KR102139024B1 - Lava seawater hydrophobic protein coating paraprobiotics manufacturing method and excellent lava seawater hydrophobic protein coating paraprobiotics or immune enhancing composition using the same method - Google Patents
Lava seawater hydrophobic protein coating paraprobiotics manufacturing method and excellent lava seawater hydrophobic protein coating paraprobiotics or immune enhancing composition using the same method Download PDFInfo
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Abstract
Description
본 발명은 천연 미네랄이 풍부한 용암해수를 사용하여 표면의 소수성(hydrophobicity)이 증가된 가열처리 유산균(파라프로바이오틱스, paraprobiotics, 사균)을 제조함으로써, 기존 가열처리 유산균에서 나타나는 소수성 감소 현상을 억제하여 장 점막면역세포의 부착성이 증가되고 이로 인한 면역활성이 증강된 용암해수 유래 소수성 파라프로바이오틱스의 제조방법에 관한 것이다. 본 발명은 또한 상기 방법으로 제조된 용암해수 유래 소수성 파라프로바이오틱스와 상기 파라프로바이오틱스를 함유하는 면역증강용 조성물에 관한 것이다. The present invention is prepared by using a natural mineral-rich lava seawater to increase the surface hydrophobicity (hydrophobicity) by producing a heat-treated lactic acid bacteria (paraprobiotics, paraprobiotics, dead bacteria), to suppress the hydrophobic reduction phenomenon that occurs in the existing heat-treated lactic acid bacteria intestinal mucosa The present invention relates to a method for preparing hydrophobic paraprobiotics derived from lava seawater with increased adhesion of immune cells and enhanced immune activity. The present invention also relates to a hydrophobic paraprobiotic derived from lava seawater prepared by the above method and a composition for enhancing immunity containing the paraprobiotic.
프로바이오틱스(Probiotics)는 라틴어로 ‘생명을 위한(for life)’의 뜻으로 주로 생균(live cells)을 의미하며, 적당량을 섭취하였을 때 숙주의 건강에 도움을 주는 미생물을 일컫는다. 대표적으로 락토바실러스 속(Lactobacillus sp.), 비피도박테리움 속(Bifidobacterium sp.), 스트렙토코커스 속(Streptococcus sp.), 바실러스 속(Bacillus sp.), 사카로마이세스 속(Saccharomyces sp.)의 미생물들이 있다. 프로바이오틱스 효능 가이드라인으로는 인간 유래의 균주이어야 하며, 인체에 안전해야 하며, 내산성 내담즙산성이 있어야 하고 점막표면과 친화적인 부착능력을 비롯한 바람직한 기능성을 가지고 있어야 한다. 이렇듯, 프로바이오틱스의 대표적인 균주군인 유산균이 장내에서 면역학적 기능을 발휘하기 위해서는 장 점막에 존재하는 면역세포들과의 상호작용이 중요하며 이를 위해서는 일차적으로 장 점막에 잘 부착이 되어야 한다. 장 점막은 일반적으로 소수성(hydrophobicity)를 나타내며 최외곽에 외부 항원이나 미생물에 의한 방어를 담당하는 점액층(mucus layer)으로 구성되어 있으며, 이 점액층은 장내세균총이 주로 상재하는 외부 점액층과 극소수의 혐기성 세균이 서식하는 내부 점액층으로 구성된다. 장 점막을 구성하는 장상피세포(intestinal epithelial cell, IEC)와 수지상세포(dendritic cell, DC)는 면역세포로서 선천면역을 유도할 수 있는 pathogen-associated molecular pattern (PAMP)이 세포표면에 발현되어 있어 외부 항원이나 미생물 세포벽 성분들을 인지하여 면역반응을 유도하게 된다. 유산균은 그람양성 세균으로 세포막의 바깥에 세포벽이 존재하며 세포벽은 펩티도글리칸, 지질단백질, 테이코익산의 복합적인 층구조를 이루고 있다. 이 세포벽 구성성분들은 인간의 장내 점막에서 발현되는 특정 패턴인식수용체(parrern recongnition receptor, PRR)에 의해 인식되는 microbe associated pattern (MAMP)과 결합하여 면역반응을 일으킨다. 장상피세포, 수지상세포, 대식세포등의 면역세포들은 외부항원 감지를 톨 유사 수용체(toll-like receptors, TLRs)에 의해 매개되며 그 종류에 따라 Th1, Th2, Th17 cell을 활성화시킨다. 톨 유사 수용체는 항원의 소수성 리간드(hydrophobic ligand)가 결합하게 되면 선천적 면역반응을 유도하게 되고 이를 인식한 장상피세포, 수지상세포, 대식세포들은 면역을 활성화시키는 사이토카인(cytokines)이나 인터페론(interferon)등을 분비하게 된다. Probiotics (Probiotics) in Latin means'for life', mainly means live cells, and refers to microorganisms that help the host's health when consumed in an appropriate amount. Typically, Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Bacillus sp., Saccharomyces sp. There are microorganisms. As a guideline for probiotic efficacy, it should be a human-derived strain, be safe for the human body, have acid-resistant bile acid