KR100808910B1 - Novel lactic acid bacteria having antimicrobial and antiviral effects and compositions comprising the same - Google Patents
Novel lactic acid bacteria having antimicrobial and antiviral effects and compositions comprising the same Download PDFInfo
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- KR100808910B1 KR100808910B1 KR1020060101881A KR20060101881A KR100808910B1 KR 100808910 B1 KR100808910 B1 KR 100808910B1 KR 1020060101881 A KR1020060101881 A KR 1020060101881A KR 20060101881 A KR20060101881 A KR 20060101881A KR 100808910 B1 KR100808910 B1 KR 100808910B1
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- South Korea
- Prior art keywords
- lactic acid
- acid bacteria
- accession
- lactobacillus
- clum0617
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Abstract
본 발명은 항균 및 항바이러스 효과를 가진 신규한 유산균에 관한 것으로서, 본 발명은 유해 미생물의 생육 억제 활성 및 조류독감 바이러스에 대한 억제 활성을 가지는 신규한 미생물인 류코노스톡 메젠테로이드 CLUM0617, 류코노스톡 시트레움 CLUC0620, 락토바실러스 플란타룸 CLP0611 및 락토바실러스 파라카제이 CLPC0603와 이러한 유산균 또는 유산균 배양물을 포함하는 조성물을 제공한다. 본 발명에 따른 신규한 유산균 또는 이 유산균의 배양물은 정장용, 생균용, 항균용, 항바이러스용 등의 용도로 사료, 발효제품, 식품, 의약품 등에 유용하게 사용될 수 있다.The present invention relates to a novel lactic acid bacteria having antimicrobial and antiviral effects, the present invention is a novel microorganism having a growth inhibitory activity of harmful microorganisms and an inhibitory activity against avian influenza virus, leuconosstock methylene terroid CLUM0617, leukonostock Provided are a composition comprising Citrium CLUC0620, Lactobacillus plantarum CLP0611 and Lactobacillus paracasei CLPC0603 and such lactic or lactic acid bacteria cultures. The novel lactic acid bacteria or cultures of the lactic acid bacteria according to the present invention can be usefully used for feed, fermented products, foods, medicines, etc. for the purpose of dressing, living bacteria, antibacterial, antiviral and the like.
류코노스톡 메젠테로이드, 류코노스톡 시트레움, 락토바실러스 플란타룸, 락토바실러스 파라카제이, 항균, 항바이러스, 조류독감 바이러스 Leuconosestock megentoids, Leuconosestock citrium, Lactobacillus plantarum, Lactobacillus paracaze, Antibacterial, Antiviral, Avian flu virus
Description
도 1은 류코노스톡 메젠테로이드(Leuconostoc mesenteroides) CLUM0617 (수탁번호: KCCM10771P)의 성장곡선을 나타내는 그래프이다.1 is a Leuconostoc mesenteroides ) A graph showing the growth curve of CLUM0617 (Accession Number: KCCM10771P).
도 2는 류코노스톡 시트레움(Leuconostoc citreum) CLUC0620 (수탁번호: KCCM10772P)의 성장곡선을 나타내는 그래프이다.2 is Leuconostoc citreum ) Graph showing the growth curve of CLUC0620 (Accession Number: KCCM10772P).
도 3은 락토바실러스 플란타룸(Lactobacillus plantarum) CLP0611 (수탁번호: KCCM10773P)의 성장곡선을 나타내는 그래프이다.Figure 3 is Lactobacillus plantarum ( Lactobacillus plantarum ) is a graph showing the growth curve of CLP0611 (Accession Number: KCCM10773P).
도 4는 락토바실러스 파라카제이(Lactobacillus paracasei) CLPC0603 (수탁번호: KCCM10774P)의 성장곡선을 나타내는 그래프이다.Figure 4 is Lactobacillus paracasei ( Lactobacillus paracasei ) is a graph showing the growth curve of CLPC0603 (Accession Number: KCCM10774P).
도 5는 본 발명의 일 실시예에 따른 혼합유산균배양액의 항균 활성을 나타내는 그래프이다.Figure 5 is a graph showing the antimicrobial activity of the mixed lactic acid bacteria culture medium according to an embodiment of the present invention.
본 발명은 유해 미생물에 대한 항균력 및 조류독감과 같은 바이러스에 대한 항바이러스력을 가진 신규한 유산균, 그 배양액 및 그러한 유산균 또는 유산균의 배양액을 조성물에 관한 것이다.The present invention relates to a composition for a novel lactic acid bacterium having an antimicrobial activity against harmful microorganisms and an antiviral activity against a virus such as avian influenza, a culture medium thereof and a culture solution of such lactic acid bacteria or lactic acid bacteria.
1983년 벨기에, 프랑스 등 유럽에서 발생하기 시작한 이래 2004년 현재까지 세계 각국에서 문제가 되고 있는 조류독감은 조류에 감염되는 급성 바이러스성 전염병으로 주로 닭, 칠면조 등의 가금류에 많은 해를 입힌다. 병원성(病原性)에 따라 고(高)병원성, 약(弱)병원성 및 비(非)병원성 3종류로 구분되며, 이 가운데 고병원성은 인간에게도 감염되어 1997년 홍콩에서 6명이 사망하였고, 2004년 베트남에서는 16명이 사망하기도 하였다.Avian influenza, which has been a problem in countries around the world since 2004, began to occur in Europe such as Belgium and France in 2004, is an acute viral infectious disease that infects birds. It mainly causes poultry such as chickens and turkeys. According to the pathogenicity, it is classified into three types of high pathogenic, weak pathogenic, and non-pathogenic. Among them, the high pathogenicity is also infected with humans, killing 6 people in Hong Kong in 1997, Vietnam in 2004 Esau also killed 16 people.
감염은 조류의 분비물을 직접 접촉할 때 주로 일어나며, 비말(飛沫), 물, 사람의 발, 사료차, 기구, 장비, 알 겉면에 묻은 분변 등에 의해서도 전파된다. 증상은 감염된 바이러스의 병원성에 따라 다양하지만 대체로 호흡기 증상과 설사, 급격한 산란율의 감소가 나타난다. 경우에 따라 볏 등 머리 부위에 청색증이 나타나고, 안면에 부종이 생기거나 깃털이 한 곳으로 모이는 현상이 나타나기도 한다. 폐사율도 병원성에 따라 0~100%로 다양하다.Infection occurs mainly in direct contact with bird secretions and is also spread by splashes, water, human feet, feed cars, appliances, equipment, and feces on the outside of eggs. Symptoms vary depending on the pathogenicity of the infected virus, but usually include respiratory symptoms, diarrhea and a sharp decrease in egg production. Occasionally, cyanosis appears on the head of the crest, edema on the face, or feathers gather in one place. Mortality rates vary from 0 to 100% depending on pathogenicity.
조류독감이 발생하면 전 세계 대부분의 국가에서는 전량 도살 처분하며, 발생국가에서는 양계산물을 수출할 수 없게 된다. 한국에서도 1996년에 이어 2003년에 조류독감이 발생해 전국적으로 확산하였다. 비록 약병원성으로 인체에는 전염되지 않는 것으로 확인되었으나, 우리나라도 조류독감의 피해로부터 결코 안전하다 할 수는 없을 것이다.If bird flu occurs, most of the world's slaughterhouses will be slaughtered, and the producing country will not be able to export poultry products. In Korea, avian influenza occurred in 2003, following 1996, and spread throughout the country. Although it has been confirmed that it is not transmitted to the human body due to medicinal pathogenicity, Korea can never be safe from the damage of bird flu.
