showing the antifungal activity of Lactobacillus plantarum KCC-24 against food fungi, and the inhibition rates of KCC-24 against A. fumigatus , P. chrysogenum , P. roqueforti , B. elliptica , and F. oxysporum .
Figure 2 shows the ability of the L. plantarum KCC-24 culture to grow in the bile salt.
FIG. 3 is a photograph showing the proteolytic activity of L. plantarum KCC-24 by forming a clear zone on the skim milk medium.
Figure 4 shows the percentage of self-aggregation with time in L. plantarum KCC-24 and hydrophobicity in xylene and chloroform.
5a shows the DPPH radical scavenging activity of L. plantarum KCC-24 cell-free extract, and b shows the effect of hydrogen peroxide on the activity of L. plantarum KCC-24.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.
L. plantarumL. plantarum 의 샘플 수집 및 분리Sample collection and separation
사료는 천안 다양한 장소로부터 수득하였으며, 잠재적 생균제로써 L. plantarum 박테리아를 생산하는 젖산의 분리를 위해 연구실에 보관하였다. 분리된 Lactobacillus 균주 데이터는 추가적인 참고를 위해 연구실에 기록하였다. 분리를 위해, 샘플을 증류수와 혼합하였고 무거운 입자를 제거하기 위해 15분 동안 8000 rpm에서 원심분리(Hanil centrifuge MF 300, Korea)하였다. 상청액을 증류수로 연속적으로 희석하였으며, MRS agar 패트리 디쉬 플레이트에 따라 48시간 동안 30 °C에서 배양하였다. 배양 이후, 정제를 위한 형태학(morphology)에 기초하여 하나의 균주를 분리하였다. 정제된 균주를 KCC-24로 표시하였고 추가적인 사용을 위해 40% 글리세롤과 함께 MRS broth에서 -80°C로 저장하였다. 배양은 사용 전 MRS 액체배지 내 두 번 배양하였다.Feeds were obtained from various sites in Cheonan and stored in a laboratory for the isolation of lactic acid producing L. plantarum bacteria as a potential probiotic. Separated Lactobacillus strain data were recorded in the laboratory for further reference. For separation, the sample was mixed with distilled water and centrifuged (Hanil centrifuge MF 300, Korea) at 8000 rpm for 15 minutes to remove heavy particles. The supernatant was continuously diluted with distilled water and cultured at 30 ° C for 48 hours according to an MRS agar Patridish dish. After incubation, one strain was isolated based on the morphology for purification. The purified strain was designated KCC-24 and was stored at -80 ° C in MRS broth with 40% glycerol for further use. Cultures were cultured twice in MRS liquid medium before use.
16S 16S rRNA의rRNA PCR 증폭 및 PCR amplification and LactobacillusLactobacillus 균주의 분자의 식별 Identification of the molecules of the strain
16S 리보솜 DNA 유전자 시퀀싱(sequencing)은 Sanger, F., Nicklen, S., and Coulson, A.: DNA sequencing with chain terminating inhibitors, Proc. Natl. Acd. Sci. USA., 74, 5463-5467 (1977)에 개시된 방법으로 Sol-Gent Co (Seoul, South Korea)에서 수행하였다. 유전체 DNA를 L. plantarum KCC-24 로부터 분리하였으며, PCR 반응을 27 정방향 프라이머 (5' AGA GTT TGA TCG TGG CTC AG 3') 및 1492 역방향 프라이머(3' GCT TAC CTT GTT ACG ACT T 5')를 사용하여 Taq DNA 중합효소와 함께 수행하였다. 써모사이클러(thermocycler) 조건은 다음과 같다; 표적 DNA의 초기 변성(denaturation)을 95 °C에서 10분 동안 30 사이클 증폭, 1분 동안 58°C에서 어닐링(annealing), 2분 동안 72°C에서 연장(elongation) 및 4°C에서 냉각. PCR 증폭된 rDNA는 QIAquick PCR 정제 키트(Qiagen Ltd., Crawley, UK)를 사용하여 정제하였으며, 앰플리콘(amplicons)을 서열화 하였다. 정제된 서열은 BLAST 정렬(alignment)을 하였으며, 서열 유사성의 검색은 NCBI에서 수행하였다. 데이터베이스는 NCBI Genbank에 보관하였으며, 할당된 NCBI 접근 번호는 KM396462이다.16S ribosomal DNA gene sequencing was performed by Sanger, F., Nicklen, S., and Coulson, A .: DNA sequencing with chain terminating inhibitors, Proc. Natl. Acd. Sci. USA, 74, 5463-5467 (1977) by Sol-Gent Co (Seoul, South Korea). The genomic DNA was isolated from L. plantarum KCC-24 and the PCR reaction was carried out using 27 forward primers (5 'AGA GTT TGA TCG TGG CTC AG 3') and 1492 reverse primer (3 'GCT TAC CTT GTT ACG ACT T 5') With Taq DNA polymerase. The thermocycler conditions are as follows; The initial denaturation of the target DNA was amplified for 30 minutes at 95 ° C for 10 minutes, annealing at 58 ° C for 1 minute, elongation at 72 ° C for 2 minutes and cooling at 4 ° C. The PCR amplified rDNA was purified using a QIAquick PCR purification kit (Qiagen Ltd., Crawley, UK) and sequenced the amplicons. The purified sequence was BLAST aligned and the search for sequence similarity was performed in NCBI. The database was kept in NCBI Genbank and the assigned NCBI access number is KM396462.
