KR20230024155A - Novel Lactococcus lactis subs. cremoris and Use there of - Google Patents
Novel Lactococcus lactis subs. cremoris and Use there of Download PDFInfo
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- KR20230024155A KR20230024155A KR1020210106326A KR20210106326A KR20230024155A KR 20230024155 A KR20230024155 A KR 20230024155A KR 1020210106326 A KR1020210106326 A KR 1020210106326A KR 20210106326 A KR20210106326 A KR 20210106326A KR 20230024155 A KR20230024155 A KR 20230024155A
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- South Korea
- Prior art keywords
- strain
- cremoris
- lactococcus lactis
- present
- composition
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Abstract
Description
본 발명은 새롭게 분리된 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스 균주 및 이의 용도에 대한 것으로, 보다 상세하게는 악취제거, 장관 면역 및 소화기능 개선을 갖는 신규한 유산균 균주 및 이를 이용한 악취제거제 조성물, 사료첨가제, 보조사료, 약학조성물 및 건강기능식품에 대한 것이다. The present invention relates to a newly isolated novel strain of Lactococcus lactis subspacesis cremoris and its use, and more particularly, to a novel strain of lactic acid bacteria having odor removal, intestinal immunity and digestive function improvement, and a odor remover using the same Compositions, feed additives, feed supplements, pharmaceutical compositions and health functional foods.
유산균은 당류를 발효하여 에너지를 획득하고 다량의 젖산을 생성하는 세균을 총칭하는 것으로, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Bifidobacterium 등의 균속으로 정의된다. 이중 락토코쿠스 속 균주들은 대표적인 유산균으로 각종 발효식품에 사용되고, 유제품 특히 치즈생산에 많이 사용되어 오고 있다. 그리고 사람이나 동물의 구강, 장관, 소의 제1위(rumen)까지 자연계에 널리 분포하고, 또한 각 균종의 확실한 분포 서식처로 분류되는 것으로 알려져 있다. Lactobacillus is a general term for bacteria that obtain energy by fermenting sugars and produce a large amount of lactic acid, and are defined as genera such as Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Bifidobacterium. Among them, strains of the genus Lactococcus are representative lactic acid bacteria and are used in various fermented foods, and have been widely used in dairy products, particularly cheese production. And it is known that it is widely distributed in the natural world, including the oral cavity, intestinal tract of humans and animals, and the rumen of cattle, and is also classified as a clear distribution habitat for each fungal species.
이러한 락토코쿠스 속 균주의 새로운 용도를 제공하고자 하는 시도들이 보고되고 있으며, 락토코쿠스 락티스 서브스페이시스 크레모리스를 이용하여 니신(Nisin) 생산량을 높여 항균물질, 식품보존 등의 효능을 높이고자 한 시도는 있으나 악취제거, 장관면역 및 소화기능개선 등의 효과는 보고된 바 없다. Attempts to provide new uses for strains of the genus Lactococcus have been reported, and to increase the efficacy of antibacterial substances and food preservation by increasing the production of Nisin using Lactococcus lactis subspaces cremoris There have been attempts, but the effects such as odor removal, intestinal immunity and digestive function improvement have not been reported.
한편, 기존의 미생물 악취제거제의 경유 유산균 배양액을 사용하기 때문에 암모니아를 대표로 하는 염기성 악취물질는 효과적으로 제거, 억제하는 성능을 가진 반면 황화수소를 대표로 하는 산성 악취물질은 제거, 억제가 되지 않는 단점이 있다. On the other hand, since the existing microbial odor remover uses a lactobacillus culture solution, it has the ability to effectively remove and suppress basic odor substances represented by ammonia, while acidic odor substances represented by hydrogen sulfide cannot be removed or suppressed. .
또한 일시적인 스트레스나 질병 그리고 노화로 인한 장관 기능저하 및 소화불량으로 인한 문제는 일종의 질환으로서 간주된다. 이러한 질환은 장내 세균군의 변화로 인한 기능저하로서 유익균을 섭취하면 저하된 기능을 복원할 수 있다. 이러한 원리를 바탕으로 상당수의 유산균류가 연구되어 장 기능개선 및 소화기능을 개선시키기 위한 노력이 있어 왔다. 그러나 이러한 기존의 유산균들은 대부분 그 효능이 미진하거나 효과가 지속적이지 않는 등 여러 가지 문제점을 가지고 있었다.In addition, temporary stress, disease, and problems caused by intestinal dysfunction and indigestion due to aging are regarded as a kind of disease. These diseases are functional decline due to changes in the intestinal flora, and the reduced function can be restored by ingesting beneficial bacteria. Based on this principle, a significant number of lactic acid bacteria have been studied and efforts have been made to improve intestinal function and digestive function. However, most of these existing lactic acid bacteria had various problems, such as insufficient efficacy or non-continuous effect.
이에 본 발명자들은 기존 악취제거제의 성능을 획기적으로 개선하고, 악취제거 및 소화기능향상 효과가 우수한 천연물 유래 조성물을 개발하고자 예의 노력한 결과, 신규 분리된 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactoccus latis subs. cremoris) RPG-HL-0136 균주가 악취제거, 장관면역 및 소화기능향상에 있어 뛰어난 효과를 가진다는 것을 확인하고, 본 발명을 완성하였다.Accordingly, the inventors of the present invention have made diligent efforts to dramatically improve the performance of existing odor removers and develop a natural product-derived composition with excellent odor removal and digestive function improvement effects, and as a result, a new isolated Lactococcus lactis subspaces cremoris (Lactoccus latis subs. cremoris) RPG-HL-0136 strain was confirmed to have excellent effects in removing odor, improving intestinal immunity and digestive function, and completed the present invention.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술을 형성하는 정보를 포함하지 않을 수 있다. The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms prior art known to those skilled in the art to which the present invention belongs. may not be
본 발명의 목적은 악취제거, 장관 면역 및 소화기능 개선 효능을 갖는 신규한 유산균 균주 및 이를 이용한 악취제거제, 사료첨가제, 보조사료, 약학조성물 및 건강기능식품을 제공하는데 있다. An object of the present invention is to provide a novel lactic acid bacteria strain having odor removal, intestinal immunity and digestive function improvement effects, and odor removers, feed additives, supplementary feeds, pharmaceutical compositions and health functional foods using the same.
상기 목적을 달성하기 위하여, 본 발명은 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스균주 및 이의 용도를 제공한다. In order to achieve the above object, the present invention provides a novel Lactococcus lactis subspaces cremoris strain and uses thereof.
구체적으로, 본 발명은 서열번호 1의 16S rRNA 서열 단편을 가지는 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris)균주를 제공한다. Specifically, the present invention provides a strain of Lactococcus lactis subs. cremoris having the 16S rRNA sequence fragment of SEQ ID NO: 1.
본 발명은 또한, 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 (기탁번호 KCCM13024P) 균주를 제공한다. The present invention also provides a Lactococcus lactis subspace cremoris RPG-HL-0136 (Accession No. KCCM13024P) strain.
본 발명은 또한, 상기 균주, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 포함하는 악취제거제, 사료첨가제, 보조사료, 약학조성물 및 건강기능식품을 제공한다. The present invention also provides odor removers, feed additives, supplementary feeds, pharmaceutical compositions and health functional foods containing any one or more of the above strains, their lysates and their cultures.
본 발명은 악취제거, 장관면역 및 소화기능 향상 효능을 갖는 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스 균주 및 이를 이용한 악취제거제 조성물, 사료첨가제 조성물, 보조사료 조성물, 약학조성물 및 건강기능식품 조성물을 제공한다. The present invention is a novel Lactococcus lactis subspaces cremoris strain having malodor removal, intestinal immunity and digestive function improvement effects, and a deodorant composition, feed additive composition, supplementary feed composition, pharmaceutical composition and health functional food composition using the same provides
본 발명에 따른 균주, 그 파쇄물 및 그 배양물은 기존 악취제거제, 사료첨가제, 보조사료, 약학조성물 및 건강기능식품의 단점을 최소화하여 안전하면서도, 산성 및 염기성 악취물질을 효과적으로 제거 또는 저감하고, 장관면역 저하 및 소화기능의 약화로 인해 초래되는 질환을 개선할 수 있다. 아울러, 효과적으로 면역인자의 발현이 증가하게 하여 면역증강 및 개선에 있어 또한 유용하다. The strain, its lysate and its culture according to the present invention minimize the disadvantages of existing odor removers, feed additives, supplementary feeds, pharmaceutical compositions, and health functional foods, and are safe, effectively remove or reduce acidic and basic odorous substances, and enter the intestines. It can improve diseases caused by weakened immunity and digestive function. In addition, it is also useful for enhancing and improving immunity by effectively increasing the expression of immune factors.