resistance, and have desirable functions, including mucosal surface and friendly adhesion. As such, interaction with immune cells present in the intestinal mucosa is important for probiotic lactic acid bacteria, a representative strain of probiotics, to exert immunological functions in the intestine, and for this purpose, it must first be well attached to the intestinal mucosa. The intestinal mucosa generally exhibits hydrophobicity and consists of a mucus layer responsible for defense by external antigens or microorganisms on the outermost layer, and this mucous layer is composed of an external mucus layer and a very small number of anaerobic bacteria. This habitat consists of an inner mucus layer. Intestinal epithelial cells (IEC) and dendritic cells (DC) constituting the intestinal mucosa are immune cells, and a pathogen-associated molecular pattern (PAMP) capable of inducing innate immunity is expressed on the cell surface. It recognizes foreign antigens or microbial cell wall components and induces an immune response. Lactobacillus is a gram-positive bacterium with a cell wall on the outside of the cell membrane, and the cell wall has a complex layered structure of peptidoglycan, lipoprotein, and teicoic acid. These cell wall components combine with the microbe associated pattern (MAMP) recognized by a specific pattern recognition receptor (PRR) expressed in the intestinal mucosa of humans to produce an immune response. Immune cells, such as intestinal epithelial cells, dendritic cells, and macrophages, are mediated by toll-like receptors (TLLs) to detect external antigens and activate Th1, Th2, and Th17 cells according to their type. Toll-like receptors induce an innate immune response when a hydrophobic ligand of an antigen is bound. Recognized intestinal epithelial cells, dendritic cells and macrophages activate cytokines or interferons that activate immunity. It secretes the back.
유산균의 제형적 한 예로 생균제제는 특성상 위장관에서의 생존율, 장내환경에서의 정착률, 기존의 장내 상재균총과 장점막 부위를 차지하기 위한 부착경쟁(competitive adhesion)으로 인해 투여 농도 대비 면역학적 개선 효과는 미미하다는 한계점이 있다. 이러한 유산균 생균제의 면역활성에 대한 약물동력학적 한계점을 파라프로바이오틱스(paraprobiotics, 사균)의 신제형으로 극복하려는 노력이 있었다. 2002년 WHO/FAO에서 개념적 정의를 통해 내려진 프로바이오틱스를 실질적인 연구를 통해 반드시 살아있는 균이 아니더라도 프로바이오틱스의 특징을 나타낼 수 있다는 연구결과들이 체계화되면서 프로바이오틱스에 대응되는 파라프로바이오틱스라는 개념이 정립되었다. 면역학적 효능을 높이기 위해서 세포표면의 소수성을 증가시켜야하는데 종래의 기술에서는 세포벽이 파괴되면서 생균 표면의 자가응집력 소수성 구조가 깨지면서 마이쎌(micelle)구조와 같은 세포응집 현상이 나타나며, 응집된 응집체 내부는 소수성이 강해지고 외부는 친수성이 강해져 섭취시 소수성 특성의 장 점막에 부착이 저해된다(대한민국 등록특허 제10-1425712). 따라서 이러한 파라프로바이오틱스의 인체 내 면역활성을 높이기 위해서는 생균의 사균화 제조과정의 문제점을 해결하는 것이 중요한 것이라 여겨지고 있다. 이러한 문제점을 단순 해결하기 위해 산업적으로는 1조단위 이상의 사균을 제조하는 방식으로 장 점막과의 부착기회를 높이려는 시도가 있었으나, 1조개의 사균체를 제조하기 위해서는 가공비의 증가 대비 효능이 미비하여 생균체를 제조 및 섭취하는 것에 비해 경제성이 떨어지는 방법으로 지적되어 왔다. As an example of the formulation of lactic acid bacteria, the probiotic is insignificant due to its characteristics such as survival rate in the gastrointestinal tract, settling rate in the intestinal environment, and competive adhesion to occupy the existing intestinal flora and mesenteric membrane area. There are limitations. There has been an effort to overcome the pharmacokinetic limitations of the immune activity of these lactic acid bacteria probiotics with new formulations of paraprobiotics. In 2002, WHO/FAO established the concept of paraprobiotics, which corresponds to probiotics, as research