분리된 Isolated L. plantarumL. plantarum KCC-24의 항진균 활성의 스크리닝 Screening of antifungal activity of KCC-24
분리된 L. plantarum KCC-24의 항진균 활성을 측정하기 위해, 일부 변형된 Smania, A. J., Delle Monache, F., Smania, E. F. A., and Cuneo, R. S.: Antibacterial activity of steroidal compounds isolated from Ganoderma applanatum (Pers) Pat fruit body, Int. J. Med. Mush., 1, 325-330 (1999)에 개시된 포어(pore) 플레이트 방법을 수행하였다. 페트리디쉬 플레이트는 30ml 살균된 MRS 배지로 제조되어 MRS 배지의 표면에 분리된 L. plantarum를 밤새 배양하고, 콜로니 발달을 위해 48시간 30°C에서 배양하였다. 그에따라 50 μL의 진균 현탁액을 포함하는 10 mL의 감자 한천 배지를 동일한 플레이트에 MRS 배지에 주입했다. 플레이트를 호기성 조건에 72시간 동안 30°C에서 배양하고, 클리어(clear) 억제 존을 기록하였다. 실험을 3회 반복하였다.To determine the antifungal activity of isolated L. plantarum KCC-24, some modified Smania, AJ, Delle Monache, F., Smania, EFA, and Cuneo, RS: Antibacterial activity of steroidal compounds isolated from Ganoderma applanatum (Pers) Pat fruit body, Int. J. Med. Mush., 1, 325-330 (1999). Petri dishes were prepared with 30 ml sterilized MRS medium, and L. plantarum isolated on the surface of MRS medium was cultured overnight and cultured at 30 ° C for 48 hours for colony development. 10 mL of potato agar medium containing 50 μL of the fungal suspension was injected into the MRS medium on the same plate. Plates were incubated at 30 ° C for 72 hours under aerobic conditions and clear inhibition zones were recorded. The experiment was repeated three times.
API 50 CHB 시스템에 의해 분리된
분리된 L.plantarum KCC-24의 하룻밤 배양은 이의 생화학적 및 생리학적 특징을 분석하는데 사용되었다. 표현형을 API 50 CH system (bioMerieux, Inc, USA)으로 분석하였고, 스트립(strip)을 제조자의 지침에 따라 제조하여 사용하였다. 스트립은 48시간 동안 30°C에서 보관하였다. 배양 이후 결과를 기록하였다.An overnight culture of isolated L. plantarum KCC-24 was used to analyze its biochemical and physiological characteristics. The phenotype was analyzed by
분리된 Isolated L. plantarum L. plantarum KCC-24의 KCC-24's 항생물질Antibiotic 감수성 분석 Susceptibility analysis
항생제 감수성 및 저항성은 Bauer, A. W., Kirby, W. M. M., Sherris, J. C., and Turck, M.: Antibiotic susceptibility testing by a standardized single disc method, Am. J. Clin. Pathol., 45, 493-496 (1996)의 한천 확산 방법에 따라 분석하였다. 멸균 MRS 배지의 25 ml를 페트리디쉬 플레이트에 따르고 10분동안 두었다. 이후 L. plantarum KCC-24의 하룻밤 배양물을 MRS 배지에 스왑(swabbed) 하였다. 배지 고체화 이후, 항생 디스크를 MRS 배지의 위에 놓았으며 항생제 확산을 위해 상온에 30분 동안 두었다. 플레이트를 48시간 동안 30°C에서 배양하고, 배양 이후 억제 존(zones)을 관찰하였다.Antimicrobial susceptibility and resistance were assessed by Bauer, AW, Kirby, WMM, Sherris, JC, and Turck, M .: Antibiotic susceptibility testing by a standardized single disc method, Am. J. Clin. Pathol., 45, 493-496 (1996). 25 ml of sterile MRS medium was placed in a petri dish for 10 minutes. The overnight culture of L. plantarum KCC-24 was then swabbed in MRS medium. After solidification of the medium, an antibiotic disc was placed on the MRS medium and allowed to stand at room temperature for 30 minutes for antibiotic diffusion. Plates were incubated at 30 ° C for 48 hours and inhibition zones were observed after incubation.