도 1은 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0135균주의 계통도이다(Raphagen HL-0136: 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0135).
도 2는 본 발명에 따른 균주를 실험용기에 조성한 암모니아(100ppm)와 황화수소(50ppm)에 살포하여 30분, 1시간, 2시간, 3시간, 6시간 간격으로 암모니아와 황화수소의 농도를 측정한 그래프이다.
도 3는 본 발명에 따른 균주를 실험용 마우스에게 30일간 대조군, 저용량군, 고용량군으로 나누어 먹인 후 장내에 있는 미생물군중 유익균을 대표하여 Lactobacillus와 Bifidobacterium을 유해균을 대표하여 Beteroides와 Enterobacter를 선별배양을 통하여 균수를 측정한 그래프이다.
도 4은 본 발명에 따른 균주를 실험용 마우스에게 30일간 대조군, 저용량군, 고용량군으로 먹인 후 수집된 분변에서 단쇄지방산(Short chine fatty acid)의 양을 측정한 그래프이다. (control,
도 5는 본 발명에 따른 균주를 실험용 마우스에게 30일간 대조군, 저용량군, 고용량군으로 먹인 후 수집된 분변에서 pH와 수분을 측정한 그래프이다.
도 6은 수집된 실험용 마우스의 분변내에 함유되어 있는 암모니아와 황화수소의 양을 각각의 군별로 측정한 그래프이다.
도 7은 수집된 실험용 마우스의 분변내 소화효소의 전분, 지방, 단백질 분대사율을 각각의 군별로 측정한 그래프이다.
도 8은 실험용 마우스에서 채혈된 혈액내의 면역글로브린G와 장관의 점막층에 존재하는 분비성 면역글로블린A를 각각의 군별로 측정한 그래프이다.
도 9는 수집된 실험용 마우스의 장조직에서 측정한 염증성 사이토카인의 mRNA 발현량을 각각의 군별로 측정한 그래프이다. (염증성 사이토카인 : TNF-α, IL-6, IL-1β, IL-12)
도 10은 수집된 실험용 마우스의 장조직에서 측정한 면역관련 인자의 mRNA 발현량을 각각의 군별로 측정한 그래프이다. (면역인자 : MUC1, MUC2, Reg3γ) 1 is a schematic diagram of the strain RPG-HL-0135 of Lactococcus lactis subs. cremoris according to the present invention (Raphagen HL-0136: Lactococcus lactis subs. cremoris) RPG-HL-0135).
Figure 2 is a graph measuring the concentration of ammonia and hydrogen sulfide at intervals of 30 minutes, 1 hour, 2 hours, 3 hours, and 6 hours by spraying the strain according to the present invention to ammonia (100 ppm) and hydrogen sulfide (50 ppm) prepared in an experimental container. am.
Figure 3 shows that the strain according to the present invention was divided into a control group, a low-dose group, and a high-dose group for 30 days to experimental mice, and then Lactobacillus and Bifidobacterium, representing beneficial bacteria among the microorganisms in the intestine, and Beteroides and Enterobacter, representing harmful bacteria, were selectively cultured. It is a graph measuring the number of bacteria.
Figure 4 is a graph measuring the amount of short chine fatty acid (Short chine fatty acid) in the feces collected after feeding the strain according to the present invention to experimental mice as a control group, a low dose group, and a high dose group for 30 days. (control,
Figure 5 is a graph measuring pH and moisture in feces collected after feeding the strain according to the present invention to experimental mice as a control group, low dose group, and high dose group for 30 days.
Figure 6 is a graph measuring the amount of ammonia and hydrogen sulfide contained in the feces of collected experimental mice for each group.
Figure 7 is a graph measuring the starch, fat, and protein component metabolism rate of digestive enzymes in the feces of the collected experimental mice for each group.
8 is a graph showing the measurement of immunoglobulin G in blood collected from experimental mice and secreted immunoglobulin A present in the mucosal layer of the intestinal tract for each group.
Figure 9 is a graph measuring the mRNA expression level of inflammatory cytokines measured in the intestinal tissue of collected experimental mice for each group. (Inflammatory cytokines: TNF-α, IL-6, IL-1β, IL-12)
Figure 10 is a graph measuring the mRNA expression level of immune-related factors measured in the intestinal tissue of the collected experimental mice for each group. (Immune factors: MUC1, MUC2, Reg3γ)
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.
본 발명의 상세한 설명 등에서 사용되는 주요 용어의 정의는 다음과 같다.Definitions of key terms used in the detailed description of the present invention are as follows.
본 발명에서의 용어 "파쇄물"이란 균주를 detergent 등을 이용한 화학적 방법 또는 물리적 방법 등으로 파쇄하여 얻은 세포 용해물을 의미하며, 균주의 "배양액"이란 균주를 배양시킨 다음, 균주를 제외하고 남은 세포 배양용액을 의미한다. 추가적으로 본 발명에서 "배양물"이란 배양액 또는 배양된 균주를 포함하는 물질이다. The term "lysate" in the present invention means a cell lysate obtained by crushing a strain by a chemical method or a physical method using a detergent, etc., and the "culture medium" of a strain means the remaining cells after culturing the strain, excluding the strain. Means the culture solution. Additionally, in the present invention, "culture" is a material containing a culture medium or a cultured strain.
이하, 구체적으로 본 발명을 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 일 관점에서, 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) 균주에 관한 것이다. 본 발명의 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스 균주는 토양에서 분리한 유산균이다. In one aspect, the present invention relates to a novel Lactococcus lactis subspace cremoris strain. The novel Lactococcus lactis subspaces cremoris strain of the present invention is a lactic acid bacterium isolated from soil.
본 발명에 따른 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스 균주는 서열번호 1의 16S rRNA 서열 단편을 가지는 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) 균주임을 특징으로 한다. 한국미생물 보존센터(KFCC)에 미생물 분석을 의뢰한 결과, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 균주는 서열번호 1의 16S rRNA 서열 단편을 가지는 신규 균주로서 도 1과 같이 계통상 락토코쿠스 락티스 서브스페이시스 크레모리스 AB100802 균주와 가장 근접하게 나타나 락토코쿠스 락티스 서브스페이시스 크레모리스 종으로 분류할 수는 있으나 이와 99%의 homology를 보일 뿐인바 새로운 균주임을 확인할 수 있었다. 아울러, 분리된 신규 균주는 배양 온도 등 균주 특성이나 효능 면에서도 기존 균주와 상이한 효과를 보임을 확인하였다. The novel Lactococcus lactis subspace cremoris strain according to the present invention is characterized by being a Lactococcus lactis subs. cremoris strain having the 16S rRNA sequence fragment of SEQ ID NO: 1 . As a result of requesting microbial analysis to the Korea Microorganism Conservation Center (KFCC), the Lactococcus lactis subspaces cremoris strain according to the present invention is a new strain having a 16S rRNA sequence fragment of SEQ ID NO: 1, and is phylogenetically as shown in FIG. It appeared closest to the Lactococcus lactis subspaces cremoris AB100802 strain and could be classified as a Lactococcus lactis subspaces cremoris species, but only showed 99% homology with it, confirming that it was a new strain. In addition, it was confirmed that the isolated new strain showed different effects from the existing strain in terms of strain characteristics and efficacy, such as incubation temperature.
이에 본 발명자는 분리된 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스 균주를 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136라 명명하고, 한국미생물보존센터 (Korean Culture Center of Microorganisms)에 2021년 07월 27일자로 기탁하였다(기탁번호: KCCM13024P). 이에 본 발명은 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 (기탁번호 KCCM13024P) 균주를 제공한다. Accordingly, the present inventors named the isolated novel Lactococcus lactis subspaces cremoris strain as Lactococcus lactis subs. cremoris RPG-HL-0136, and the Korea Microorganism Conservation Center (Korean Culture Center of Microorganisms) on July 27, 2021 (accession number: KCCM13024P). Accordingly, the present invention provides a strain of Lactococcus lactis subs. cremoris RPG-HL-0136 (accession number KCCM13024P).