results that probiotics, which were created through conceptual definition, can represent the characteristics of probiotics even though they are not necessarily living bacteria, have been systematized. In order to increase the immunological efficacy, the hydrophobicity of the cell surface must be increased. In the conventional technique, the cell wall is destroyed and the self-aggregating hydrophobic structure of the surface of the live cell is broken, resulting in cell aggregation phenomenon such as a micelle structure. The hydrophobicity becomes stronger and the hydrophilicity becomes stronger on the outside, which inhibits adhesion to the intestinal mucosa of hydrophobic nature when ingested (Republic of Korea Patent No. 10-1425712). Therefore, in order to increase the immune activity in the human body of such paraprobiotics, it is considered that it is important to solve the problem of the production process of the sterilization of live bacteria. In order to solve this problem simply, industrially, attempts were made to increase the chance of adhesion to the intestinal mucosa by producing more than 1 trillion units of fungi, but to produce 1 trillion fungi, the efficacy compared to the increase in processing cost was insufficient. It has been pointed out as a less economical method than the production and consumption of living cells.
이에 본 발명에서는 천연 미네랄이 풍부한 용암해수를 배양수로 사용하여 유산균을 고농도로 배양하고 용암해수가 포함된 배지의 멸균과정에서 생성되는 소수성 응집단백질이 유산균 생균체에 코팅되어 이를 사균화 시의 가열처리로 인해 발생되는 세포표면의 소수성 감소 증상을 억제하는 것을 확인하였다. 이렇게 증가된 세포표면의 소수성을 통해 파라프로바이오틱스의 자가응집현상을 방해하여 장내 부착성을 활성화하면서 면역활성이 우수한 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스를 제조하게 되었다. Accordingly, in the present invention, lactic acid bacteria are cultured at a high concentration using lava seawater rich in natural minerals as culture water, and a hydrophobic aggregated protein produced during sterilization of the medium containing lava seawater is coated on the lactic acid bacteria living cells and heated during fertilization. It was confirmed that the symptoms of the decrease in the hydrophobicity of the cell surface caused by the treatment were suppressed. This increased hydrophobicity of the cell surface prevents the self-aggregation of paraprobiotics, thereby activating adhesion in the intestine, while producing hydrophobic protein-coated paraprobiotics derived from lava seawater having excellent immune activity.
본 발명이 목적은 천연 미네랄이 풍부한 용암해수를 사용하여 표면의 소수성(hydrophobicity)이 증가된 가열처리 유산균(파라프로바이오틱스, paraprobiotics) 을 제조함으로써, 기존의 소수성이 감소된 가열처리 유산균과 달리 상기 소수성으로 인해 장 점막면역세포의 부착성이 증가되고 이로 인한 면역활성이 증강된 용암해수 유래 소수성 파라프로바이오틱스의 제조방법, 상기 방법으로 제조된 용암해수 유래 소수성 파라프로바이오틱스와 상기 파라프로바이오틱스를 함유하는 면역증강용 조성물을 제공하는 데에 있다. The object of the present invention is to produce a heat-treated lactic acid bacteria (paraprobiotics, paraprobiotics) with increased hydrophobicity of the surface by using lava seawater rich in natural minerals, unlike the existing heat-treated lactic acid bacteria with reduced hydrophobicity. Due to the increased adhesion of the intestinal mucosal immune cells and the resulting increased immune activity, the method for preparing hydrophobic paraprobiotics derived from lava seawater, the composition for immunopotentiation containing hydrophobic paraprobiotics derived from lava seawater prepared by the above method and the paraprobiotics In providing.