또한 양계산업에 있어서 바이러스성 질병뿐 아니라 세균 감염에 의한 닭 호 흡기성 마이코플라스마병(CRD), 대장균증, 살모넬라증(가금디프스, 추백리) 등의 세균성 질병도 빈번히 발생하는데, 닭이 세균에 감염되었을 경우 저항력이 떨어져 다른 질병에도 보다 취약해질 수 있다. 이러한 조류질병으로부터의 피해를 막기 위해 양계 농가에서는 1차적으로는 방역 및 위생관리, 2차적으로는 백신이나 항생제를 사용한다. 하지만 실제 백신의 사용에는 많은 어려움이 따르고, 무절제한 항생제의 사용 또한 바람직하지 않다. 따라서 병원성 세균이나 바이러스에 대한 억제 효과를 가지면서 닭의 면역시스템을 강화시킬 수 있는 복합적인 치료예방제가 필요하다.In addition, in the poultry industry, not only viral diseases but also bacterial diseases such as chicken respiratory mycoplasma disease (CRD), Escherichia coli and Salmonella (poultry diffus, Chubaekli) caused by bacterial infection are frequently caused. If you are less resistant you can be more vulnerable to other diseases. In order to prevent the damage from bird disease, poultry farmers primarily use protection and hygiene, and secondly vaccines and antibiotics. However, the actual use of vaccines has a lot of difficulties, and the use of uncontrolled antibiotics is also undesirable. Therefore, there is a need for a complex therapeutic agent that can enhance the chicken's immune system while having an inhibitory effect on pathogenic bacteria or viruses.
유산균은 자연계에 널리 존재하며 탄수화물을 혐기적으로 이용하여 유산을 생산한다. 유산균이 발견되는 자연환경은 다양한데, 사람이나 동물의 장내에 존재할 뿐 아니라, 다양한 채소와 과일에서도 발견되며 요구르트, 우리나라의 김치나 독일의 사우어크라우트(sauerkraut)와 같은 발효식품에서 그 발효과정에 중요한 역할을 담당한다. 이러한 유산균으로는 스트렙토코커스 속(Streptococcus sp .), 페디오코커스 속(Pediococcus sp .), 류코노스톡 속(Leuconostoc sp .), 락토바실러스 속(Lactobacillus sp .), 스포로락토바실러스 속(Sporolactobacilus sp .), 비피도박테리움 속(Bifidobacterium sp .) 등이 있다.Lactobacillus is widely present in nature and uses carbohydrates anaerobicly to produce lactic acid. The natural environment in which lactobacillus is found is diverse, not only in the gut of humans or animals, but also in various vegetables and fruits. In charge of. These lactic acid bacteria are Streptococcus genus (Streptococcus sp . Pediococcus sp . ), Leuconostoc sp . ), Lactobacillus sp . , Sporolactobacilus sp . ), Bifidobacterium (Bifidobacterium sp . ).
이들 유산균은 장내로 유입된 후 장내 상피세포에 착생하게 되어 유해 미생물의 장 정착을 방지하고 항균 물질을 분비함으로써 유해 미생물의 생육을 억제하고 설사와 변비를 개선할 뿐만 아니라, 면역활성 증진, 항암 작용 등의 효능을 가지고 있다. 특히 동물에게 먹이는 경우, 사료의 소화식이를 돕고, 면역력을 강화시 켜 생균제로서 많이 사용되고 있다.These lactic acid bacteria enters the intestinal epithelial cells and enters the intestinal epithelial cells to prevent intestinal settlement of harmful microorganisms and secrete antimicrobial substances, thereby inhibiting the growth of harmful microorganisms and improving diarrhea and constipation, as well as enhancing immune activity and anticancer activity. It has the effect of back. In particular, when feeding to animals, it is used as a probiotic to help the digestion of the feed, strengthen the immunity.
또한, 항바이러스 효능이 있는 유산균도 많이 보고되어 있는데, 이러한 유산균들은 체내에서 면역활성의 증진과 함께 직접적으로 바이러스의 감염을 억제함으로써 바이러스 감염에 대한 효과적인 예방효과를 기대할 수 있다.In addition, there are many reports of lactic acid bacteria having antiviral efficacy, these lactic acid bacteria can be expected to effectively prevent the viral infection by enhancing the immune activity in the body and directly inhibit the infection of the virus.
따라서 본 발명이 이루고자 하는 기술적 과제는 유해 세균 및 바이러스에 대한 억제효능이 뛰어난 신규한 유산균, 및 이러한 유산균 또는 유산균 배양물을 포함하는 조성물을 제공하는 것이다.Therefore, the technical problem to be achieved by the present invention is to provide a novel lactic acid bacteria having excellent inhibitory effect against harmful bacteria and viruses, and a composition comprising such lactic acid bacteria or lactic acid bacteria culture.
상기 기술적 과제를 달성하기 위하여, 본 발명은 항균 및 항바이러스 효과를 갖는 신규한 유산균인 류코노스톡 메젠테로이드(Leuconostoc mesenteroides) CLUM0617 (수탁번호: KCCM10771P), 류코노스톡 시트레움(Leuconostoc citreum) CLUC0620 (수탁번호: KCCM10772P), 락토바실러스 플란타룸(Lactobacillus plantarum) CLP0611 (수탁번호: KCCM10773P) 또는 락토바실러스 파라카제이(Lactobacillus paracasei) CLPC0603 (수탁번호: KCCM10774P)을 제공한다.In order to achieve the above technical problem, the present invention is a novel lactic acid bacterium having a antimicrobial and antiviral effect Leukonostoc ( Leuconostoc) mesenteroides ) CLUM0617 (Accession No .: KCCM10771P), Leuconostoc citreum) CLUC0620 (accession number: KCCM10772P), Lactobacillus Planta Room (Lactobacillus plantarum ) CLP0611 (Accession No .: KCCM10773P) or Lactobacillus paracasei CLPC0603 (Accession No .: KCCM10774P).
본 발명은 또한 상기 유산균(들) 또는 유산균(들)의 배양물을 포함하는 항균용, 항바이러스용, 정장용, 생균제용 또는 사료첨가용 조성물과 발효제품을 제공한다.The present invention also provides antimicrobial, antiviral, formal, probiotic or feed additive compositions and fermentation products comprising the culture of the lactic acid bacteria (s) or lactic acid bacteria (s).
본 발명자들은 채소와 과일로부터 천연적으로 존재하는 유산균들 중 항균력 및 항바이러스 능력이 우수한 유산균들을 새롭게 분리, 동정하였다. 본 발명자들이 새롭게 분리 및 동정한 유산균들은 유해 세균에 대한 항균력이 우수하며, 조류독감 바이러스 등 바이러스에 대한 억제 효능이 우수하여 유해 미생물의 생육 억제 및 조류독감에 대한 예방효과를 기대할 수 있다.The present inventors newly isolated and identified lactic acid bacteria excellent in antibacterial activity and antiviral ability among naturally occurring lactic acid bacteria from vegetables and fruits. The lactic acid bacteria newly isolated and identified by the present inventors are excellent in antibacterial activity against harmful bacteria, and have an excellent inhibitory effect against viruses such as avian influenza virus, and thus can be expected to inhibit growth of harmful microorganisms and prevent avian influenza.