본 발명의 일 실시예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 악취제거, 장관면역 및 소화기능 개선 효과를 가짐을 확인하였다. 먼저, 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 악취를 제거할 수 있는 지를 확인하기 위하여, 산성계열 악취물질을 대표로 황화수소(H2S)와 염성기 악취물질을 대표로 암모니아(NH3)를 탈취제 관련 환경부의 환경표지 인증기준(EL608)에 의하여 각각의 악취 물질에 대한 저감을 측정하였다. 그 결과, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주 배양액을 처리한 실험에서 암모니아와 황화수소가 급격하게 저감되는 것을 확인 하였다. 기존의 악취제거용 미생물제제는 화학제제에 비해 토양, 대기, 가축 등에 피해가 없는 매우튼 강점을 가지고 있다. 그러나 기존의 악취제거용 미생물제제가 pH에 의한 중화방식으로 사용되는 유산균의 낮은 pH에 의한 악취 물질을 저감하는 방식이었다. 그러므로 염기성 악취는 상대적으로 효과적인 반면 산성계열의 악취는 제거하지 못하는 큰 단점을 가지고 있다. 즉, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 악취제거용 조성물로서 매우 유용한 것으로 확인되었다. In one embodiment of the present invention, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention has odor removal, intestinal immunity and digestive function improvement effects. First, in order to confirm whether the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain can remove the odor, hydrogen sulfide (H2S) as a representative of acidic odorous substances and ammonia as representative of basic odorous substances (NH3) was measured for the reduction of each odorous substance according to the environmental label certification standard (EL608) of the Ministry of Environment related to deodorant. As a result, it was confirmed that ammonia and hydrogen sulfide were rapidly reduced in an experiment in which the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain culture medium according to the present invention was treated. Compared to chemical preparations, existing microbial preparations for odor removal have a very strong advantage of not causing damage to soil, air, and livestock. However, the conventional microbial preparation for removing odor was a method of reducing odor substances by low pH of lactic acid bacteria used in a neutralization method by pH. Therefore, while the basic odor is relatively effective, it has a great disadvantage that it cannot remove the acidic odor. That is, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention is very useful as a composition for removing odor.
따라서, 본 발명은 다른 관점에서, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 포함하는 악취제거용 조성물에 대한 것이다. 이때, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주 및 그 파쇄물이나 배양물은 악취제거제, 퇴비부숙제의 형태로도 제공될 수 있다.Therefore, from another point of view, the present invention relates to a composition for removing odor comprising at least one of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain, a lysate thereof, and a culture thereof according to the present invention. . At this time, the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain and its lysate or culture according to the present invention may also be provided in the form of an odor remover and compost.
본 발명에 따른 악취제거제 조성물은 다음에 의하여 제공될 수 있다. The malodor remover composition according to the present invention can be provided by the following.
본 발명의 악취제거제 조성물에 포함되는 성분은 상기 유효 성분 이외에 악취제거제 조성물에 통상적으로 이용되는 성분들을 포함할 수 있다. 본 발명의 악취제거제 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어 용액, 현탁액, 유탁액, 분말 등으로 제형화 될 수 있으나, 이에 한정되는 것은 아니다. Ingredients included in the malodor remover composition of the present invention may include ingredients commonly used in malodor remover compositions in addition to the above active ingredients. The odor remover composition of the present invention may be prepared in any formulation commonly prepared in the art, and may be formulated into, for example, a solution, suspension, emulsion, powder, etc., but is not limited thereto.
본 발명의 악취제거제 조성물은 단독 또는 중복하여 사용하거나, 본 발명 이외의 다른 악취제거 조성물과 중복하여 사용할 수 있다. 또한 본 발명에 따른 악취제거제 조성물은 통상적인 사용방법에 따라 사용될 수 있으며, 사용자의 사용 환경 또는 장소에 따라 그 사용횟수를 달리할 수 있다.The malodor removing composition of the present invention may be used alone or overlapping, or may be used overlapping with other malodor removing compositions other than the present invention. In addition, the malodor remover composition according to the present invention may be used according to a conventional method of use, and the number of times of use may vary depending on the environment or place of use of the user.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 장관 유익균 증가시키고, 유해균을 감소시켜 장기능 활성을 가짐을 확인하였다. 즉 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 유의하게 유익균 증가시키고, 유해균을 감소킴을 알 수 있었다. In another embodiment of the present invention, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention increases intestinal beneficial bacteria and reduces harmful bacteria to have long-term functional activity. That is, it was found that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention significantly increased beneficial bacteria and decreased harmful bacteria.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 분변내 단쇄지방산(short chain fatty acid)을 유의미하게 증가시키는 것을 확인하였다. 장내 단쇄지방산은 장내의 pH를 낮춤으로서 장의 연동운동 증가 및 유해균 억제 등 다양한 효과를 가지는 것으로 알려져 있다. In another embodiment of the present invention, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention significantly increased short chain fatty acids in feces. Intestinal short-chain fatty acids are known to have various effects such as increasing intestinal peristalsis and inhibiting harmful bacteria by lowering the pH of the intestine.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 분변내 pH를 유의미하게 낮게 만들고, 수분은 유의미하게 증가시키는 것을 확인하였다. 이는 앞선 다른 실시 예에서 단쇄지방산의 증가로 인한 pH의 저하로 확인되었으며, 장내 단쇄지방산의 증가로 인한 장내 연동운동증가로 분변내 수분이 증가하는 것으로 확인 되었다. In another embodiment of the present invention, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention significantly lowers fecal pH and significantly increases moisture. This was confirmed as a decrease in pH due to an increase in short-chain fatty acids in the previous example, and it was confirmed that water in the feces increased due to an increase in intestinal peristalsis due to an increase in short-chain fatty acids in the intestine.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 분변내 악취물질인 암모니아와 황화수소의 농도를 유의미하게 낮추는 것을 확인하였다. 앞선 다른 실시 예에 의한 유익균의 증가와 유해균의 억제가 분변내 악취물질인 암모니아와 황화수소의 농도를 유의미하게 낮춘 것이 확인 되었다. In another embodiment of the present invention, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention significantly lowers the concentrations of ammonia and hydrogen sulfide, which are odorous substances in feces. It was confirmed that the increase in beneficial bacteria and the suppression of harmful bacteria according to the previous example significantly lowered the concentrations of ammonia and hydrogen sulfide, which are odorous substances in feces.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 분변내 소화효소의 영양성분(전분, 지방, 단백질) 대사율을 측정한 결과 유의미하게 각각 영양성분에 대하여 대사율을 크게 증가시켰다. 즉 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 영양성분에 대한 대사율을 증가시켜 소화효율을 증가 시켰음을 알 수 있었다. In another embodiment of the present invention, the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention measured the metabolic rate of nutrients (starch, fat, protein) of digestive enzymes in feces. The metabolic rate was greatly increased for each nutrient component. That is, it was found that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention increased the metabolic rate for nutrients and increased the digestion efficiency.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 쥐의 혈액내 면역 글로블린G와 실험용쥐의 대장조직에 장점막 분비성 면역글로블리A를 측정한 결과 유의미하게 증가한 것을 확인하였다. 즉 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 면역력을 증가시켰음을 알 수 있었다. In another embodiment of the present invention, the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention is secreted by immunoglobulin G in the blood of laboratory mice and intestinal secretion immunoglobulin in the colon tissue of laboratory mice. As a result of measuring Bl A, it was confirmed that it significantly increased. That is, it was found that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention increased the immunity of the experimental mice.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 쥐의 혈액내 면역 글로블린G와 실험용쥐의 대장조직에 장점막 분비성 면역글로블리A를 측정한 결과 유의미하게 증가한 것을 확인하였다. 즉 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 면역력을 증가시켰음을 알 수 있었다. In another embodiment of the present invention, the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention is secreted by immunoglobulin G in the blood of laboratory mice and intestinal secretion immunoglobulin in the colon tissue of laboratory mice. As a result of measuring Bl A, it was confirmed that it significantly increased. That is, it was found that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention increased the immunity of the experimental mice.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 쥐의 장 조직내에서 염증성 사이토카인 (염증성 사이토카인 : TNF-α, IL-6, IL-1β, IL-12)을 발현하는 mRNA의 양에는 영향을 주지 않을 것으로 확인 되었다. 즉 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스에서 염증반응을 증가시키지 않았음을 알 수 있었다. In another embodiment of the present invention, the strain of Lactococcus lactis subspaces cremoris RPG-HL-0136 according to the present invention can induce inflammatory cytokines (inflammatory cytokines: TNF-α, IL- 6, IL-1β, IL-12) was confirmed not to affect the amount of mRNA. That is, it was found that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention did not increase the inflammatory response in the experimental mice.
본 발명의 다른 실시 예에서는, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 쥐의 장 조직내에서 면역인자(면역인자 : MUC1, MUC2, Reg3γ)을 발현하는 mRNA의 양을 증가시켰음을 확인하였다. 즉 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스에서 면역반응을 증가시켰음을 알 수 있었다. In another embodiment of the present invention, the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention expresses immune factors (immune factors: MUC1, MUC2, Reg3γ) in the intestinal tissues of laboratory mice. It was confirmed that the amount of mRNA to be increased. That is, it was found that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention increased the immune response in the experimental mice.