분리된 Isolated L. plantarumL. plantarum KCC-24의 생균제 특성의 평가 Evaluation of probiotic properties of KCC-24
낮은 pH에 내성Resistant to low pH
L. plantarum 의 낮은 pH 내성을 Tambekar, D. H., and Bhutada, S. A.: Studies on antimicrobial activity and characteristics of bacteriocins produced by Lactobacillus strains isolated from milk of domestic animals, Int. J. Microbiol., 8, 1-6 (2010)에 설명된 방법에 따라 측정하였다. 간단하게 분리된 박테리아 배양물을 다양한 pH (2, 3, 4, 및 5.7)의 멸균된 MRS 액체배지(broth)에 접종하고, 액체배지를 48시간 동안 37°C에서 배양하였다. 배양 이후, 각 튜브로부터 100μL의 접종물을 주입(pour) 플레이트법에 따라 MRS 배지에 주입했다. 플레이트를 48시간 동안 30°C에서 배양하고 콜로니 개수를 세었다.The low pH tolerance of L. plantarum was investigated by Tambekar, DH, and Bhutada, SA: Studies on antimicrobial activity and characteristics of bacteriocins produced by Lactobacillus strains isolated from milk of domestic animals, Int. J. Microbiol., 8, 1-6 (2010). Briefly isolated bacterial cultures were inoculated into sterile MRS liquid broth at various pHs (2, 3, 4, and 5.7) and the liquid medium was incubated at 37 ° C for 48 hours. After incubation, 100 μL of inoculum from each tube was injected into the MRS medium according to the pour plate method. Plates were incubated at 30 ° C for 48 hours and the number of colonies counted.
자극된 위액에 내성Resistant to stimulated gastric juice
자극된 위액에 대한 L. plantarum KCC-24의 내성 분석을 Charteris, W. P., Kelly, P.M., Morelli, L., Collins, J.K.: Development and application of an in-vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract, J. Appl. Microbiol., 84, 759-768 (1998)의 방법에 의해 측정하였다. 자극된 위액을 펩신 3 mg/mL와 함께 준비하였고 0.5% w/v 소듐 클로라이드, 물, 및 pH는 2 및 3으로 조정하였다. 하룻밤 박테리아 배양물의 30 밀리리터를 20분동안 6000 rpm으로 원심분리하였다. 원심분리 이후, 상청액을 제거하고 세포는 50 mM K2HPO4 의 10mL로 두 번 세척하고 3mL K2HPO4로 재현탁하였다. 다양한 pH를 갖는 위액의 9mL를 세포 현탁액 1mL와 혼합하고 3시간 동안 37°C에서 배양하였다. 이후 100μL의 세포 현탁을 다른 시간 간격으로 MRS 배지에 따르고 플레이트를 48시간 동안 30°C에서 배양하였다. 총 살아있는 세포의 수를 세었다.The tolerance of L. plantarum KCC-24 to stimulated gastric juice was investigated by Charteris, WP, Kelly, PM, Morelli, L., Collins, JK: Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract, J. Appl. Microbiol., 84, 759-768 (1998). Stimulated gastric juice was prepared with 3 mg / mL pepsin and 0.5% w / v sodium chloride, water, and pH adjusted to 2 and 3. 30 milliliters of overnight bacterial culture was centrifuged at 6000 rpm for 20 minutes. After centrifugation, the supernatant was removed and the cells were washed twice with 10 mL of 50 mM K 2 HPO 4 and resuspended in 3 mL K 2 HPO 4 . 9 mL of gastric juice with varying pH was mixed with 1 mL of cell suspension and incubated at 37 ° C for 3 hours. 100 μL of cell suspension was then added to the MRS medium at different time intervals and the plate was incubated at 30 ° C for 48 hours. The total number of living cells was counted.