본 발명은 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법에 관한 것이며, 더 자세하게는 용암해수가 포함된 물을 배양용수로 하는 배지 제조 중에 형성된 침전단백질을 가열처리 유산균에 코팅함으로써 소수성이 부가된 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법에 관한 것이다. 상기 제조방법은 하기와 같다. The present invention relates to a method for preparing a hydrophobic protein-coated paraprobiotics derived from lava seawater, and more specifically, lava seawater to which hydrophobicity is added by coating a precipitated protein formed during production of a medium containing water containing lava seawater as culture water to a heat-treated lactic acid bacteria. It relates to a method for producing a derived hydrophobic protein-coated paraprobiotics. The manufacturing method is as follows.
(제1단계) 용암해수가 30~70%(v/v) 포함된 물을 배양용수로 이용하여 소수성 단백질이 형성된 유산균 배양용 배지를 준비하는 단계; (First step) preparing a culture medium for lactic acid bacteria in which hydrophobic protein is formed by using water containing 30 to 70% (v/v) of lava seawater as culture water;
(제2단계) 상기 유산균 배양용 배지에 유산균을 접종하고 배양하여 배양액을 얻고 가열처리하는 단계; 및,(Second step) Inoculating and culturing the lactic acid bacteria in the culture medium for lactic acid bacteria to obtain a culture solution and heat treatment; And,
(제3단계) 가열처리된 배양액을 농축하여 배지성분을 제거하면서 파라프로바이오틱스 외부에 용암해수 유래 소수성 단백질을 코팅하고, 농축이 완료된 파라프로바이오틱스 농축물을 건조하여 분말을 수득하는 단계; 를 포함할 수 있다. (Step 3) Concentrating the heat-treated culture medium to remove the medium component, coating the hydrophobic protein derived from lava seawater outside the paraprobiotics, and drying the concentrated paraprobiotics concentrate to obtain a powder; It may include.
상기 제1단계의 배지는 바람직하게는, 유산균의 배양이 가능한 모든 배지일 수 있으나, 더 바람직하게는, 구성 성분으로, 포도당, 효모추출물, 소이펩톤 및 카제인이 포함되고, 상기 구성 성분을 용암해수가 30~70%(v/v) 포함된 물에 용해하고 멸균하여 준비된 유산균 배양용 배지일 수 있다. The medium of the first step may be any medium capable of culturing lactic acid bacteria, but more preferably, as a component, glucose, yeast extract, soypeptone, and casein are included, and the component is lava seawater. It may be a medium for lactic acid bacteria culture prepared by dissolving and sterilizing 30-70% (v/v) contained water.
보다 더 바람직하게는 상기 제1단계의 배지는 총 부피 1ℓ 기준, 포도당 1~5%(w/v), 효모추출물 0.5~5%(w/v), 소이펩톤 0.5~5%(w/v) 및 카제인 0.5~3%(w/v)가 되도록 각 성분을 용암해수가 30~70%(v/v) 포함된 물에 용해하고 멸균하여 준비된 유산균 배양용 배지일 수 있다. Even more preferably, the medium of the first step is based on a total volume of 1 ℓ, glucose 1-5% (w/v), yeast extract 0.5-5% (w/v), soypeptone 0.5-5% (w/v) ) And casein 0.5 to 3% (w/v), each component may be a medium for lactic acid bacteria culture prepared by dissolving and sterilizing lava seawater in water containing 30 to 70% (v/v).
상기 제1단계의 배지 멸균은 95~123℃, 0.12~0.30MPa에서 30~200분간 수행하는 것이 좋다. The sterilization of the medium in the first step is preferably performed at 95 to 123°C and 0.12 to 0.30 MPa for 30 to 200 minutes.
상기 제2단계의 가열처리는 95~123℃, 0.12~0.30MPa에서 30~200분간 수행할 수 있다. The heat treatment in the second step may be performed at 95 to 123°C and 0.12 to 0.30 MPa for 30 to 200 minutes.