따라서, 본 발명은 또 다른 관점에서, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 포함하는 장관기능 개선 및 소화기능 향상을 위한 사료첨가제, 보조사료, 건강기능식품에 대한 것이다. Therefore, from another aspect, the present invention improves intestinal function and digestive function including any one or more of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain, its lysate and its culture according to the present invention. It is about feed additives, supplementary feeds, and health functional foods for improvement.
이때, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주 및 그 파쇄물이나 배양물은 장관기능 개선 및 소화기능 향상을 위한 사료첨가제, 보조사료, 건강기능식품의 형태로도 제공될 수 있다. At this time, the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain and its lysates or cultures according to the present invention are in the form of feed additives, supplementary feeds, and health functional foods for improving intestinal function and digestive function. may also be provided.
본 발명에 따른 장관기능 개선 및 소화기능 조성물은 다음에 의하여 제공될 수 있다. The intestinal function improvement and digestive function composition according to the present invention may be provided by the following.
즉, 본 발명에 따르면, 상기 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주 및 그 파쇄물이나 배양물은 장관기능 개선 및 소화기능 조성물의 전체 중량에 대해서 0.0001 ~ 30.0 중량% 함유될 수 있으며, 더욱 바람직하게는, 상기 균주 등은 사료첨가제, 보조사료, 건강기능식품 조성물의 전체 중량에 대해서 0.001 ~ 10 중량% 함유되는 것을 특징으로 한다. 상기 균주 등의 함량이 0.0001 중량% 미만인 경우에는 장관기능 개선 및 소화기능 효과가 나타나지 않으며, 30.0 중량% 를 초과하는 경우에는 함유량 증가에 대한 효과 증대 정도가 미미하며, 제형상의 안전 및 안정성에 문제가 있으며 경제적이지도 못하다.That is, according to the present invention, the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain and its lysate or culture according to the present invention are 0.0001 to 30.0 based on the total weight of the intestinal function improvement and digestive function composition. It may be contained in % by weight, and more preferably, the strain etc. is characterized in that it is contained in 0.001 to 10% by weight based on the total weight of the feed additive, supplementary feed, and health functional food composition. If the content of the above strain is less than 0.0001% by weight, the effect of improving intestinal function and digestive function does not appear, and if it exceeds 30.0% by weight, the degree of increase in the effect of the increased content is insignificant, and the safety and stability of the formulation are problematic and is not economical.
본 발명의 악취제거제 조성물에 포함되는 성분은 상기 유효 성분 이외에 악취제거제 조성물에 통상적으로 이용되는 성분들을 포함할 수 있다. 본 발명의 악취제거제 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어 용액, 현탁액, 유탁액, 분말 등으로 제형화 될 수 있으나, 이에 한정되는 것은 아니다. Ingredients included in the malodor remover composition of the present invention may include ingredients commonly used in malodor remover compositions in addition to the above active ingredients. The odor remover composition of the present invention may be prepared in any formulation commonly prepared in the art, and may be formulated into, for example, a solution, suspension, emulsion, powder, etc., but is not limited thereto.
본 발명의 사료첨가제, 보조사료 조성물은 단독 또는 중복하여 사용하거나, 본 발명 이외의 다른 사료첨가제, 보조사료 조성물과 중복하여 사용할 수 있다. 또한 본 발명에 따른 사료첨가제, 보조사료 조성물은 통상적인 사용방법에 따라 사용될 수 있으며, 사용자의 사용 환경 또는 축종에 따라 그 사용용량을 달리할 수 있다.The feed additive and feed supplement composition of the present invention may be used alone or overlapping, or may be used overlapping with other feed additives and supplementary feed compositions other than the present invention. In addition, the feed additive and supplementary feed composition according to the present invention can be used according to a conventional method of use, and the amount of use can be varied according to the user's use environment or livestock species.
한편, 본 발명에 따른 약학 조성물은 다음에 의하여 제공될 수 있다. Meanwhile, the pharmaceutical composition according to the present invention may be provided by the following.
즉, 본 발명에 따른 균주, 그 파쇄물 및 그 배양물 중 어느 하나 이상을 유효성분으로 함유하는 약학 조성물은 이들을 각각 단독으로 포함하거나 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함하여 약학 조성물로 제공될 수 있으며, 상기 균주 등은 질환 및 이의 중증정도, 환자의 연령, 체중, 건강상태, 성별, 투여 경로 및 치료 기간 등에 따라 적절한 약학적으로 유효한 양으로 약학 조성물에 포함될 수 있다.That is, the pharmaceutical composition containing at least one of the strain, its lysate and its culture according to the present invention as an active ingredient includes each of them alone or includes one or more pharmaceutically acceptable carriers, excipients or diluents. It can be provided as, and the strain and the like can be included in the pharmaceutical composition in an appropriate pharmaceutically effective amount depending on the disease and its severity, the patient's age, weight, health condition, sex, administration route and treatment period.
상기에서 "약학적으로 허용되는"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 슈크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리톤, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.In the above, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolithone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
상기 약학 조성물은 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다. 또한 본 발명의 약학 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사 용액, 멸균 분말의 형태일 수 있다.The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives. In addition, the pharmaceutical composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The dosage form may be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder.
아울러, 본원에서 '기능성 식품'이란, 일반 식품에 본 발명에 따른 균주 또는 그 파쇄물 및 그 배양물 중 어느 하나 이상을 첨가함으로써 식품의 기능성을 향상시킨 것을 의미한다.In addition, 'functional food' herein means that the functionality of food is improved by adding any one or more of the strain or its lysate and its culture according to the present invention to general food.
본 발명의 기능성 식품은 약제, 식품 및 음료 등에 다양하게 이용될 수 있다. The functional food of the present invention can be used in various ways such as medicine, food and beverage.
본 발명의 기능성 식품은, 예를 들어, 각종 식품류, 캔디, 초콜릿, 음료, 껌, 차, 비타민 복합제, 건강 보조 식품류 등이 있고, 분말, 과립, 정제, 캡슐 또는 음료인 형태로 사용할 수 있다.Functional foods of the present invention include, for example, various foods, candy, chocolate, beverages, gum, tea, vitamin complexes, health supplements, etc., and can be used in the form of powders, granules, tablets, capsules or beverages.
이때, 식품 또는 음료 중의 상기 균주 등의 양은 일반적으로 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ml를 기준으로 0.02 내지 10 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다. At this time, the amount of the strain, etc. in the food or beverage can generally be added in 0.01 to 15% by weight of the total food weight, and the health drink composition is 0.02 to 10 g, preferably 0.3 to 1 g based on 100 ml. can be applied to
본 발명의 건강 음료 조성물은 지시된 비율로 필수 성분으로서 상기 균주 등을 함유하는 것 외에 액체 성분에는 특별한 제한점은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등의 디사카라이드, 예를 들어 말토스, 슈크로스 등의 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 화합물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ml당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The health drink composition of the present invention has no particular limitations on liquid components other than containing the above strains as essential components in the indicated proportions, and may contain various flavors or natural carbohydrates as additional components like conventional beverages. . Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, such as maltose, sucrose, etc., and polysaccharides such as dextrins, cyclodextrins, and the like. It is sugar and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia compounds (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 분리 및 시료의 준비Isolation of Novel Lactococcus lactis subspace cremoris RPG-HL-0136 Strain and Preparation of Samples
1-1: 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 분리1-1: Isolation of novel Lactococcus lactis subs. cremoris RPG-HL-0136 strain
토양에서 다수개의 균주 분리 후 선별과정을 거쳐 새로운 균주 RPG-HL-0136을 분리하였다. 이는 한국미생물 보존센터(KCCM)에 미생물 분석을 의뢰한 결과, 서열번호 1의 16S rRNA 서열 단편을 가지는 신규 균주로서 도 1과 같이 계통상 락토코쿠스 락티스 서브스페이시스 크레모리스 AB100802 균주와 가장 근접하게 나타났다. 이에 이에 본 발명자는 분리된 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스 균주를 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136라 명명하였다. 그러나, 신규 분리된 균주는 가장 근접하게 나타난 락토코쿠스 락티스 서브스페이시스 크레모리스 AB100802 균주와도 99%의 homology를 보일 뿐인바 새로운 균주임을 확인할 수 있었다. A new strain, RPG-HL-0136, was isolated through a selection process after isolating a number of strains from the soil. As a result of requesting microbial analysis to the Korea Center for Microbial Conservation (KCCM), this is a new strain having a 16S rRNA sequence fragment of SEQ ID NO: 1, and as shown in FIG. appeared Accordingly, the present inventors named the isolated novel strain of Lactococcus lactis subspace cremoris as Lactococcus lactis subs. cremoris RPG-HL-0136. However, the newly isolated strain showed only 99% homology with the Lactococcus lactis subspaces cremoris AB100802 strain, which appeared the closest, and it was confirmed that it was a new strain.