담즙산염에In the bile salt 내성 tolerance
두 담즙염에 대한 분리된 L. plantarum KCC-24 의 성장 내성의 측정은 Vinderola, C. G., and Reinheimer, J. A.: Lactic acid and probiotic bacteria: a comparative in-vitro study of probiotic characteristicsand biological barrier resistance, Food. Res. Int., 36, 895-904 (2003)의 변형된 방법에 따라 수행하였다. MRS 액체배지의 두 종류를 0.3% 우담(oxgall) 및 0.3% 소듐 티오글리콜레이트로 제조하고, 이들을 1% 박테리아 배양 현탁액으로 접종하고 24시간 동안 37°C에서 배양하였다. 배양 기간 이후, 600 nm에서 세포 배양의 광학 밀도를 대조군(담즙 염 없는)과 비교하였다. 결과는 대조군에 비해 성장 내성의 백분율로 표현하였다.Growth measurement of resistance of the
분리된 Isolated L. plantarumL. plantarum KCC-24의 단백질 분해 활성 Proteolytic activity of KCC-24
단백질가수분해 활성은 Rajagopal, S. N., and Sandine, W.E.: Associative growth and proteolysis of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk, J. Dairy Sci., 73, 894-899 (1990) 방법에 따라 분석하였다. 하룻밤 박테리아 배양물의 3 밀리리터를 10분 동안 4000 rpm(4 °C) 에서 원심분리하였다. 수득한 박테리아를 본 분석에 단백질분해효소의 원천으로 사용하였다. 한천 플레이트를 1% 한천, 0.5% 펩톤, 0.3% 쇠고기 추출물, 및 0.5% 탈지 우유로 제조하였다. 박테리아 5 마이크로리터를 한천 배지에 스폿(spot)하고 플레이트를 48시간 동안 30°C에서 배양하였다. 클리어 존은 단백질분해효소를 생산하는 분리된 박테리아의 능력을 나타낸다.Protein hydrolysis activity was determined by Rajagopal, SN, and Sandine, WE: Associative growth and proteolysis of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk, J. Dairy Sci., 73, 894-899 (1990). Three milliliters of overnight bacterial culture was centrifuged at 4000 rpm (4 ° C) for 10 minutes. The bacteria obtained were used as a source of proteolytic enzymes in this analysis. Agar plates were prepared with 1% agar, 0.5% peptone, 0.3% beef extract, and 0.5% skim milk. Five microliters of bacteria were spotted in the agar medium and the plates were incubated at 30 ° C for 48 hours. Clear Zones represent the ability of isolated bacteria to produce proteolytic enzymes.
자가-응집(aggregation) 분석Self-aggregation analysis
일부 셀 간의 자가응집은 일부 변형된 Del Re, B., Sgorbati, B., Miglioli, M., and Palenzona, D.: Adhesion, auto-aggregation and hydrophobicityof 13 strains of Bifidobacterium longum, Lett. Appl. Microbiol., 31, 438-442 (2000)에 설명된 방법에 따라 수행하였다. MRS 액체배지 박테리아 분리물을 10분 동안 8000 rpm으로 원심분리하고, 펠렛을 약 108 CFU ml-1의 농도에 PBS(pH7)로 재현탁하였다. 세포 현탁액의 4 밀리리터를 10 초 동안 볼텍싱(vortexing)하여 혼합하고 두 번째 자가 응집물을 실온에서 3시간 동안 배양하여 분석하였다. 각 시간마다, 위쪽 세포 현탁액의 100 μL를 PBS 3.9 mL와 혼합하고 600 nm에서 흡수를 측정하였다.Self-aggregation between some cells can be induced by some modified Del Re, B., Sgorbati, B., Miglioli, M., and Palenzona, D .: Adhesion, auto-aggregation and hydrophobicity 13 strains of Bifidobacterium longum , Lett. Appl. Microbiol., 31, 438-442 (2000). A MRS broth bacterial isolates for 10 minutes and centrifuged at 8000 rpm and the pellet was resuspended in PBS (pH7) to a concentration of approximately 10 8 CFU ml -1. Four milliliters of the cell suspension was mixed by vortexing for 10 seconds and the second autogenous agglutinates were analyzed by incubation at room temperature for 3 hours. At each time point, 100 μL of the upper cell suspension was mixed with 3.9 mL of PBS and absorbance was measured at 600 nm.
자가 응집은 하기 식에 따라 계산하였다:Self-aggregation was calculated according to the following formula:
1-(At/A0) x 10, At는 시간 t= 1, 2 및 3 및에 흡수를 A0는 t=0에 흡수를 나타냄.1 (A t / A 0 ) x 10, A t absorbs at time t = 1, 2 and 3 and A 0 represents absorption at t = 0.