상기 제2단계의 유산균은 락토바실러스 속 (Lactobacillus sp.), 비피도박테리움 속 (Bifidobacterium sp.), 스트렙토코커스 속 (Streptococcus sp.), 락토코커스 속 (Lactococcus sp.), 엔테로코커스 속 (Enterococcus sp.), 페디오코커스 속 (Pediococcus sp.), 바이셀라 속 (Weissella sp.), 사카로마이세스 속 (Saccharomyces sp.), 바실러스 속 (Bacillus sp.)으로 이루어진 군 중에서 1종 이상 선택될 수 있다. Lactobacillus sp. ( Lactobacillus sp.), Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp.), Pediococcus sp., Weissella sp., Saccharomyces sp., Bacillus sp. Can.
상기 제2단계의 유산균 배양조건은 70~150rpm, 30~37℃ 조건으로 18~24시간인 것이 바람직하다. 상기 제2단계의 유산균 배양은 유가식배양(fed-batch culture) 방법으로 수행되는 것이 특징이다. 이를 위해, 10 내지 50%(w/v)의 포도당 수용액 및 10 내지 50%(w/v)의 수산화나트륨 수용액을 혼합한 용해액을 적하하여 배양 중 pH를 6.0~7.5로 유지하는 방법을 이용할 수 있다. 이 혼합 용해액은 각각 포도당 용액과 수산화나트륨 용액을 1:0.5 내지 1:2의 부피비로 혼합한 것일 수 있다. The culture conditions of the lactic acid bacteria in the second step are preferably 18 to 24 hours under conditions of 70 to 150 rpm and 30 to 37°C. The lactic acid bacteria culture in the second step is characterized in that it is performed by fed-batch culture method. To this end, a solution of 10-50% (w/v) glucose solution and 10-50% (w/v) sodium hydroxide aqueous solution is added dropwise to maintain a pH of 6.0 to 7.5 during culture. Can. The mixed solution may be a mixture of glucose solution and sodium hydroxide solution in a volume ratio of 1:0.5 to 1:2, respectively.
상기 제3단계에서 파라프로바이오틱스가 포함된 배양액을 농축함으로써 파라프로바이오틱스에 소수성을 갖는 염석 단백질이 코팅된다.In the third step, the hydrochloride protein having hydrophobicity is coated on the paraprobiotics by concentrating the culture solution containing the paraprobiotics.
즉, 상기 제3단계에서 농축하여 얻은 농축물은 제1단계의 배지준비 시에 용암해수의 첨가로 인해 침전된 소수성을 갖는 염석 단백질이 균체와 불균일하게 혼합된 상태인 것을(단순 혼합 과정), 이러한 농축 과정을 거쳐 소수성을 갖는 염석 단백질이 코팅된 파라프로바이오틱스를 얻는 것이다. In other words, the concentrate obtained by concentration in the third step is in a state in which the salting-out protein having hydrophobicity precipitated due to the addition of lava seawater during the preparation of the medium in the first step is in an unevenly mixed state with the cells (simple mixing process), Through this concentration process, it is to obtain paraprobiotics coated with hydrochloric acid protein having hydrophobicity.
상기 제3단계의 건조는 동결건조, 분무건조 또는 진공건조를 통해 수행가능하다. The drying in the third step can be performed through freeze drying, spray drying or vacuum drying.
본 발명은 상기 제조방법으로 제조된 용암해수 유래 천연 미네랄이 코팅된 파라프로바이오틱스를 제공한다. The present invention provides para-probiotics coated with natural minerals derived from lava seawater prepared by the above manufacturing method.
상기 파라프로바이오틱스 내에는 칼슘 0.2~2.0mg/g, 마그네슘 0.5~2.0mg/g, 셀레늄 0.000001~0.00001mg/g이 대략 포함되는 것을 확인하였다.It was confirmed that the paraprobiotics contained approximately 0.2 to 2.0 mg/g of calcium, 0.5 to 2.0 mg/g of magnesium, and 0.000001 to 0.00001 mg/g of selenium.
상기 파라프로바이오틱스는 뮤신에 대한 부착성이 있는 것을 특징으로 한다. The paraprobiotics are characterized by having adhesion to mucin.