분리된 신규 균주는 다음의 표 1과 같은 특성을 보였다. The isolated new strain showed the characteristics shown in Table 1 below.
본 발명에서 분리된 신규 균주는 특히 일반적인 락토바실러스 속 균주들이 24~30도에서 배양되는 것과 달리, 고온에서 배양되는 특성을 보였다. 즉, 31 내지 45도에서, 바람직하게는 36도 정도에서 생육되는 고온성 특성을 보였다. 이외에도 발효 진행, 맛, 미생물 군집 형태에서도 상이한 모습을 나타내었다. The novel strain isolated in the present invention showed characteristics of being cultured at a high temperature, unlike general strains of the genus Lactobacillus being cultured at 24 to 30 degrees. That is, it showed high-temperature characteristics growing at 31 to 45 degrees, preferably at about 36 degrees. In addition, fermentation progress, taste, and microbial community morphology also showed different appearances.
이에 본 발명자는 분리된 신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주를 한국미생물보존센터 (Korean Culture Center of Microorganisms)에 2021년 07월 28일자로 기탁하였다(기탁번호: KCCM13024P). Accordingly, the present inventors reported the isolated novel Lactococcus lactis subspace cremoris RPG-HL-0136 strain to the Korean Culture Center of Microorganisms on July 28, 2021. It was deposited as (accession number: KCCM13024P).
1-2: 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136균주 시료의 준비1-2: Preparation of Lactococcus lactis subs. cremoris RPG-HL-0136 strain sample
실시예 1-1에서 분리된 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 효능을 추가적으로 테스트하기 위하여 다음과 같이 시료를 준비하였다. In order to additionally test the efficacy of the Lactococcus lactis subs. cremoris RPG-HL-0136 strain isolated in Example 1-1, samples were prepared as follows.
즉, TS broth(BD, USA)에서 분리된 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주를 16시간 배양한 다음 120도에서 15분간 열처리한 후 냉각하여 사균화한 뒤, 1.0 x 105 ~1.0 x 106 cells/ml로 희석하여 시료를 준비하였다. That is, the Lactococcus lactis subs. cremoris RPG-HL-0136 strain isolated from TS broth (BD, USA) was incubated for 16 hours, then heat-treated at 120 degrees for 15 minutes and then cooled. After inactivation, samples were prepared by diluting to 1.0 x 10 5 to 1.0 x 10 6 cells/ml.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 악취제거 효능 확인Novel Lactococcus lactis subspace cremoris (Lactococcus lactis subs. cremoris) RPG-HL-0136 odor removal efficacy confirmed
2-1 : 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 암모니아 제거 효과2-1: Ammonia removal effect of Lactococcus lactis subs. cremoris strain RPG-HL-0136
본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 대기중에 있는 암모니아를 제거 할 수 있는지 확인하기 위하여, 다음의 실험을 수행하였다. In order to confirm whether the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention can remove ammonia in the air, the following experiment was performed.
먼저, 암모니아를 환경부의 환경표지 인증기준(EL608)에 의하여 밀폐된 11리터 실험용기에 100ppm농도로 만들었다. First, ammonia was made at a concentration of 100 ppm in an 11-liter test vessel sealed according to the environmental label certification standards (EL608) of the Ministry of Environment.
그 다음, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 배양액을 20ml를 실험용기에 분무한 후 30분, 1시간, 2시간, 3시간 6시간에 암모니아 검지관을 이용하여 실험용기내의 암모니아에 농도를 측정하였다. 그 결과를 도 2에 나타내었다.Then, after spraying 20 ml of the culture solution of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention into the test container, ammonia detection was performed at 30 minutes, 1 hour, 2 hours, 3 hours and 6 hours. The concentration of ammonia in the test vessel was measured using a tube. The results are shown in FIG. 2 .
2-2 : 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 황화수소 제거 효과2-2: Hydrogen sulfide removal effect of Lactococcus lactis subs. cremoris RPG-HL-0136 strain
본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 대기중에 있는 암모니아를 제거 할 수 있는지 확인하기 위하여, 다음의 실험을 수행하였다. In order to confirm whether the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention can remove ammonia in the air, the following experiment was performed.
먼저, 황화수소를 환경부의 환경표지 인증기준(EL608)에 의하여 밀폐된 11리터 실험용기에 50ppm농도로 만들었다. First, hydrogen sulfide was made at a concentration of 50 ppm in an 11-liter test container sealed according to the environmental label certification standards (EL608) of the Ministry of Environment.
그 다음, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 배양액을 20ml를 실험용기에 분무한 후 30분, 1시간, 2시간, 3시간 6시간에 황화수소 검지관을 이용하여 실험용기내의 황화수소에 농도를 측정하였다. 그 결과를 도 2에 나타내었다.Then, after spraying 20 ml of the culture solution of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention into an experiment container, hydrogen sulfide detection was performed at 30 minutes, 1 hour, 2 hours, 3 hours and 6 hours. The concentration of hydrogen sulfide in the test vessel was measured using a tube. The results are shown in FIG. 2 .
즉, 본 발명에 따른 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 배양액이 암모니아와 황화수소를 효과적으로 제거하였음으로 악취제거제용 조성물로서 매우 유용한 것으로 확인되었다. That is, since the culture solution of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention effectively removed ammonia and hydrogen sulfide, it was confirmed to be very useful as a composition for odor removal.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 실험용 마우스 장내 세균군 개선Improvement of the intestinal flora of laboratory mice of novel Lactococcus lactis subs. cremoris strain RPG-HL-0136
락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주를 30일간 먹인 후 실험용 마우스를 희생시키고, 장내에 남아있는 분변에 수집하여 장내 세균을 분석하였다.After feeding the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain for 30 days, the experimental mice were sacrificed, and feces remaining in the intestine were collected to analyze intestinal bacteria.
유익균을 대표하여 Lactobacillus 와 Bifidobacterium을 선택배지에서 선택배양하여 숫자를 추정하였으며, 유해균을 대표하여 Beteroides와 Enterobacter을 선택배지에서 선택배양하여 숫자를 추정하였다. Representing beneficial bacteria, Lactobacillus and Bifidobacterium were selectively cultured on a selective medium to estimate their numbers, and representing harmful bacteria, Beteroides and Enterobacter were selectively cultured on a selective medium to estimate their numbers.
Lactobacillus는 LBS배지(BD Difco. Co., Ltd MD, USA)를 사용하였고, Bifidobacterium은 BBL 한천배지(BD Difco. Co., Ltd MD, USA)를 사용하여 배양하였다. Beteroides의 배양에는 GAM배지(BD Difco. Co., Ltd MD, USA)가 사용되었고, Enterobacter의 배양에는 에오신-메틸렌블루 한천배지(BD Difco. Co., Ltd MD, USA)가 사용되었으며, 각각은 37℃에서 48시간 동안 배양되었다. 배양후 미생물의 개수는 평판측정법에 의해 진행되었다. Lactobacillus was cultured using LBS medium (BD Difco. Co., Ltd MD, USA), and Bifidobacterium was cultured using BBL agar medium (BD Difco. Co., Ltd MD, USA). GAM medium (BD Difco. Co., Ltd MD, USA) was used for culturing Beteroides, and Eosin-Methylene Blue agar medium (BD Difco. Co., Ltd MD, USA) was used for culturing Enterobacter. Incubated for 48 hours at 37°C. After incubation, the number of microorganisms was performed by a plate measurement method.
그 결과 , 도3에 나타난 바와 같이, 유익균을 대표하는 Lactobacillus 와 Bifidobacterium의 숫자는 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미하게 증가하였다. 그리고 유해균을 대표하는 Beteroides와 Enterobacter의 숫자는 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미하게 감소하는 것을 확인 하였다. As a result, as shown in FIG. 3, the numbers of Lactobacillus and Bifidobacterium representing beneficial bacteria increased significantly in a dose-dependent manner of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention. In addition, it was confirmed that the number of Beteroides and Enterobacter, which represent harmful bacteria, decreased significantly in a dose-dependent manner of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention.