세포 표면 소수성의 결정Determination of cell surface hydrophobicity
L. plantarum KCC-24의 소수성을 결정하기 위해 인산염 버퍼 용액으로부터 탄화수소에 결합하는 미생물의 능력을 측정하는 소수성 분석을 Rosenberg, M., Gutnick, D., and Rosenberg, E.: Adherence of bacteria to hydrocarbons: a simple method for measuring cell surface hydrophobicity, FEMS. Microbiol. Lett., 9, 29-33 (1980)의 프로토콜에 따라 수행하였다. 분리된 박테리아 배양물 1일령을 15분 동안 5000 rpm에서 원심분리 하였다. 세포 현탁액을 수집하고 PBS(pH 7)로 세척하였다. 흡수는 540 nm에서 측정하였다. 세포 현탁액의 1 밀리리터를 동일한 부피의 탄화수소(자일렌 및 클로로폼)와 혼합하였고 540 nm에서 흡수를 측정하였다. 30초 이후 수용액을 측정하였고 초기 측정치와 비교하였다. 소수성은 하기 식을 사용하여 계산하였다:M. Rosenberg, M., Gutnick, D., and Rosenberg, E .: Adherence of bacteria to hydrocarbons (Lactobacillus acidophilus) to determine the hydrophobicity of L. plantarum KCC-24 by measuring the ability of microorganisms to bind to hydrocarbons from phosphate buffer solutions. : a simple method for measuring cell surface hydrophobicity, FEMS. Microbiol. Lett., 9, 29-33 (1980). One day of isolated bacterial culture was centrifuged at 5000 rpm for 15 minutes. The cell suspension was collected and washed with PBS (pH 7). Absorption was measured at 540 nm. One milliliter of the cell suspension was mixed with the same volume of hydrocarbons (xylene and chloroform) and absorbance was measured at 540 nm. After 30 seconds, the aqueous solution was measured and compared with the initial measurements. The hydrophobicity was calculated using the following formula:
소수성% = (A540 초기 A540 수상)/A540 초기 x 100Hydrophobic% = (A 540 Initial A 540 Award) / A 540 Initial x 100
분리된 Isolated L. plantarumL. plantarum KCC-24의 체외 항산화 특성 In vitro antioxidant properties of KCC-24
DPPH 자유 라디칼 제거 활성의 결정Determination of DPPH free radical scavenging activity
분리된 L. plantarum KCC-24 의 DPPH 라디칼 제거 능력은 Chen, F. A., Wu, A. B., Shieh, P.C., Kuo, D. H., and Hsieh, C. Y.: Evaluation of antioxidant activity of Ruellia tuberosa, Food Chem., 94, 14-18 (2006)의 변형된 방법에 따라 측정하였다. 간단하게, 새로 제조된 분리된 세포(109 CFU/ml)의 다양한 10, 20, 30, 40 및 50 μL 농도를 (0.05 mM) DPPH 용액의 1ml와 혼합하고 30분 동안 실온에서 암실에 배양하였다. DPPH 용액은 대조군으로 사용하였다. 메탄올 및 세포의 조합을 블랭크(blank)로 사용하였다. 흡수는 517nm에서 측정하였다.The DPPH radical scavenging ability of the isolated L. plantarum KCC-24 was evaluated by the method of Chen, FA, Wu, AB, Shieh, PC, Kuo, DH, and Hsieh, CY: Evaluation of antioxidant activity of Ruellia tuberosa, -18 (2006). Briefly, various 10, 20, 30, 40 and 50 μL concentrations of freshly prepared isolated cells (10 9 CFU / ml) were mixed with 1 ml of (0.05 mM) DPPH solution and incubated in the dark room at room temperature for 30 minutes . DPPH solution was used as a control. The combination of methanol and cells was used as a blank. Absorption was measured at 517 nm.
DPPH 제거 능력은 하기 식을 사용하여 계산하였다:The DPPH removal capacity was calculated using the following equation:
DPPH 제거 능력 (%) = [(A샘플-A블랭크)/A대조군] x 100DPPH removal capacity (%) = [(A sample- A blank ) / A control group ] x 100
과산화수소에 대한 저항성의 결정Determination of resistance to hydrogen peroxide
이러한 분석은 약간 변형된 Buchmeier, N., Bossie, S., Chen, C.Y., Fang, F. C., Guiney, D. G., and Libby, S. J.: SlyA, a transcriptional regulator of salmonella typhimurium is required for resistance to oxidative stress and is expressed in the intra cellular environment of macrophages, Infec. Immun., 65, 3725-3730 (1997)의 방법에 따라 수행하였다. 간단하게 밤새 배양한 박테리아 배양물의 10mL를 원심분리하고, 펠렛을 과산화수소의 다양한 농도(0.5, 1.0 및 1.5 mM)의 다양한 플라스크 내 0.9% NaCl의 10ml와 혼합하였다. 박테리아 세포 활성은 샘플의 각각의 희석액으로 BCP 한천 플레이트를 사용하여 37°C에서 30분 배양 이후 처음 측정하였다. 실험을 3회 반복하였다.This analysis is based on a somewhat modified version of Buchmeier, N., Bossie, S., Chen, CY, Fang, FC, Guiney, DG, and Libby, SJ: SlyA, a transcriptional regulator of salmonella typhimurium is required for resistance to oxidative stress and is Expressed in the cellular environment of macrophages, Infec. Immun., 65, 3725-3730 (1997). Briefly, 10 mL of overnight cultured bacterial culture was centrifuged and the pellet mixed with 10 mL of 0.9% NaCl in various flasks at various concentrations of hydrogen peroxide (0.5, 1.0 and 1.5 mM). Bacterial cell activity was first measured after incubation at 37 ° C for 30 min using a BCP agar plate as each dilution of the sample. The experiment was repeated three times.