이하, 본 발명의 항균 및 항바이러스 효과를 가진 신규한 유산균 및 이를 포함하는 조성물에 대해 보다 상세히 설명한다.Hereinafter, a novel lactic acid bacteria having an antimicrobial and antiviral effect of the present invention and a composition comprising the same will be described in more detail.
본 발명은 항균 및 항바이러스 효과가 뛰어나 생균제용, 항균용, 항바이러스용, 사료용 등의 조성물에 사용되기에 유용한 신규한 유산균인 류코노스톡 메젠테로이드(Leuconostoc mesenteroides) CLUM0617 (수탁번호: KCCM10771P), 류코노스톡 시트레움(Leuconostoc citreum) CLUC0620 (수탁번호: KCCM10772P), 락토바실러스 플란타룸(Lactobacillus plantarum) CLP0611 (수탁번호: KCCM10773P) 또는 락토바실러스 파라카제이(Lactobacillus paracasei) CLPC0603 (수탁번호: KCCM10774P)를 제공한다. Leuconostoc mesenteroides CLUM0617 (Accession No .: KCCM10771P), a novel lactic acid bacterium useful for use in compositions such as probiotics, antimicrobials, antivirals, feeds, etc., having excellent antibacterial and antiviral effects. Leuconostoc citreum CLUC0620 (Accession No .: KCCM10772P), Lactobacillus plantarum CLP0611 (Accession No .: KCCM10773P) or Lactobacillus Paracaze ( Lactobacillus) paracasei ) provides CLPC0603 (Accession No .: KCCM10774P).
본 발명에 따른 유산균들은 다음과 같은 방법에 의해 분리 및 동정되었다. 본 발명자들은 먼저, 과일 및 채소로부터 유산균 선별 배지를 이용하여 유산균만을 분리하고 이 중에서 항균력이 우수한 균주를 분리하였다(하기 표 3 참조). 그 다음 상기 분리된 균주의 조류독감 바이러스에 대한 항바이러스 효능이 우수한 균주를 4종 분리하였다(하기 표 4 참조).Lactic acid bacteria according to the present invention were isolated and identified by the following method. The present inventors, first, only lactic acid bacteria were isolated from the fruits and vegetables using lactic acid bacteria selection medium, and among them, strains excellent in antibacterial activity were separated (see Table 3 below). Then, four strains having excellent antiviral efficacy against the avian influenza virus of the isolated strain were isolated (see Table 4 below).
최종 선발된 유산균 4종은 각각 16S rDNA 염기서열에 기초한 분자계통분류학적 분석을 실시하였다. 그 결과, 본 발명에서 분리한 균주 CLUM0617은 류코노스톡 메젠테로이드로, CLUC0620은 류코노스톡 시트레움으로, CLP0611은 락토바실러스 플 란타룸으로, CLPC0603은 락토바실러스 파라카제이로 각각 동정되었다. 본 발명자들은 본 발명에서 분리된 균주를 각각 류코노스톡 메젠테로이드 CLUM0617, 류코노스톡 시트레움 CLUC0620, 락토바실러스 플란타룸 CLP0611, 및 락토바실러스 파라카제이 CLPC0603이라 명명하고 2006년 9월 18일자로 부다페스트 조약하의 국제기탁기관인 대한민국 서울시 서대문구 홍제1동 361-221 유림빌딩에 소재하는 한국미생물보존센터(KCCM)에 수탁번호 KCCM10771P, KCCM10772P, KCCM10773P 및 KCCM10774P로 각각 기탁하였다.The final four selected lactic acid bacteria were subjected to molecular lineage analysis based on the 16S rDNA sequence. As a result, the strain CLUM0617 isolated from the present invention was identified as a leuconosstock megentoid, CLUC0620 as a leuconosstock citrium, CLP0611 as a Lactobacillus plantarum, and CLPC0603 as a Lactobacillus paracaze. The present inventors named the strains isolated in the present invention, respectively, the leukonostock mementoidoid CLUM0617, the leukonostock citrium CLUC0620, the Lactobacillus plantarum CLP0611, and the Lactobacillus paracaze CLPC0603, and on September 18, 2006, Budapest It was deposited with the accession numbers KCCM10771P, KCCM10772P, KCCM10773P and KCCM10774P to the Korea Microorganism Conservation Center (KCCM) located in Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea.
본 발명에서 처음으로 분리 및 동정된 유산균 CLUM0617, CLUC0620, CLP0611, 및 CLPC0603은 다음과 같은 특성을 지닌다.The lactic acid bacteria CLUM0617, CLUC0620, CLP0611, and CLPC0603 isolated and identified for the first time in the present invention have the following characteristics.
첫째, 본 발명에 따른 유산균들은 유해 미생물을 억제하는 효능을 가지며(하기 표 3 참조), 10% 농도로 처리했을 경우 조류독감 바이러스를 억제하는 효과를 나타낸다(하기 표 4 참조).First, the lactic acid bacteria according to the present invention have the effect of inhibiting harmful microorganisms (see Table 3 below), and when treated at a concentration of 10% shows an effect of inhibiting avian influenza virus (see Table 4 below).
둘째, 본 발명에 따른 유산균들은 혼합배양이 가능하며, 그 혼합배양액은 2.5% 이상의 희석농도에서 유해미생물들의 성장을 억제하는 효능을 가진다(하기 도 3 참조). 본 발명에 따른 신규 유산균으로 성장이 억제될 수 있는 유해 미생물로는 살모넬라 티피무리움(Salmonella typhimurium), 스타필로코커스 아우레우스(Staphylococcus aureus), 대장균(E. coli), 크레브시엘라 뉴모니아에(Klebsiella pneumoniae), 바실러스 세레우스(Bacillus cereus), 엔테로박터 크로아세(Enterobacter cloacae) 등이 있으나, 이에 한정되는 것은 아니다(하기 표 8 참조).Second, the lactic acid bacteria according to the present invention can be mixed culture, the mixed culture medium has the effect of inhibiting the growth of harmful microorganisms at a dilution concentration of 2.5% or more (see Figure 3 below). Harmful microorganisms that can be inhibited growth by the novel lactic acid bacteria according to the present invention ( Salmonella typhimurium) typhimurium ), Staphylococcus aureus ), Escherichia coli ( E. coli ), Klebsiella pneumoniae ), Bacillus cereus , Enterobacter cloacae and the like, but are not limited thereto (see Table 8 below).