이러한 결과로 미루어 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 장내에 세균군중에 유익균을 증가시키고, 유해균을 억제하는 것을 확인하였다. Based on these results, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain increased beneficial bacteria and suppressed harmful bacteria in the bacterial population in the intestine of the experimental mouse.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 실험용 마우스 분변내 단쇄지방산 증가효과 Effect of increasing short-chain fatty acids in feces of experimental mice of novel Lactococcus lactis subs. cremoris RPG-HL-0136 strain
락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주를 30일간 먹인 후 실험용 마우스를 희생시키기 전 분변을 모아, 분변내에 존재하는 단쇄지방산의 함량을 측정하였다. After feeding the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain for 30 days, feces were collected before sacrificing the experimental mice, and the content of short-chain fatty acids present in the feces was measured.
측정된 단쇄지방산은 acetic acid, propionic acid 및 butryic acid로 대표적인 단쇄지방산이다. 단쇄지방산은 장내에서 pH를 조절에 관여하여, 유익균을 증가시키고, 유해균을 억제하며 염증을 억제하는 기능한다. The measured short-chain fatty acids are acetic acid, propionic acid, and butryic acid, which are typical short-chain fatty acids. Short-chain fatty acids are involved in regulating pH in the intestine, increasing beneficial bacteria, suppressing harmful bacteria, and suppressing inflammation.
분변내 단쇄지방산의 함량 측정은 수집된 분변 0.5g을 증류수 1mL를 넣고잘 섞은 다음 탈 이온수에 3 시간 동안 담그고, 12,000 rpm으로 10분 동안 원심분리후 상등액을 분리하여 0.22μm 마이크로필터로 여과한 후 High Performance Liquid Chromatography(HPLC Waters e2695 HPLC, photodiode Array detector, *?*USA)로 측정되었다. 사용된 컬럼은 Innocolumn C18 4.6mm ID × 250mm L (YoungJin Biochrom Co., Ltd. Korea)이였으며, 이동상은 pH 2.7 인 20mM/L NaH2PO4,주입량은 20μL, flow 0.8mL/m였다. 흡광도는 210nm였고, co컬럼의 온도는 30 ℃였다. To measure the content of short-chain fatty acids in feces, 0.5 g of the collected feces was added to 1 mL of distilled water, mixed well, soaked in deionized water for 3 hours, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was separated and filtered through a 0.22 μm microfilter. It was measured by High Performance Liquid Chromatography (HPLC Waters e2695 HPLC, photodiode array detector, *?*USA). The column used was Innocolumn C18 4.6mm ID × 250mm L (YoungJin Biochrom Co., Ltd. Korea), the mobile phase was 20mM/L NaH 2 PO 4 at pH 2.7, the injection amount was 20μL, and the flow was 0.8mL/m. The absorbance was 210 nm, and the temperature of the co column was 30 °C.
그 결과 , 도4에 나타난 바와 같이, 대표적인 단쇄지방산인 acetic acid, propionic acid 및 butryic acid의 함량은 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미하게 증가하였다. As a result, as shown in FIG. 4, the contents of acetic acid, propionic acid and butryic acid, which are representative short-chain fatty acids, are significant in a dose-dependent manner of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention. has increased
이러한 결과로 미루어 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 분변내의 단쇄지방산 함량을 증가시킨 것을 확인하였다. Based on these results, it was confirmed that the strain Lactococcus lactis subspaces cremoris RPG-HL-0136 increased the content of short-chain fatty acids in the feces of experimental mice.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 실험용 마우스 분변내 pH 및 수분함량 분석 Analysis of pH and moisture content in feces of laboratory mice of novel Lactococcus lactis subs. cremoris RPG-HL-0136 strain
락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주를 30일간 먹인 후 실험용 마우스를 희생시키기 전 분변을 모아, 분변내의 pH와 수분을 측정하였다. After feeding the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain for 30 days, feces were collected before sacrificing experimental mice, and pH and moisture in the feces were measured.
분변의 pH측정은 수집된 분변 2g을 증류수 20ml에 녹여 pH측정기(Starter 3100, OHAUS, NJ, USA)로 측정되었다. 또한 분변의 수분측정은 수집된 분변을 수분측정기(MB23, OHAUS, NJ, USA)로 측정되었다. The pH of feces was measured by dissolving 2 g of collected feces in 20 ml of distilled water and using a pH meter (Starter 3100, OHAUS, NJ, USA). In addition, fecal moisture was measured with a moisture meter (MB23, OHAUS, NJ, USA) for the collected feces.
그 결과 , 도5에 나타난 바와 같이, pH는 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미하게 낮아지는 것을 확인하였다. 또한 수분의 경우 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미하게 높아지는 것을 확인하였다. As a result, as shown in FIG. 5, it was confirmed that the pH was significantly lowered in a dose-dependent manner of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention. In addition, it was confirmed that water content was significantly increased in a dose-dependent manner of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention.
이러한 결과로 미루어 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 분변내의 pH를 낮추고, 수분함량을 높이는 것을 확인하였다. Based on these results, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain lowered the pH and increased the water content in the feces of experimental mice.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 실험용 마우스 분변내 암모니아 황화수소 함량 분석 Analysis of ammonia hydrogen sulfide content in feces of experimental mice of novel Lactococcus lactis subs. cremoris strain RPG-HL-0136
락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주를 30일간 먹인 후 실험용 마우스를 희생시키기 전 분변을 모아, 분변내의 암모니아와 황화수소의 함량을 측정하였다. After feeding the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain for 30 days, feces were collected before sacrificing the experimental mice, and the contents of ammonia and hydrogen sulfide in the feces were measured.
분변의 H2S 농도는 분변 현탁액 100 ml을 Alka-Seltzer tablets와 넣고 2분간 방치한 후 H2S detectionkit (Model NI-SA, HACH, *?*USA)를 사용하여 측정되였다. The fecal H 2 S concentration was measured using an H 2 S detectionkit (Model NI-SA, HACH, *?*USA) after putting 100 ml of the fecal suspension into Alka-Seltzer tablets and leaving it for 2 minutes.
분변내의 암모니아의*?*농도측정을 위해 암모니아검출키트*?*(Model NI-SA, HACH, USA)가 사용되었으며, 분변현탁액 5 ml를 ammonia salicylate reagent p분말을 첨가하여 3분간 반응시켰다. 그리고 여기에 ammonia cyanurate reagent 분말을 첨가하여 15분간 더 반응시켰고, 시약이 녹색으로 변한 후 colorcomparator box을 이용하여 결과 값을 측정하였다.An ammonia detection kit (Model NI-SA, HACH, USA) was used to measure the *?* concentration of ammonia in feces, and 5 ml of the fecal suspension was reacted for 3 minutes by adding ammonia salicylate reagent p powder. Then, ammonia cyanurate reagent powder was added and reacted for 15 minutes. After the reagent turned green, the result value was measured using a colorcomparator box.
그 결과 , 도6에 나타난 바와 같이, 분변내 암모니아와 황화수소의 함량은 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미하게 낮아지는 것을 확인하였다. As a result, as shown in FIG. 6, it was confirmed that the contents of ammonia and hydrogen sulfide in feces were significantly lowered in a dose-dependent manner of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention.
이러한 결과로 미루어 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 분변내의 암모니아와 황화수소의 함량을 낮추는 것을 확인하였다. Based on these results, it was confirmed that the strain Lactococcus lactis subspaces cremoris RPG-HL-0136 lowered the contents of ammonia and hydrogen sulfide in the feces of experimental mice.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 실험용 마우스 분변내 영양성분 대사율 분석 Metabolic rate analysis of nutrient components in experimental mouse feces of novel Lactococcus lactis subs. cremoris RPG-HL-0136 strain
락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주를 30일간 먹인 후 실험용 마우스를 희생시키기 전 분변을 모아, 분변내의 영양성분(영양성분 : 전분, 지방, 단백질)의 대사율을 측정하였다. After feeding the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain for 30 days, feces were collected before sacrificing the experimental mice, and the metabolic rate of nutrients (nutrients: starch, fat, protein) in the feces was measured. .
실험에 사용된 시료는 MRS 배지(pH6.2)에 1% 분변 현탁액과 5%의 전분을 혼합하여 37℃에서 24시간동안 혐기배양한 시료가 사용되었다.Samples used in the experiment were mixed with 1% fecal suspension and 5% starch in MRS medium (pH 6.2) and incubated anaerobically at 37° C. for 24 hours.