통계 분석Statistical analysis
모든 수치 데이터는 세가지 독립적 실험으로부터 획득하였고, 이들 데이터 분석은 MS-Exceland SPSS 16 statistical analysis (SPSS Inc., Chicago, IL, USA)으로 수행하였다. 결과는 평균 ± 표준 오차로 나타냈다. 평균 사이에 현저한 차이는 P<0.05 또는 P<0.01의 현저한 수준으로 원-웨이(One-Way) ANOVA를 사용하여 분석하였다.All numerical data were obtained from three independent experiments, and these data analyzes were performed with MS-
결과result
L. plantarumL. plantarum KCC-24의 분리 및 선별 Isolation and selection of KCC-24
본 연구의 목적은 국내 천안에서 수집된 이탈리안 라이그라스 사료로부터 L. plantarum 을 분리하고, 생균제 특징을 확인하는 것이다. 10 락토바실러스 균주 전체는 사료로부터 분리되었다. MRS 내 성장 속도에 기초하여, 한 균주가 다양한 병원체에 대한 항진균 활성을 보이고 다른 생균제 특성을 갖는 것을 밝혔다. 박테리아 균주의 일차 스크리닝 가운데, 추가적인 분자적 확인(identification)을 위해 하나의 균주를 선택하였다. 선택된 균주의 16S rRNA를 분리 및 증폭하고, 서열을 분석하여 NCBI 데이터베이스 내 정보와 비교하였다. BLAST 결과는 L. plantarum의 분리된 박테리아 균주 KCC-24은 GeneBank 데이터베이스에 있는 다른 L. plantarum 균주에 비해 99 % 이상의 상동성을 보였다. 정제된 KCC-24의 박테리아 배양물은 농업생명공학연구원(KACC)에 기탁하여 기탁번호 KACC91962P를 부여받았으며, 16S rRNA 서열은 NCBI Genbank에 등록번호 KM396462로 등록하였다.The purpose of this study was to isolate L. plantarum from Italian ragwass feed collected in Cheonan, Korea and to identify the characteristics of probiotics. 10 whole Lactobacillus strain was isolated from feed. Based on the growth rate in MRS, one strain showed antifungal activity against various pathogens and had other probiotic properties. Among the primary screening of bacterial strains, one strain was selected for additional molecular identification. The 16S rRNA of the selected strain was isolated and amplified, and the sequences were analyzed and compared with the information in the NCBI database. BLAST results discrete bacterial strain KCC-24 of L. plantarum showed a homology of 99% or more compared to the other L. plantarum strains in the GeneBank database. A bacterial culture of purified KCC-24 was deposited with the National Institute of Agricultural Biotechnology (KACC) and assigned Accession No. KACC91962P, and the 16S rRNA sequence was registered with NCBI Genbank under registration number KM396462.
L. plantarumL. plantarum KCC- KCC- 24 의24 생리학적 및 생화학적 특성 Physiological and biochemical characteristics
48시간 배양 이후, 분리된 L. plantarum KCC-24 박테리아를 현미경을 사용하여 육안 관찰하였다. L. plantarum KCC-24은 크림 색상, 막대기 모양, 혐기성, 그람 양성, 및 비포자형성 박테리아이다. 박테리아 배양물은 API 50CH (BioMerieux, Marcy L’Etoile, France) 마이크로 테스트 스트립을 사용하여 생화학적으로 검증하였다. 탄수화물 활용 실험의 결과에 따라, 분리된 박테리아 배양물 KCC-24는 L-Arabinose, D-Ribose, D-Galactose, D-Glucose, D-Fructose, D-Mannose, D-Mannitol, Methyl- αD-mannoside, N-acetyl glucosamine, Amygdalin, Arbutin, Salicin, D-Celiobiose, D-Maltose, D-Lactose, D-Melibiose, D-Saccharose, D-Trehalose, D-Melezitose, D-Raffinose, Gentiobiose, D-Tagatose, D-Fucose, L-Fucose, Potassium gluconate, Potassium 5-Ketogluconate 발효되고, glycerol, erthritol, D-Arabinose, D-xylose, L-xylose, D-adonitol, methyl βD-xyloptranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-Sorbitol, methyl-αD-glucoside, Esculin ferric citrate, inulin, amidon, glycogen, xylitol, D-turanose, D-arabitol, L-arabitol 및 potassium 2-ketogluconate 비발효되었다(표 1).After 48 hours of incubation, the isolated L. plantarum KCC-24 bacteria was visually observed using a microscope. L. plantarum KCC-24 is cream colored, sticky, anaerobic, gram positive, and non-cell forming bacteria. Bacterial cultures were biochemically validated using API 50CH (BioMerieux, Marcy L'Etoile, France) microtest strips. According to the results of the carbohydrate utilization experiment, the isolated bacterial culture KCC-24 was incubated with L-Arabinose, D-Ribose, D-Galactose, D-Glucose, D-Fructose, D- Mannose, D-Mannitol, D-Maltose, D-Lactose, D-Melibiose, D-Saccharose, D-Trehalose, D-Melezitose, D-Raffinose, Gentiobiose, D-Tagatose, D-xylose, L-xylose, D-adonitol, methyl β-xyloptranoside, L-sorbose, L-rhamnose (Fig. 1). The results of this study were as follows: 1) Inoculation of D-arabitol, L-arabitol, and potassium 2-ketogluconate was inhibited by dulcitol, inositol, D-sorbitol, methyl-α-D-glucoside, Esculin ferric citrate, inulin, amidon, glycogen, xylitol, .