셋째, 본 발명에 따른 유산균의 혼합배양액은 조류독감바이러스에 대해 높은 저해효능을 나타내어 우수한 항바이러스 효능을 가진다(하기 표 9 참조). 본 발명의 실시예에서는 조류독감 바이러스인 H9N2를 대상으로만 항바이러스 효능을 확인하였지만, 조류독감 바이러스와 비슷한 기작으로 활동하는 바이러스에 대해서도 억제 효능을 가짐을 유추해 볼 수 있다.Third, the mixed culture solution of lactic acid bacteria according to the present invention shows a high inhibitory effect against avian influenza virus (see Table 9 below). In the embodiment of the present invention, the antiviral efficacy was confirmed only for the avian influenza virus H9N2, but it can be inferred to have an inhibitory effect on a virus acting similar to the avian influenza virus.
상기 본 발명에 따른 유산균 CLUM0617, CLUC0620, CLP0611 및 CLPC0603은 통상적인 락토바실러스 속 미생물의 배양법에 의해 대량으로 배양할 수 있다. 배양배지로는 탄소원, 질소원, 비타민 및 미네랄로 구성된 배지를 사용할 수 있다. 예를 들어, MRS 액체 배지 및 우유가 첨가된 액체 배지를 사용할 수 있다. 배양은 통상의 유산균 배양 조건으로 수행할 수 있으며, 예를 들어 약 15-45℃에서 약 10-72시간 동안 배양할 수 있다. 배양액 중의 배양배지를 제거하고 농축된 균체만을 회수하기 위해 원심분리 또는 여과과정을 거칠 수 있으며 이러한 단계는 당업자의 필요에 따라 수행할 수 있다. 농축된 균체는 통상적인 방법에 따라 냉동하거나 냉동건조하여 그 활성을 잃지 않도록 보존할 수 있다.The lactic acid bacteria CLUM0617, CLUC0620, CLP0611 and CLPC0603 according to the present invention can be cultured in large quantities by a conventional method of culturing Lactobacillus sp. The culture medium may be a medium consisting of a carbon source, nitrogen source, vitamins and minerals. For example, MRS liquid medium and liquid medium with milk added can be used. The culture can be carried out under conventional lactic acid culture conditions, for example, can be incubated for about 10-72 hours at about 15-45 ℃. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only concentrated cells, and this step may be performed according to the needs of those skilled in the art. The concentrated cells can be preserved so as not to lose their activity by freezing or lyophilizing according to a conventional method.
또한, 본 발명에 따른 미생물은 당업계에 공지된 통상적인 물리화학적 돌연변이 방법 등에 의해 이와 동등한 활성을 가지거나 또는 이보다 우수한 활성을 가지도록 개선 또는 개량될 수 있다.In addition, the microorganism according to the present invention may be improved or improved to have equivalent activity or better activity by conventional physicochemical mutation methods and the like known in the art.
한편, 본 발명의 CLUM0617, CLUC0620, CLP0611 및 CLPC0603은 유해 세균에 대한 저해효능이 우수하고, 항바이러스 효능이 있어서 동물의 건강증진을 위한 정장용 조성물, 생균제, 항균용 조성물 및 항바이러스용 조성물의 형태로 제공될 수 있다.On the other hand, CLUM0617, CLUC0620, CLP0611 and CLPC0603 of the present invention is excellent in the inhibitory effect against harmful bacteria, and has antiviral efficacy in the form of a dressing composition, a probiotic, an antimicrobial composition and an antiviral composition for animal health promotion. Can be provided.
상기 '정장용'이란 동물의 장내 세균총의 이상 발효에 의하여 야기되는 제반증상을 치료 및 개선하는 용도를 말한다. 상기 증상으로는 이에 한정되지는 않으나 예를 들어, 유해 미생물에 의한 감염성 설사, 위장염, 염증성 장질환, 신경성 장염 증후군, 소장 미생물 과성장증, 장 급이성 설사 등이 포함된다.The 'formal use' refers to the use for treating and ameliorating the symptoms caused by abnormal fermentation of the intestinal flora of the animal. The symptoms include, but are not limited to, for example, infectious diarrhea caused by harmful microorganisms, gastroenteritis, inflammatory bowel disease, neuro enteritis syndrome, small intestine microbial hypergrowth, intestinal diarrhea and the like.
상기 '생균제'는 살아 있는 균 즉, 동물이 섭취했을 때 위장관에 머물러 생존할 수 있는 미생물로서 특정 병리 상태를 예방하거나 치료할 수 있는 효과가 있는 미생물 제제를 말한다. 일반적으로 생균제는 장내 세균총의 이상 발효에 의하여 야기되는 제반 증상을 치료하고 개선하는 효과가 있으며 체내에 투여되면 장내의 소화관 벽에 밀집, 정착하여 유해 미생물이 정착하지 못하게 하는 작용을 하고 유산을 생성하여 장내 pH를 낮추어서 유해 미생물의 증식을 억제한다. 또한, 투여된 생균제는 영양분의 흡수를 담당하는 장융모의 활동을 도와주는 역할을 수행한다.The 'probiotic' refers to a microorganism that has the effect of preventing or treating a specific pathological condition as a living microorganism, that is, a microorganism that can survive in the gastrointestinal tract when ingested by an animal. In general, probiotics have the effect of treating and improving all symptoms caused by abnormal fermentation of the intestinal flora, and when administered into the body, they are concentrated and settled in the gut wall of the intestine, preventing harmful microorganisms from settling and producing lactic acid. It lowers intestinal pH to inhibit the growth of harmful microorganisms. In addition, the administered probiotics play a role in assisting the activity of the intestinal villi responsible for the absorption of nutrients.
상기 '항균용' 또는 '항바이러스용'이란 유해 세균 또는 유해 바이러스의 생육을 저해 또는 억제하는 활성을 말한다. 바람직하게는 상기 유해 미생물로는 생체 내 소화기관에 존재하면서 소화기관과 관련된 여러 질환을 유발하는 미생물일 수 있다.The 'antibacterial' or 'antiviral' refers to the activity of inhibiting or inhibiting the growth of harmful bacteria or harmful viruses. Preferably, the harmful microorganism may be a microorganism present in the digestive tract in vivo and causing various diseases related to the digestive tract.
상술한 조성물들은 본 발명에 따른 유산균의 파쇄된 세포벽 분획, 생균, 사균, 건조균 또는 이의 배양물을 유효성분으로 함유할 수 있으며, 적합한 부형제 또는 담체를 추가로 포함할 수 있다. 상기 배양물은 본 발명에 따른 유산균을 적합한 액체 배지에서 배양한 배양액 자체, 상기 배양액을 여과 또는 원심분리하여 균주를 제거한 여액(여과액 또는 원심분리한 상등액), 상기 배양액을 초음파 처리하거나 상기 배양액에 용해효소(lysozyme)를 처리하여 수득한 세포 파쇄액 등을 포함하나 이에 한정되는 것은 아니다.The above-mentioned compositions may contain the crushed cell wall fraction of lactic acid bacteria, live bacteria, dead bacteria, dried bacteria or cultures thereof according to the present invention as an active ingredient, and may further include a suitable excipient or carrier. The culture is culture medium itself incubated with lactic acid bacteria according to the present invention in a suitable liquid medium, the filtrate (filtered or centrifuged supernatant) from which the strain was removed by filtration or centrifugation, the culture solution by sonication or to the culture solution Cell lysates obtained by treating lysozyme, but are not limited thereto.