분변 미생물군의 전분분해능력의 측정은 시료를 24시간동안 방치후 1ml 혼합액에 1 ml 에탄올과 5ml NaOH(1M) 혼합하고, 혼합액을 항온수조에서 10분 동안 끓이여 전분을 완전히 아교상태로 만든 후 혼합액에 증류수 43 ml를 투입하였다. 혼합액에서 5ml 취하여 50 ml 병에 넣고, 1ml 아세트산(1M)과 1 ml 요드혼합물(0.2g I2 +2 g KI + 250ml ddH2O) 넣어 30℃에서 20분 동안 방치한 후 흡광도 620nm에서 OD값으로 측정되었다.*?*To measure the starch degrading ability of fecal microbes, the sample was left for 24 hours, mixed with 1 ml ethanol and 5 ml NaOH (1M) in a 1 ml mixture, boiled for 10 minutes in a constant temperature water bath to completely glue the starch, 43 ml of distilled water was added to the mixture. Take 5ml from the mixed solution, put it in a 50ml bottle, add 1ml acetic acid (1M) and 1ml iodine mixture (0.2g I 2 +2 g KI + 250ml ddH 2 O), and leave it at 30℃ for 20 minutes. OD value at absorbance 620nm was measured as *?*
분변내의 세균군의 지방분해율의 측정은 시료에 디클로로메탄을 넣고 용해되지 않는 지방을 용해시킨 후 *?*흡광도 420nm로 OD값으로 측정되었다. To measure the lipolysis rate of the bacterial flora in feces, dichloromethane was added to the sample and insoluble fat was dissolved, and *** absorbance was measured at 420 nm as an OD value.
분변내의 세균군의 단백질분해율의 측정은 MRS배지(pH6.2)에 1% 분변현탁액과 1% Ovalbumin 혼합 후 37℃에서 24시간동안 혐기배양하여 마이크로플레이트리더로 남은Ovalbumin양의 측정을 통해 측정되었다. The protein degradation rate of the bacterial flora in feces was measured by mixing 1% fecal suspension and 1% Ovalbumin in MRS medium (pH 6.2), followed by anaerobic culture at 37 ° C for 24 hours, and measuring the amount of Ovalbumin remaining with a microplate reader. .
그 결과 , 도7에 나타난 바와 같이, 분변내 세균군의 영양성분(영양성분 : 전분, 지방, 단백질)에 대한 대사율은 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미하게 높아지는 것을 확인하였다. As a result, as shown in FIG. 7, the metabolic rate for nutritional components (nutritional components: starch, fat, protein) of fecal bacterial flora according to the present invention Lactococcus lactis subspacesis Cremoris RPG-HL-0136 It was confirmed that the strain increased significantly in a dose-dependent manner.
이러한 결과로 미루어 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 영양성분에 대사능력을 증가시켜 소화기능을 개선한 것을 확인하였다. Based on these results, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain improved the digestive function by increasing the metabolic ability of the experimental mice to nutrients.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 실험용 마우스 혈액내 면역글로블린G와 장관점막의 분비성 면역글로블린A의 함량분석 Analysis of Immunoglobulin G in Blood and Secreted Immunoglobulin A in the Intestinal Mucosa of Experimental Mouse of Novel Lactococcus lactis subs. cremoris RPG-HL-0136 Strain
락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주를 30일간 먹인 후 실험용 마우스를 희생시킨후 후대정맥에서 채혈하고, 장조직을 잘라 혈액내 면역글로블린G와 장관점막의 분비성 면역글로블린A를 측정하였다. After feeding the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain for 30 days, the experimental mice were sacrificed, blood was collected from the posterior vena cava, and the intestinal tissue was cut to obtain immunoglobulin G in the blood and secreted immunoglobulin in the intestinal mucosa. A was measured.
실험용 마우스를 희생시키면서 대장조직과 대장내 분변을 수집하였고, 혈액은 하대정맥에서 채혈 되었다. 채혈된 혈액를 4℃에서 5,000rpm으로 5분간 원심분리하여 혈장을 확보하였고, 마우스 면역글로블린G ELSIA kit(R&D systems MN USA)를 사용하여 마우스 혈장의 면역글로블린G를 측정하였다. 측정은 동일 혈장 10μl씩 3회 반복되었다. While the experimental mice were sacrificed, colon tissue and feces were collected, and blood was collected from the inferior vena cava. Plasma was obtained by centrifuging the collected blood at 5,000 rpm for 5 minutes at 4° C., and immunoglobulin G in mouse plasma was measured using a mouse immunoglobulin G ELSIA kit (R&D systems MN USA). The measurement was repeated 3 times with 10 μl of the same plasma.
분비성 면역글로블린A는 확보된 장 조직을 실온에서 5ml의 PBS1X로 세척하고, 세척액은 4℃에서 10분간 1,000rpm에서 원심분리하여, 분리된 상등액과 m마우스 분비성 면역글로블린A ELISA kit(QuickDetect, BioVision Inc. CA USA)를 이용하여 측정하였다. 면역글로블린G와 분비성 면역글로블린A의 측정에는 마이크로플레이트리더(iMark, Bio-rad Laboratories, Inc. CA USA)가 사용되었다. For secretory immunoglobulin A, the obtained intestinal tissue was washed with 5 ml of PBS1X at room temperature, and the washing solution was centrifuged at 1,000 rpm for 10 minutes at 4°C, and the separated supernatant and mmouse secretory immunoglobulin A ELISA kit (QuickDetect, It was measured using BioVision Inc. CA USA). A microplate reader (iMark, Bio-rad Laboratories, Inc. CA USA) was used to measure immunoglobulin G and secreted immunoglobulin A.
그 결과 , 도8에 나타난 바와 같이, 마우스의 혈액내 면역글로블린G와 장 점막조직의 분비성 면역글로블린A의 양은 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미하게 높아지는 것을 확인하였다. As a result, as shown in FIG. 8, the amount of immunoglobulin G in blood and secreted immunoglobulin A in the intestinal mucosal tissue of mice was the same as that of Lactococcus lactis subspaces Cremoris RPG-HL-0136 strain according to the present invention. It was confirmed that it was significantly increased in a dose-dependent manner.
이러한 결과로 미루어 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 혈액내 면역글로블린G와 장 점막조직의 분비성 면역글로블린A의 양을 증가시켜 면역력을 강화한 것을 확인하였다. Based on these results, it was confirmed that the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain enhanced immunity by increasing the amount of immunoglobulin G in blood and secreted immunoglobulin A in intestinal mucosal tissue of experimental mice. .
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 실험용 마우스 장 조직내의 염증성 사이토카인의 함량분석 Analysis of Inflammatory Cytokine Content in Intestinal Tissues of Experimental Mouse of Novel Lactococcus lactis subs. cremoris RPG-HL-0136 Strain
락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주를 30일간 먹인 후 실험용 마우스를 희생시킨후 장조직을 잘라 장조직내의 염증성 사이토카인 (염증성 사이토카인 : TNF-α, IL-6, IL-1β, IL-12)의 mRNA 발현량을 측정하였다. After feeding the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain for 30 days, the experimental mice were sacrificed, and the intestinal tissue was cut to detect inflammatory cytokines (inflammatory cytokines: TNF-α, IL-6, IL-1β, IL-12) mRNA expression levels were measured.
마우스이 장 조직에서 대표적인 염증성 사이토카인인 TNF-α, IL-1b, IL-6, IL-12의 mRNA 발현량을 측정하기 위하여 확보된 장 조직을 universal RNA extraction kit (Bioneer Co.,ltd, Deajeon Korea) 와 automated nucleic acid extractor (ExiPrep16 Plus, Bioneer Co., ltd, Deajeon Korea)를 이용하여 mRNA를 추출 후 q-PCR(Exicycler™96 V4 Real-Time qPCR, BioneerCo., ltd, Deajeon Korea)로 분석하였다. *?*q-PCR을 위한 parimer는 코스모진텍(Seoul Korea)에서 구매되었다.In order to measure the mRNA expression levels of representative inflammatory cytokines, TNF-α, IL-1b, IL-6, and IL-12 in mouse intestinal tissue, the obtained intestinal tissue was prepared using a universal RNA extraction kit (Bioneer Co.,ltd, Deajeon Korea ) and automated nucleic acid extractor (ExiPrep16 Plus, Bioneer Co., ltd, Deajeon Korea) after extracting mRNA, it was analyzed by q-PCR (Exicycler™96 V4 Real-Time qPCR, Bioneer Co., ltd, Deajeon Korea) . *?*parimers for q-PCR were purchased from Cosmogenetech (Seoul Korea).
그 결과 , 도9에 나타난 바와 같이, 마우스의 장 조직내의 염증성 사이토카인의 mRNA 발현량은 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량과는 관계없이 일정수준을 유지하였다. As a result, as shown in Figure 9, the mRNA expression level of inflammatory cytokines in the intestinal tissue of mice is constant regardless of the amount of Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention. level was maintained.
이러한 결과로 미루어 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 장 조직 내에서 염증반응을 촉진하지 않았음을 확인하였다. Based on these results, it was confirmed that the strain Lactococcus lactis subspaces cremoris RPG-HL-0136 did not promote an inflammatory response in the intestinal tissue of the experimental mice.