분리된 Isolated L. plantarumL. plantarum KCC-24의 KCC-24's 항곰팡이Antifungal 활성 activation
분리된 L. plantarum KCC-24 균주 액체 배양물의 항진균 활성은 다양한 곰팡이 균주 또는 속에 대하여 결정되었다. 연구 결과, 포어 플레이트 방법의 결과는 분리된 박테리아 배양물은 시각적으로 관찰되는 진균 병원체의 성장을 억제할 수 있것으로 나타났다. 마이크로 희석 방법의 결과는 또한 A. fumigatus (73.43 ± 0.96), P. chrysogenum (59.04 ± 0.74), P. roqueforti (56.67 ± 0.72), B. elliptica (40.23 ± 0.43), 및 F. oxysporum (52.47 ± 0.68)에 대한 항진균 활성을 확인하였다(도 1). 이들 억제는 항진균 화합물 및 유기산의 생산에 기인한 것이다.The antifungal activity of the isolated L. plantarum KCC-24 strain liquid culture was determined against various fungal strains or genera. Studies have shown that the results of the forepart method show that isolated bacterial cultures can inhibit the growth of visually observed fungal pathogens. The results of the micro-dilution method were also found to be in the order of A. fumigatus (73.43 ± 0.96) , P. chrysogenum (59.04 ± 0.74) , P. roqueforti (56.67 ± 0.72) , B. elliptica (40.23 ± 0.43), and F. oxysporum (52.47 ± 0.68). These inhibitions are due to the production of antifungal compounds and organic acids.
항생물질에On antibiotics L. plantarumL. plantarum 의 민감성Sensitivity
생균제 균주의 중요한 점은 그들은 항생제 내성을 부여하는 유전자를 갖지 않는 것이다. 분리된 KCC-24 균주의 항생제 민감성은 다양한 종류의 항생제를 사용하여 결정하였다. 실험 결과는, 분리된 KCC-24 균주는 chloramphenicol, nitrofurantoin, tetracycline, streptomycin, dicloxacillin, ampicillin, cefalexin, cefuroxime에 민감성, kanamycin, sulphafurazole, colistin methane sulphonate, amikacin, gentamicin, cefoxitin, 및 co-Trimoxazole에 내성인 것으로 나타났다(표 2).The important point of the probiotic strains is that they do not have genes that confer antibiotic resistance. Antibiotic susceptibility of isolated KCC-24 strains was determined using various antibiotics. The results showed that the isolated KCC-24 strain was resistant to chloramphenicol, nitrofurantoin, tetracycline, streptomycin, dicloxacillin, ampicillin, cefalexin, cefuroxime, kanamycin, sulphafurazole, colistin methane sulphonate, amikacin, gentamicin, cefoxitin, and co- (Table 2).
(Disc potency) (μg)Disk potency
(Disc potency) (μg)
분리된 Isolated L. plantarum L. plantarum KCC-24의 생균제 특성Probiotic characteristics of KCC-24
위장관 내 분리된 L. plantarum KCC-24의 활성에 산도의 효과는 박테리아의 생균제 특성의 중요한 결정인자 중의 하나이다. 따라서 낮은 pH, 위액, 및 담즙염에 내성을 측정하였다. L. plantarum KCC- 24는 pH 5.7, 4.0, 3.0, 및 2.0의 산성 조건에서 3시간 배양 이후에도 생존하였다. pH 1.5에서 살아있는 세포는 발견되지 않았으며, 박테리아 생장은 강하게 억제되었다. 그러나, 박테리아 수는 pH 5.7 내지 2.0에서 1시간 내에 변하지 않았다. L. plantarum KCC-24 박테리아 배양물은 위액에 대한 내성을 보였다. 생리학적으로 담즙염은 일반적으로 인체 내 0.3 내지 0.5% 사이의 범위이다. 담즙염 존재하에 생존 능력을 측정하기 위해 우담(0.3%) 및 소듐 타우로클로레이트(0.3%)를 사용하였다. 결과는 분리된 박테리아 배양물이 담즙염 내 생장하는 능력을 보였다(도 2a-c).The effect of acidity on the activity of isolated L. plantarum KCC-24 in the gastrointestinal tract is one of the important determinants of the probiotic properties of bacteria. Therefore, low pH, gastric juice, and bile salt tolerance were measured. L. plantarum KCC-24 survived after 3 hours incubation at acidic conditions of pH 5.7, 4.0, 3.0, and 2.0. No viable cells were found at pH 1.5, and bacterial growth was strongly inhibited. However, the bacterial counts did not change within one hour at pH 5.7 to 2.0. The culture of L. plantarum KCC-24 bacteria showed resistance to gastric juice. Physiologically, bile salts generally range between 0.3 and 0.5% in the human body. (0.3%) and sodium taurocholate (0.3%) were used to measure viability in the presence of bile salts. The results showed the ability of the isolated bacterial culture to grow in the bile salt (Fig. 2a-c).