또한, 상기 조성물은 당업계에 공지된 방법에 따라 다양한 제형과 방법으로 제조 및 투여될 수 있다. 예를 들어, 본 발명의 유산균 또는 이의 배양물은 약제학적 분야에서 통상적으로 사용되는 담체와 혼합하여 정제(tablet), 트로키(troche), 캡슐(capsule), 엘릭시르(elixir), 시럽(syrup), 산제(powder), 현탁제(suspension), 과립제(granule) 등의 형태로 제조되어 투여될 수 있다. 상기 담체로는 예를 들어, 이에 한정되지는 않으나 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소 및 향료가 포함된다.In addition, the composition may be prepared and administered in various formulations and methods according to methods known in the art. For example, the lactic acid bacterium of the present invention or its culture may be mixed with a carrier commonly used in the pharmaceutical field, such as tablets, troches, capsules, elixirs, syrups. It may be prepared and administered in the form of a powder, a suspension, a granule, or the like. Such carriers include, but are not limited to, binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavorings.
예를 들어, 산제는 본 발명의 유산균과 유당, 전분, 미결정셀룰로오스 등 약제학적으로 허용되는 적당한 부형제를 단순 혼합함으로써 제조될 수 있다. 과립제는 본 발명의 유산균; 약제학적으로 허용되는 적당한 부형제; 및 폴리비닐피롤리돈, 히드록시프로필셀룰로오스 등의 약제학적으로 허용되는 적당한 결합제를 혼합한 후, 물, 에탄올, 이소프로판올 등의 용매를 이용한 습식과립법 또는 압축력을 이용한 건식과립법을 이용하여 제조될 수 있다. 또한 정제는 상기 과립제를 마그네슘스테아레이트 등의 약제학적으로 허용되는 적당한 활택제화 혼합한 후, 타정기를 이용하여 타정함으로써 제조될 수 있다.For example, powders may be prepared by simply mixing the lactic acid bacteria of the present invention with a suitable pharmaceutically acceptable excipient such as lactose, starch, microcrystalline cellulose. Granules are lactic acid bacteria of the present invention; Pharmaceutically acceptable excipients; And a pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropyl cellulose, and then prepared by using a wet granulation method using a solvent such as water, ethanol, isopropanol, or a dry granulation method using a compressive force. Can be. Tablets may also be prepared by mixing the granules with a suitable pharmaceutically acceptable glidant such as magnesium stearate and then tableting using a tableting machine.
상기 조성물의 투여방법으로는 경구투여 방법 또는 정맥내, 근육내, 피하, 국소, 설하, 비강내, 복강내, 직장내 등을 포함하는 다양한 비경구적 방법에 의해 투여될 수 있다.The composition may be administered by oral administration or by various parenteral methods including intravenous, intramuscular, subcutaneous, topical, sublingual, intranasal, intraperitoneal, rectal, and the like.
또한, 투여 용량은 체내에서의 활성성분의 흡수도, 불활성율, 배설속도, 피투여자의 연령, 성별, 상태, 질병의 중증정도 등에 따라 적절히 선택할 수 있다. 생균제용 조성물의 경우에는 조성물 내 생균수가 1×105 내지 1×1011cells/g, 바람직하게는 1×106cells/g 이상이 되도록 투여할 수 있다.In addition, the dosage may be appropriately selected depending on the degree of absorption, inactivation rate, excretion rate, age, sex, condition, severity of disease, etc. of the active ingredient in the body. In the case of a probiotic composition, the number of viable cells in the composition may be administered to 1 × 10 5 to 1 × 10 11 cells / g, preferably 1 × 10 6 cells / g or more.
또한, 본 발명에 따른 CLUM0617, CLUC0620, CLP0611, CLPC0603 또는 이의 개별 혹은 혼합배양물은 사료 조성물의 형태로 제공될 수 있다. 상기 사료 조성물은 기존 항생제의 대체용으로 사용되어 장내 유해균의 생육을 억제하며 장내 균총을 안정되게 유지하여 가축의 건강상태를 양호하게 하여 가축의 증체량과 육질을 개선시키고 산유량 및 면역력을 증가시킬 수 있다. 본 발명의 사료 조성물은 이에 한정되지는 않으나 발효사료, 배합사료, 펠렛 형태, 사일레지(silage) 등의 형태로 제조될 수 있다. 상기 발효사료는 본 발명의 유산균과 여러 가지 미생물 또는 효소를 첨가함으로써 유기물을 발효시켜 제조할 수 있으며, 상기 배합사료는 여러 종류의 일반사료와 본 발명의 유산균을 혼합하여 제조할 수 있다. 또한, 상기 펠렛 형태의 사료는 상기 발효사료 또는 배합사료를 펠렛기에서 열과 압력을 가하여 제조할 수 있으며, 사일레지는 청예사료를 본 발명의 유산균으로 발효시킴으로써 제조할 수 있다.In addition, CLUM0617, CLUC0620, CLP0611, CLPC0603 or individual or mixed cultures thereof according to the present invention may be provided in the form of a feed composition. The feed composition can be used as a substitute for the existing antibiotics to inhibit the growth of harmful intestinal bacteria and maintain the intestinal flora in a stable condition to improve the health of the livestock, improve the weight gain and meat quality of the livestock and increase the milk yield and immunity . Feed composition of the present invention is not limited thereto, but may be prepared in the form of fermented feed, compound feed, pellet form, silage (silage) and the like. The fermented feed may be prepared by fermenting an organic material by adding the lactic acid bacteria and various microorganisms or enzymes of the present invention, the blended feed may be prepared by mixing various kinds of lactic acid bacteria of the general feed and the present invention. In addition, the feed of the pellet form can be prepared by applying heat and pressure to the fermented feed or blended feed in a pellet machine, silage can be prepared by fermenting the green food feed with lactic acid bacteria of the present invention.
또한, 상기 본 발명에 따른 조성물들은 본 발명의 유산균과 함께 동물이 섭취하기에 적합하고 섭취시 유해 미생물의 생육을 억제하며 장내 균총의 균형을 개 선시키는 활성을 가지는 다른 종류의 공지된 미생물을 추가로 포함할 수 있다. 이러한 미생물의 예로는 이에 한정되지는 않으나, 사카로마이세스 세레비지에(Saccharomyces cerevisease), 바실러스 코아굴란스(Bacillus coagulans), 바실러스 리케니포르미스(Bacillus licheniformis), 비피도박테리움 롱검(Bifidobacterium longum), 락토바실러스 사케(Lactobacillus sake), 엔테로코커스 파에칼리스(Enterococcus faecalis), 락토바실러스 아리멘타리우스(Lactobacillus alimentarius), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 루테리(Lactobacillus reuteri), 락토코커스 락티스(Lactococcus lactis) 등을 들 수 있다.In addition, the compositions according to the present invention, along with the lactic acid bacteria of the present invention, adds other known microorganisms that are suitable for ingestion by animals and have the activity of inhibiting the growth of harmful microorganisms and improving the balance of the intestinal flora. It can be included as. Examples of such microorganisms include, but are not limited to, Saccharomyces cerevisease , Bacillus cocillus. coagulans ), Bacillus rickenformis ( Bacillus) licheniformis), ronggeom Bifidobacterium (Bifidobacterium longum), Lactobacillus sake (Lactobacillus sake), Enterococcus faecalis par (Enterococcus faecalis), Lactobacillus Ari menta Julius (Lactobacillus alimentarius), Lactobacillus casei (Lactobacillus casei ), Lactobacillus reuteri , Lactococcus lactis ), and the like.