상기 파라프로바이오틱스는 뮤신에 부착되어 TNF-α(tumor necrosis factor-α), IL-6(interleukin-6), IL-1β(interleukin-1β), NOS(nitric oxide synthase) 및 COX-2(cyclooxygenase-2)로 이루어진 군 중에서 1종 이상 선택되는 면역 개시 인자 또는 면역 활성화 효소의 유전자 발현 또는 단백질 발현을 증가시키는 것을 특징으로 한다. The paraprobiotics are attached to mucin, TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), IL-1β (interleukin-1β), NOS (nitric oxide synthase) and COX-2 (cyclooxygenase- 2) is characterized by increasing the gene expression or protein expression of one or more immune initiation factors or immune activating enzymes selected from the group consisting of.
또 다른 양태에서 본 발명은 상기 파라프로바이오틱스를 함유하는 면역 증강용 건강기능식품을 제공한다. In another aspect, the present invention provides a health functional food for enhancing immunity containing the paraprobiotics.
본 발명은 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법 및 상기 방법으로 제조한 소수성 단백질 코팅 파라프로바이오틱스를 함유하는 면역증강용 조성물에 관한 것이다. 본 발명은 용암해수를 이용하여 제조한 배지 내에 생성된 염석이 소수성을 갖는 것을 확인하여, 이 소수성 단백질을 가열처리된 균체에 코팅하고, 상기 가열처리된 균체인 파라프로바이오틱스가 뮤신 부착력이 증강되어 면역증강 기능이 있음을 제시한다. 이러한 방법을 통해 생균제 배양시 균체를 고농도로 배양할 수 있어 대량의 파라프로바이오틱스를 획득할 수 있으며, 일반 생균제는 세포 표면의 소수성이 제조과정에서 약화되어 장내세포의 점액질을 구성하는 뮤신에 결합할 수 있는 소수성이 저하됨으로 인해 장내 부착성이 감소되는 단점을 개선하여 장내 부착성이 향상되어 면역 기능성이 우수한 파라프로바이오틱스를 함유한 건강기능식품을 대량 제공할 수 있다. The present invention relates to a method for preparing hydrophobic protein-coated paraprobiotics derived from lava seawater and a composition for enhancing immunity containing hydrophobic protein-coated paraprobiotics prepared by the above method. The present invention confirms that the salt salt generated in the medium prepared by using lava seawater has hydrophobicity, and coats the hydrophobic protein with the heated cells, and the paraprobiotics, the heated cells, have enhanced mucin adhesion and immunity. It suggests that there is an augmentation function. In this way, when the probiotic is cultured, the cells can be cultured at a high concentration to obtain a large amount of paraprobiotics, and the general probiotic can bind to mucin, which makes up the mucus of intestinal cells because the hydrophobicity of the cell surface is weakened during the manufacturing process. By improving the disadvantage that the intestinal adhesion is reduced due to the decreased hydrophobicity, the intestinal adhesion is improved, and thus a large number of health functional foods containing paraprobiotics having excellent immune function can be provided.
도 1은 배양용수로서 사용된 용암해수 농도에 따라 멸균 배지 내에 생성된 염석 단백질양 내의 단백질 함량을 비교한 결과이다.
도 2는 용암해수 농도별로 제조된 멸균 배지에서 제조된 파라프로바이오틱스의 개수를 비교한 결과이다.
도 3은 상수와 용암해수를 배양용수로서 사용하여 제조된 파라프로바이오틱스의 TNF-a 면역활성 비교 결과이다.
도 4는 상수와 용암해수를 배양용수로서 사용하여 제조된 파라프로바이오틱스의 IL-6 면역활성 비교 결과이다.
도 5는 상수와 용암해수를 배양용수로서 사용하여 제조된 파라프로바이오틱스의 IL-1β 면역활성 비교 결과이다.
도 6은 상수와 용암해수를 배양용수로서 사용하여 제조된 파라프로바이오틱스의 iNOS 면역활성 비교 결과이다.
도 7은 상수와 용암해수를 배양용수로서 사용하여 제조된 파라프로바이오틱스의 COX-2 면역활성 비교 결과이다.
도 8은 상수와 용암해수를 배양용수로서 사용하여 제조된 파라프로바이오틱스의 TNF-a 면역활성 비교 결과이다.