신규한 락토코쿠스 락티스 서브스페이시스 크레모리스(Lactococcus lactis subs. cremoris) RPG-HL-0136 균주의 실험용 마우스 장 점막조직내의 면역인자의 함량분석 Analysis of Immune Factors in the Intestinal Mucosal Tissue of Experimental Mouse of Novel Lactococcus lactis subs. cremoris RPG-HL-0136 Strain
락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주를 30일간 먹인 후 실험용 마우스를 희생시킨 후 장조직을 잘라 장 점막조직 내의 면역인자(면역인자 : MUC1, MUC2, Reg3γ)의 mRNA 발현량을 측정하였다. After feeding the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain for 30 days, the experimental mice were sacrificed, and the intestinal tissue was cut to express mRNA expression of immune factors (immune factors: MUC1, MUC2, Reg3γ) in the intestinal mucosal tissue. amount was measured.
마우스이 장 조직에서 대표적인 면역인자인 MUC1, MUC2, Reg3γ의 mRNA 발현량을 측정하기 위하여 확보된 장 조직을 universal RNA extraction kit (Bioneer Co.,ltd, Deajeon Korea) 와 automated nucleic acid extractor (ExiPrep16 Plus, Bioneer Co., ltd, Deajeon Korea)를 이용하여 mRNA를 추출 후 q-PCR(Exicycler™96 V4 Real-Time qPCR, BioneerCo., ltd, Deajeon Korea)로 분석하였다. *?*q-PCR을 위한 parimer는 코스모진텍(Seoul Korea)에서 구매되었다.In order to measure the mRNA expression levels of representative immune factors, MUC1, MUC2, and Reg3γ in mouse intestinal tissue, the obtained intestinal tissue was used with universal RNA extraction kit (Bioneer Co.,ltd, Deajeon Korea) and automated nucleic acid extractor (ExiPrep16 Plus, Bioneer Co., ltd, Deajeon Korea) after extracting mRNA, it was analyzed by q-PCR (Exicycler™96 V4 Real-Time qPCR, Bioneer Co., ltd, Deajeon Korea). *?*parimers for q-PCR were purchased from Cosmogenetech (Seoul Korea).
그 결과 , 도10에 나타난 바와 같이, 마우스의 장 점막조직내의 면역인자의 mRNA 발현량은 본 발명에 따른 락토쿠쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주의 사용량 의존적으로 유의미한 큰 증가를 확인하였다. As a result, as shown in FIG. 10, the mRNA expression level of immune factors in the intestinal mucosal tissue of mice significantly increased in a dose-dependent manner of the Lactococcus lactis subspaces cremoris RPG-HL-0136 strain according to the present invention. confirmed.
이러한 결과로 미루어 락토코쿠스 락티스 서브스페이시스 크레모리스 RPG-HL-0136 균주가 실험용 마우스의 장 점막조직 내에서 면역인자을 촉진하여, 장관 면역을 증가시켰음을 확인하였다. Based on these results, it was confirmed that the strain Lactococcus lactis subspaces cremoris RPG-HL-0136 stimulated immune factors in the intestinal mucosal tissue of experimental mice and increased intestinal immunity.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the invention will be defined by the appended claims and their equivalents.
부호 없음unsigned
<110> PARK, byounghee PARK, jungkuk <120> Novel Lactococuus lactis subs. cremoris and use there of <130> rpg0136 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1456 <212> RNA <213> Lactococcus sp. <400> 1 ttgattcttg ctcaggacga acgctggcgg cgtgcctaat acatgcaagt tgagcgatga 60 agattggtgc ttgcaccaat ttgaagagca gcgaacgggt gagtaacgcg tggggaatct 120 gcctttgagc gggggacaac atttggaaac gaatgctaat accgcataac aactttaaac 180 ataagtttta agtttgaaag atgcaattgc atcactcaaa gatgatcccg cgttgtatta 240 gctagttggt gaggtaaagg ctcaccaagg cgatgataca tagccgacct gagagggtga 300 tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc 360 ttcggcaatg gacgaaagtc tgaccgagca acgccgcgtg agtgaagaag gttttcggat 420 cgtaaaactc tgttggtaga gaagaacgtt ggtgagagtg gaaagctcat caagtgacgg 480 taactaccca gaaagggacg gctaactacg tgccagcagc cgcggtaata cgtaggtccc 540 gagcgttgtc cggatttatt gggcgtaaag cgagcgcagg tggtttatta agtctggtgt 600 aaaaggcagt ggctcaacca ttgtatgcat tggaaactgg tagacttgag tgcaggagag 660 gagagtggaa ttccatgtgt agcggtgaaa tgcgtagata tatggaggaa caccggtggc 720 gaaagcggct ctctggcctg taactgacac tgaggctcga aagcgtgggg agcaaacagg 780 attagatacc ctggtagtcc acgccgtaaa cgatgagtgc tagatgtagg gagctataag 840 ttctctgtat cgcagctaac gcaataagca ctccgcctgg ggagtacgac cgcaaggttg 900 aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 960 caacgcgaag aaccttacca ggtcttgaca tactcgtgct attcctagag ataggaagtt 1020 ccttcgggac acgggataca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1080 gggttaagtc ccgcaacgag cgcaacccct attgttagtt gccatcatta agttgggcac 1140 tctaacgaga ctgccggtga taaaccggag gaaggtgggg atgacgtcaa atcatcatgc 1200 cccttatgac ctgggctaca cacgtgctac aatggatggt acaacgagtc gcgagacagt 1260 gatgtttagc taatctctta aaaccattct cagttcggat tgtaggctgc aactcgccta 1320 catgaagtcg gaatcgctag taatcgcgga taaacacgcc gcggtgaata cgttcccggg 1380 ccttgtacac accgcccgtc acaccacggg agttgggagt acccgaagta ggttgcctaa 1440 ccgcaaggag ggcgct 1456 <110> PARK, byounghee PARK, jungkuk <120> Novel Lactococcus lactis subs. cremoris and use there of <130> rpg0136 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1456 <212> RNA <213> Lactococcus sp. <400> 1 ttgattcttg ctcaggacga acgctggcgg cgtgcctaat acatgcaagt tgagcgatga 60 agattggtgc ttgcaccaat ttgaagagca gcgaacgggt gagtaacgcg tggggaatct 120 gcctttgagc gggggacaac atttggaaac gaatgctaat accgcataac aactttaaac 180 ataagtttta agtttgaaag atgcaattgc atcactcaaa gatgatcccg cgttgtatta 240 gctagttggt gaggtaaagg ctcaccaagg cgatgataca tagccgacct gagagggtga 300 tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc 360 ttcggcaatg gacgaaagtc tgaccgagca acgccgcgtg agtgaagaag gttttcggat 420 cgtaaaactc tgttggtaga gaagaacgtt ggtgagagtg gaaagctcat caagtgacgg 480 taactaccca gaaagggacg gctaactacg tgccagcagc cgcggtaata cgtaggtccc 540 gagcgttgtc cggatttatt gggcgtaaag cgagcgcagg tggtttatta agtctggtgt 600 aaaaggcagt ggctcaacca ttgtatgcat tggaaactgg tagacttgag tgcaggagag 660 gagagtgggaa ttccatgtgt agcggtgaaa tgcgtagata tatggaggaa caccggtggc 720 gaaagcggct ctctggcctg taactgacac tgaggctcga aagcgtgggg agcaaacagg 780 attagatacc ctggtagtcc acgccgtaaa cgatgagtgc tagatgtagg gagctataag 840 ttctctgtat cgcagctaac gcaataagca ctccgcctgg ggagtacgac cgcaaggttg 900 aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 960 caacgcgaag aaccttacca ggtcttgaca tactcgtgct attcctagag ataggaagtt 1020 ccttcgggac acgggataca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1080 gggttaagtc ccgcaacgag cgcaacccct attgttagtt gccatcatta agttgggcac 1140 tctaacgaga ctgccggtga taaaccggag gaaggtgggg atgacgtcaa atcatcatgc 1200 cccttatgac ctgggctaca cacgtgctac aatggatggt acaacgagtc gcgagacagt 1260 gatgtttagc taatctctta aaaccattct cagttcggat tgtaggctgc aactcgccta 1320 catgaagtcg gaatcgctag taatcgcgga taaacacgcc gcggtgaata cgttcccggg 1380 ccttgtacac accgcccgtc acaccacggg agttgggagt acccgaagta ggttgcctaa 1440 ccgcaaggag ggcgct 1456
Claims (8)
Claims 1 to 3 of any one of the strain, its lysate and its culture, including any one or more feed additives and auxiliary feed composition for removing malodor.
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