기타, 본 발명의 유산균 또는 이의 배양물은 유해 세균 또는 바이러스의 성장을 억제하기 위하여 식품, 화장품, 의약품 등에 첨가제로 사용될 수 있다.In addition, the lactic acid bacteria or cultures thereof of the present invention can be used as additives in food, cosmetics, pharmaceuticals, etc. in order to inhibit the growth of harmful bacteria or viruses.
이하, 본 발명을 보다 구체적으로 설명하기 위하여 하기 실시예 등을 들어 설명한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며 본 발명의 범위가 아래에서 상술하는 실시예 들에 한정되는 것으로 해석돼서는 안 된다. 본 발명의 실시예들은 본 발명의 구체적 이해를 돕기 위해 예시적으로 제공되는 것이다.Hereinafter, the following examples and the like will be described in order to describe the present invention in more detail. However, embodiments according to the present invention may be modified in many different forms and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided by way of example in order to facilitate a specific understanding of the present invention.
<실시예 1> 유산균의 분리 및 항균력 확인Example 1 Separation and Confirmation of Antibacterial Activity of Lactic Acid Bacteria
항균력 및 항바이러스능이 있는 유산균을 분리하기 위해 야채 및 과일로부터 다양한 종의 유산균을 분리하였다. 분리한 유산균들을 멸균한 하기 표 1의 MRS 액 체 배지에 접종하여 37℃에서 24시간 배양하였다. 이후 배양액을 6000rpm으로 5분간 원심분리하여 균체를 제거한 후 0.22um 필터로 여과하여 배양액을 제조하였다.To isolate lactic acid bacteria having antibacterial and antiviral activity, lactic acid bacteria of various species were isolated from vegetables and fruits. The isolated lactic acid bacteria were inoculated into sterilized MRS liquid medium of Table 1 and incubated at 37 ° C. for 24 hours. Thereafter, the culture solution was centrifuged at 6000 rpm for 5 minutes to remove the cells, and then filtered through a 0.22um filter to prepare a culture solution.
항균력이 우수한 유산균주를 선별을 위해 제조한 유산균배양액으로 다음과 같은 방법으로 항균력 측정을 하였다.Lactobacillus culture solution prepared for screening lactic acid bacteria with excellent antibacterial activity was measured by the following method.
하기 표 2의 항균활성 측정용 지시균들을 각각 Tryptic soy 액체 배지에 접종하여 37℃에서 하룻밤동안 배양하였다. 멸균하여 45℃로 식힌 소프트 아가 배지(0.8% 아가)에 각 지시균 배양액을 1% 접종하였다. 지시균이 접종된 소프트 아가 배지를 일회용 배지접시에 부어 식혀 평판배지를 제조하였다. 제조된 평판 배지 위에 종이 디스크를 올려놓고 종이 디스크에 제조한 유산균 배양액을 100ul씩 점적한 후, 각 지시균의 최적성장온도에서 24시간 배양하였다. 배양이 끝난 후 각 지시균에 대한 생육억제환(Clear zone)의 지름을 측정함으로써 각 유산균들의 항균활성을 측정하였다.The indicators for measuring the antimicrobial activity of Table 2 were then inoculated in Tryptic soy liquid medium, and then incubated overnight at 37 ° C. 1% of each indicator culture medium was inoculated into soft agar medium (0.8% agar) cooled to 45 ° C. The soft agar medium inoculated with the indicator bacteria was poured into a disposable medium dish and cooled to prepare a plate medium. A paper disk was placed on the prepared flat medium, and the lactic acid bacteria culture solution prepared on the paper disk was instilled by 100 ul, followed by incubation for 24 hours at the optimum growth temperature of each indicator. After incubation, the antimicrobial activity of each lactic acid bacteria was measured by measuring the diameter of the growth zone (Clear zone) for each indicator.
생육억제환의 지름을 비교함으로써 각 균종별로 항균력이 높은 균주를 2종씩 선발하였다. 선발된 균주의 항균활성을 나타내는 저지환의 지름을 표 3에 나타내었다.By comparing the diameter of the growth inhibition ring, two strains with high antimicrobial activity were selected for each strain. Table 3 shows the diameter of the low-ring ring showing the antimicrobial activity of the selected strain.
<실시예 2> 선발한 유산균의 항 조류독감 바이러스 효능Example 2 Anti-Avian Flu Virus Efficacy of Selected Lactic Acid Bacteria
실시예 1에서 선발한 유산균 8종에 대해 다음과 같은 방법으로 항바이러스 효능을 실시하였다.The eight kinds of lactic acid bacteria selected in Example 1 was carried out antiviral efficacy by the following method.
공시 바이러스로는 저병원성 조류인플루엔자 H9N2 혈청형 바이러스인 A/chicken/Korea/MS96/1996 (H9N2)를 사용하였다. 바이러스 AIV MS96주를 9~10일령의 SPF 부화란에 배양하여 역가가 최소 108.0 EID50/ml이상 되도록 증식하며, 증식된 바이러스는 영하 70℃가 유지되는 냉동고에 보관하면서 사용하였다. 유산균 배양액은 증류수로 각각 10배 희석하여 4℃로 유지한 후 사용하였다.As a test virus, A / chicken / Korea / MS96 / 1996 (H9N2), a low-pathogenic avian influenza H9N2 serotype virus, was used. Virus AIV MS96 strains were grown in SPF embryonated eggs at 9-10 days of age to grow to a minimum titer of 10 8.0 EID 50 / ml. The propagated virus was used while stored in a freezer maintained at minus 70 ° C. The lactic acid bacteria culture medium was diluted 10 times with distilled water and maintained at 4 ° C.
10배 희석한 바이러스액(요막강액) 1.0ml를 24ml의 증류수와 섞어 추출물과의 반응용 바이러스를 준비하였다. 10배 희석된 유산균배양액을 2.5ml씩 시험관에 넣고, 준비된 바이러스액 2.5ml를 넣어 혼합한 다음(총 5ml), 4℃에서 정확히 45분간 반응시키며 도중에 10분마다 잘 흔들어 주었다.1.0 ml of a 10-fold diluted virus solution (uremic fluid) was mixed with 24 ml of distilled water to prepare a virus for reaction with the extract. The lactic acid bacteria culture medium diluted 10-fold was put in a test tube of 2.5ml, 2.5ml of the prepared virus solution was mixed (total 5ml), and reacted for exactly 45 minutes at 4 ° C and shaken well every 10 minutes.