L. plantarumL. plantarum KCC-24의 단백질 분해 활성 Proteolytic activity of KCC-24
분리된 L. plantarum KCC-24의 단백질분해 활성은 탈지유를 포함하는 한천 플레이트 상에 분석되었다. 본 목적을 위해 분리된 박테리아 배양물을 탈지유 상에 접종하고 플레이트를 24시간 동안 37°C에서 배양하였다. 배양 이후, L. plantarum 는 탈지유 배지 상에 클리어 존을 형성하여 단백질분해 활성을 나타냈다(도 3).The proteolytic activity of the isolated L. plantarum KCC-24 was analyzed on agar plates containing skim milk. For this purpose, isolated bacterial cultures were inoculated on skim milk and the plates were incubated at 37 ° C for 24 hours. After incubation, L. plantarum showed proteolytic activity by forming a clear zone on the skimmed milk medium (FIG. 3).
침강 속도 및 세포 표면 소수성Settling velocity and cell surface hydrophobicity
응집 및 소수성의 세포 표면 특성은 분리된 박테리아 배양물이, 상피 세포 및 목적하는 박테리아와 상호작용할 수 있는지 결정하기 위해 사용되었다. 도 4에 나타난 바와 같이, 분리된 L. plantarum KCC-24은 3시간 배양 이후 50-73%의 강한 응집 특성을 나타냈다. 나아가, 자일렌 및 클로로폼을 사용하여 세포 표면 소수성을 분석하였다. 결과를 볼 때, L. plantarum 은 자일렌(41.13%) 및 클로로폼(24.17%)에서 세포 표면 소수성의 현저한 차이를 나타냈다. 이들 결과는 L. rhamnosus ST461BZ, L. rhamnosus ST462BZ (75-80%) 및 L. plantarum ST664BZ (55%)의 소수성을 보인, Boza, a natural source ofprobiotic lactic acid bacteria(Todorov, S.D., Botes, M., Guigas, C., Cchillinger, U., Wiid, I., Wachsman, M. B., Holzapfel, W. H., and Dicks, L. M. T., J. Appl. Microbiol., 104, 465-477 (2008))에 나타난 결과에 의해 뒷받침된다.The cell surface properties of aggregation and hydrophobicity were used to determine whether the isolated bacterial culture could interact with epithelial cells and the desired bacteria. As shown in FIG. 4, the isolated L. plantarum KCC-24 exhibited strong aggregation characteristics of 50-73% after 3 hours of culture. Furthermore, cell surface hydrophobicity was analyzed using xylene and chloroform. As a result, L. plantarum showed a significant difference in cell surface hydrophobicity in xylene (41.13%) and chloroform (24.17%). These results indicate that L. rhamnosus ST461BZ, L. rhamnosus A natural source of probiotic lactic acid bacteria (Todorov, SD, Botes, M., Guigas, C., Cchillinger, U.), showing hydrophobicity of ST462BZ (75-80%) and L. plantarum ST664BZ (55% (Wiid, I., Wachsman, MB, Holzapfel, WH, and Dicks, LMT, J. Appl. Microbiol., 104, 465-477 (2008)).
L. L. plantarumplantarum 의 of 체외 항산화 활성 In vitro antioxidant activity
도 5a는 분리된 L. plantarum KCC-24 무세포 추출물의 DPPH 라디칼 제거 활성을 보여준다. 무세포 추출물의 제거 활성은 아스코르브산(P<0.05)에 비할 때, 용량 의존적으로 현저하게 증가했다. 결과로부터, L. plantarum의 DPPH 라디칼 제거 특성은 이차 대사물질의 생산에 기인할 수 있는 것으로 결론지었다. 도 5b는 분리된 박테리아 L. plantarum KCC-24의 활성에 과산화수소의 영향을 나타낸다. L. plantarum KCC-24 은 다양한 농도의 과산화수소에 보통의 내성을 보이며, 과산화수소의 0.3-1,0 mM에 생존할 수 있다. 반면, 박테리아 생장은 과산화수소 1.0 mM 존재 하에 감소하였다.Figure 5a shows DPPH radical scavenging activity of the isolated L. plantarum KCC-24 cell-free extract. The cell-free extract removal activity was significantly increased in a dose-dependent manner when compared to ascorbic acid ( P <0.05). From the results, it was concluded that the DPPH radical scavenging properties of L. plantarum can be attributed to the production of secondary metabolites. Figure 5b shows the effect of hydrogen peroxide on the activity of the isolated bacterial L. plantarum KCC-24. L. plantarum KCC-24 shows normal resistance to various concentrations of hydrogen peroxide and can survive at 0.3-1.0 mM of hydrogen peroxide. On the other hand, bacterial growth was decreased in the presence of 1.0 mM of hydrogen peroxide.
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