바이러스 증식여부의 판정을 위해 반응이 끝나면 반응액을 PBS를 사용하여 10-1, 10-2, 10-3, 10-4, 10-5, 및 10-6로 희석하고 희석배수당 4개의 10일란 발육란에 중화된 반응액 0.2ml을 요막강 내로 접종하였다. 접종 후 37℃에서 5일 동안 배양하며, 매일 검란을 실시하고, 접종 24시간 이내에 죽은 발육란은 사고사로 간주하고 시험성적에서 제외하였다. 접종 24시간 후부터 5일 이내에 죽은 접종란은 모두 4℃에 보관하였다. 접종 5일 후까지 살아남은 모든 발육란과 4℃에 보관된 죽은 접종란으로부터 요막강액을 각각 채취하였고, 혈구응집여부 시험을 실시하여 바이러스 유무를 판단하고 그 결과를 하기 표 4에 나타내었다.At the end of the reaction for determination of virus propagation, the reaction solution was diluted to 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 , and 10 −6 using PBS and 4 10 per dilution factor. 0.2 ml of neutralized reaction solution was inoculated into the ureter cavity into the embryonic egg. After inoculation, the cells were incubated at 37 ° C. for 5 days, and the eggs were examined daily, and embryonated eggs that died within 24 hours of the inoculation were considered as accidental deaths and excluded from test results. All inoculated eggs that were killed within 5 days after 24 hours of inoculation were stored at 4 ° C. Urinary fluids were collected from all embryonated eggs that survived until 5 days after inoculation and dead inoculated eggs stored at 4 ° C., respectively, and the presence or absence of virus was determined by a hemagglutination test.
종별로 항바이러스 효능을 비교하여 최종적으로 CLUM0617, CLUC0620, CLP0611 및 CLPC0603 4가지 균주를 선발하였다.Finally, four strains of CLUM0617, CLUC0620, CLP0611 and CLPC0603 were selected by comparing the antiviral efficacy of each species.
<실시예 3> 선발된 유산균의 동정Example 3 Identification of Selected Lactic Acid Bacteria
<3-1> 형태학적 분석 및 생화학적 특성<3-1> Morphological Analysis and Biochemical Properties
본 발명의 선별된 유산균들을 MRS 평판 배지에서 배양하고 콜로니의 형태를 현미경으로 관찰하였다. 선발한 유산균의 콜로니 형태는 하기 표 5에 나타내었으며, 생화학적인 특징은 하기 표 6에 나타내었다.Selected lactic acid bacteria of the present invention were cultured in MRS plate medium and the morphology of the colonies was observed under a microscope. Colony forms of the selected lactic acid bacteria are shown in Table 5, and biochemical characteristics are shown in Table 6 below.
<3-2> 당 발효 특성 분석<3-2> Sugar Fermentation Characteristics Analysis
본 발명에 따른 유산균의 당 발효 특성을 조사하였다. 당 발효 특성은 API 50 CHL 키트(Bio Merioux사)를 이용하여 공급회사의 실험방법에 따라 조사하였으며, 그 결과는 하기 표 7에 나타내었다. 당 발효 특성 분석 결과, CLUM0617은 류코노스톡 메젠테로이드(Leuconostoc mesenteroides ssp mesenteroides / dextranicum)와 젠티오바이오스 이용성에서 차이를 보이며 99.5%의 동정확률을 나타내었다. CLUC0620은 류코노스톡 시트레움(Leuconostoc citreum)과 D-자일로스, D-만노오스, 2 케토-글루코네이트 및 5 케토-글루코네이트 이용성에서 차이를 보였으며, 72.0%의 동정확률을 나타내었다. CLP0611은 락토바실러스 플란타룸(Lactobacillus plantarum)과 99.9%의 동정확률을 나타내었으며, CLPC0603은 락토바실러스 카제이(Lactobacillus paracasei ssp paracasei)와 락토오스 이용성에서 차이를 보이고, 92.1%의 동정확률을 나타내었다.The sugar fermentation characteristics of the lactic acid bacteria according to the present invention were investigated. Sugar fermentation characteristics were investigated in accordance with the experimental method of the supplier using the API 50 CHL kit (Bio Merioux), the results are shown in Table 7 below. As a result of the analysis of sugar fermentation, CLUM0617 was identified as Leuconostoc. mesenteroides ssp mesenteroides / dextranicum ) and genthiobioavailability showed 99.5%. CLUC0620 differed in Leuconostoc citreum and D-xylose, D-mannose, 2 keto-gluconate and 5 keto-gluconate availability, with a 72.0% probability of identification. CLP0611 had a 99.9% probability of identification with Lactobacillus plantarum , and CLPC0603 showed Lactobacillus paracasei ssp. paracasei ) and lactose availability showed 92.1% identification probability.
상기 표 7에서, +는 발효함을 의미하고, -는 발효하지 않음을 의미한다.In Table 7, + means fermentation,-means not fermentation.
<3-3> 16S rDNA 분석<3-3> 16S rDNA analysis
선발된 4종의 유산균을 동정하기 위해 16S rDNA 염기서열분석을 실시하였으며 그 결과는 각각 서열번호 1 내지 4에 나타내었다. 본 발명의 유산균 CLUM0617은 류코노스톡 메젠테로이드와 99.4%의 16S rDNA 상동성을 보였으며, CLUC0620은 류코노스톡 시트레움과 99.7%의 상동성을, CLP0611은 락토바실러스 플란타룸과 99.7%의 상동성을, CLPC0603은 락토바실러스 파라카제이와 99.8%의 상동성을 보였다. 본 발명자들은 이상의 실험 결과를 기초하여 본 발명에서 분리된 균주 CLUM0617은 류코노스톡 메젠테로이드(Leuconostoc mesenteroides), CLUC0620은 류코노스톡 시트레움(Leuconostoc citreum), CLP0611은 락토바실러스 플란타룸(Lactobacillus plnatarum), CLP0603은 락토바실러스 파라카제이(Lactobacillus paracasei)임을 확인하였다. 따라서, 본 발명자들은 본 발명에서 분리된 균주들을 2006년 9월 18일자로 부다페스트 조약하의 국제기탁기관인 한국미생물보존센터에 수탁번호 KCCM10771P, KCCM10772P, KCCM10773P 및 KCCM10774P로 기탁하였다.16S rDNA sequencing was performed to identify four selected lactic acid bacteria, and the results are shown in SEQ ID NOs: 1 to 4, respectively. The lactic acid bacterium CLUM0617 of the present invention showed homology of 99.4% to 16S rDNA with leukonostock mementoid, CLUC0620 showed homology of 99.7% with leukonostock citrium, and CLP0611 with 99.7% of Lactobacillus plantarum. Homogeneity, CLPC0603 showed 99.8% homology with Lactobacillus paracasei. Based on the above experimental results, the present inventors have isolated the strain CLUM0617 from the present invention, Leuconostoc. mesenteroides ), CLUC0620 was identified as Leuconostoc citreum , CLP0611 was identified as Lactobacillus plnatarum , and CLP0603 as Lactobacillus paracasei . Therefore, the inventors of the present invention deposited the strains isolated in the present invention with the accession numbers KCCM10771P, KCCM10772P, KCCM10773P and KCCM10774P to the Korea microbial conservation center under the Budapest Treaty on September 18, 2006.
<3-4> 선발된 유산균주의 배양특성<3-4> Culture Characteristics of Selected Lactic Acid Bacteria
최종 선발된 유산균 4종을 각각 멸균된 MRS broth에 접종하여 30℃에서 정치배양 하면서 균수와 pH의 변화를 확인하였다. 각 균주의 성장곡선은 각각 도 1 내지 4에 나타내었다.Four final selected lactic acid bacteria were inoculated into sterilized MRS broth, and the cultures were incubated at 30 ° C. to confirm the bacterial count and pH change. The growth curves of each strain are shown in FIGS. 1 to 4, respectively.
<실시예 4><Example 4>
<4-1> 혼합유산균 배양액의 제조<4-1> Preparation of mixed lactic acid bacteria culture medium
상기 실시예 2에서 최종 선발된 유산균 4종에 대해 혼합배양을 실시하였다. 선발된 4종의 유산균을 각각 멸균된 MRS broth에 접종하여 30℃에서 16시간 동안 종균배양을 한 후, 이 종균배양액을 각각 1%씩 멸균된 MRS broth에 혼합접종하여 30℃에서 24시간 동안 배양하였다. 이후 배양액을 6000rpm으로 5분간 원심분리하여 균체를 제거한 후 0.22um 필터로 여과하여 배양액을 제조하였다.The mixed culture was carried out for four kinds of lactic acid bacteria finally selected in Example 2. Four selected lactic acid bacteria were inoculated into sterilized MRS broth and seeded incubated for 16 hours at 30 ° C. Then, the seed culture solution was mixed and inoculated with sterilized MRS broth in 1% each for 24 hours at 30 ° C. It was. Thereafter, the culture solution was centrifuged at 6000 rpm for 5 minutes to remove the cells, and then filtered through a 0.22um filter to prepare a culture solution.
<4-2> 혼합유산균배양액의 항균 활성<4-2> Antimicrobial Activity of Mixed Lactic Acid Bacteria Culture
실시예 4-1에서 제조한 배양액의 항균활성을 측정하기 위해 다음과 같은 방법으로 MIC 시험을 하였다. 하기 표 8의 지시균을 각각 영양 액체 배지에 접종하여 37℃에서 16시간 전배양 후, 전배양액을 새로운 영양 액체 배지에 1%씩 접종하였다. 지시균이 접종된 배지 80ul와 증류수에 희석한 혼합유산균배양액 20ul를 섞어 96웰 플레이트의 각 웰에 분주하였다. 이 플레이트를 37℃에서 배양한 후 600nm에서 OD값을 측정하여 혼합유산균배양액의 지시균 성장억제 농도를 확인하였다(도 5 참조). 그 결과 2.5% 이상의 희석농도에서 모든 지시균에 대한 성장 억제 효과를 확인할 수 있었다.In order to measure the antimicrobial activity of the culture medium prepared in Example 4-1, the MIC test was carried out by the following method. The indicator bacteria of Table 8 were inoculated in the nutrient liquid medium, respectively, and pre-cultured at 37 ° C. for 16 hours, and then the preculture was inoculated in fresh nutrient liquid medium by 1%. 80ul of the medium inoculated with the indicator bacteria and 20ul of mixed lactic acid bacteria culture medium diluted in distilled water were mixed and dispensed into each well of a 96-well plate. After incubating the plate at 37 ° C., the OD value was measured at 600 nm to confirm the indicator growth inhibition concentration of the mixed lactic acid bacteria culture medium (see FIG. 5). As a result, it was confirmed that the growth inhibitory effect on all indicator bacteria at a dilution concentration of 2.5% or more.
<4-3> 혼합유산균배양액의 항 조류독감바이러스 효능<4-3> Anti-Avian Flu Virus Efficacy of Mixed Lactic Acid Bacteria Culture
실시예 4-1에서 제조한 혼합유산균배양액에 대한 항바이러스 효능을 평가하였다. 평가방법은 상기 실시예 2와 같은 방법을 사용하였으며 그 결과는 하기 표 9에 나타내었다. 혼합유산균배양액의 처리결과 처리 후의 바이러스 역가가 100.0이 됨으로써 매우 높은 항바이러스 활성을 가지고 있음을 확인할 수 있었다.The antiviral efficacy of the mixed lactic acid bacteria culture medium prepared in Example 4-1 was evaluated. Evaluation method used the same method as in Example 2 and the results are shown in Table 9 below. As a result of the treatment of the mixed lactic acid bacteria culture medium, the virus titer after the treatment was 10 0.0 , indicating that the antiviral activity was very high.
본 발명은 항균 및 항바이러스 효과를 가진 신규 유산균, 및 이러한 유산균 및/또는 유산균 배양물을 함유하는 조성물을 제공한다. 본 발명에 따른 유산균, 유산균 배양물 또는 조성물은 항균 및 항바이러스 효과가 있어 사료, 식품, 화장품, 의약품 등에 유용하게 사용될 수 있다.The present invention provides novel lactic acid bacteria having antibacterial and antiviral effects, and compositions containing such lactic acid bacteria and / or lactic acid bacteria cultures. Lactic acid bacteria, lactic acid bacteria culture or composition according to the present invention has an antibacterial and antiviral effect can be usefully used in feed, food, cosmetics, pharmaceuticals and the like.
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| KR101001767B1 (en) | 2008-11-04 | 2010-12-15 | 주식회사한국야쿠르트 | Lactobacillus luterii H. Y7501 having anti-influenza virus efficacy and product containing it as an active ingredient |
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| KR101269595B1 (en) | 2010-12-09 | 2013-06-07 | 주식회사 바이오리더스 | Novel Lactic Acid Bacteria Preventing Infection of Avian Influenza Virus, Natural Antivirus Using Thereof and Composition Containing Thereof |
| KR101378608B1 (en) | 2011-04-13 | 2014-03-27 | 씨제이제일제당 (주) | A Novel Lactobacillus fermentum with Treating Activity for animal disease caused by viruses |
| WO2014058114A1 (en) * | 2012-10-08 | 2014-04-17 | 서울대학교 산학협력단 | Composition, containing cis-cyclo(l-leu-l-pro) or cis-cyclo(l-phe-l-pro), for use as an antimicrobial and antiviral agent, and preparation method therefor |
| ITRM20120635A1 (en) * | 2012-12-12 | 2014-06-13 | Stefano Trovo' | "USEFUL COMPOSITION FOR BIOLOGICAL CHECK OF BEE DISEASES" |
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| KR101636014B1 (en) | 2014-07-24 | 2016-07-04 | 수원대학교산학협력단 | Lactobacillus sp. having anti-viral and anti-bacterial activity and use thereof |
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| WO2018231992A3 (en) * | 2017-06-14 | 2019-02-28 | Nutraferma, Inc. | Methods of using lactobacillus plantarum strains for protecting animals from pathogenic bacterial infection |
| US11186815B2 (en) | 2017-06-14 | 2021-11-30 | Nutraferma, Inc. | Methods of using Lactobacillus plantarum strains for protecting animals from pathogenic bacterial infection |
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| WO2019245225A1 (en) * | 2018-06-19 | 2019-12-26 | (주)코엔바이오 | Composition for improving intestinal function comprising leuconostoc sp. strain |
| JP2021527414A (en) * | 2018-06-19 | 2021-10-14 | コエンバイオ カンパニー リミテッド | Composition for improving intestinal function containing a strain of Leuconostocaceae |
| WO2022169337A1 (en) * | 2021-02-08 | 2022-08-11 | 주식회사 리스큐어바이오사이언시스 | Pharmaceutical composition comprising leuconostoc citreum strain as active ingredient for prevention or treatment of intestinal damage |
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