도 9는 상수와 용암해수를 배양용수로서 사용하여 제조된 파라프로바이오틱스의 IL-6 면역활성 비교 결과이다.
도 10은 상수와 용암해수를 배양용수로서 사용하여 제조된 파라프로바이오틱스의 전자현미경사진이다.
도 11은 다양한 균주로부터 제조한 파라프로바이오틱스가 소수성을 갖는 것을 보여주는 결과이다.
도 12는 다양한 균주로부터 제조한 파라프로바이오틱스가 뮤신 부착성을 갖는 것을 보여주는 결과이다. Figure 1 is a result of comparing the protein content in the amount of salt salt protein produced in a sterile medium according to the concentration of lava seawater used as culture water.
Figure 2 is a result of comparing the number of paraprobiotics prepared in a sterile medium prepared for each lava seawater concentration.
3 is a comparison result of the TNF-a immune activity of paraprobiotics prepared using water and lava seawater as culture water.
4 is a comparison result of IL-6 immunoactivity of paraprobiotics prepared using water and lava seawater as culture water.
5 is a comparison result of IL-1β immune activity of paraprobiotics prepared using water and lava seawater as culture water.
6 is a comparison result of iNOS immune activity of paraprobiotics prepared using water and lava seawater as culture water.
7 is a comparison result of COX-2 immune activity of paraprobiotics prepared using water and lava seawater as culture water.
8 is a comparison result of TNF-a immune activity of paraprobiotics prepared using water and lava seawater as culture water.
9 is a comparison result of IL-6 immunoactivity of paraprobiotics prepared using water and lava seawater as culture water.
10 is an electron micrograph of para-probiotic prepared using water and lava seawater as culture water.
11 is a result showing that the paraprobiotics prepared from various strains have hydrophobicity.
12 is a result showing that paraprobiotics prepared from various strains have mucin adhesion.
상기 용암해수 배양과정 중에 형성된 소수성 침전단백질을 이용하여 파라프로바이오틱스에 코팅함으로써 부가된 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법은 다음 단계들을 포함한다.The method for preparing a hydrophobic protein-coated paraprobiotic derived from lava seawater by coating the paraprobiotics using a hydrophobic precipitation protein formed during the lava seawater culture process includes the following steps.
(a) 염석을 통한 소수성 단백질 생산과 유산균 증식이 우수한 용암해수 농도결정의 단계;(A) step of determining the concentration of lava seawater excellent in hydrophobic protein production and lactic acid bacteria proliferation through salting;
(b) 상기 (a)단계에서 결정된 농도의 용암해수를 이용한 유산균의 배양단계;(b) culturing the lactic acid bacteria using lava seawater at the concentration determined in the step (a);
(c) 상기 (b)단계에서 배양된 유산균을 가열처리하고 균체만을 농축시키는 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법을 제공한다.(c) A method for producing a hydrophobic protein-coated paraprobiotics derived from lava seawater, which heat-treats the lactic acid bacteria cultured in the step (b) and concentrates only the cells.
또한 본 발명은 상기의 제조방법으로 제조된 면역활성이 우수한 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스 원료를 제공한다. In addition, the present invention provides a hydrophobic protein-coated paraprobiotic raw material derived from lava seawater having excellent immune activity prepared by the above-described manufacturing method.
이하, 본 발명의 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법을 상세히 설명한다. Hereinafter, a method of manufacturing a hydrophobic protein-coated paraprobiotic derived from lava seawater of the present invention will be described in detail.
(a) 염석을 통한 소수성 단백질 생산과 유산균 증식이 우수한 용암해수 농도결정의 단계;(A) step of determining the concentration of lava seawater excellent in hydrophobic protein production and lactic acid bacteria proliferation through salting;
배양배지를 조제하는 과정에서 용암해수를 배양수로 사용하여 코팅에 필요한 소수성 단백질을 제공하고 상층액부분에서 고농도 유산균 배양을 할 수 있는 최적용암해수 농도를 결정하였다. 단백질과 같은 고분자 전해질 성분은 일반 상수에서는 물에 대한 용해도가 증가하는 반면, 용암해수와 같이 염농도가 일정수준으로 높은 물에서는 용해도가 감소하여 소수성 단백질 상태로 침전이 된다. 이는 염농도 증가 시, 외부 이온의 농도가 증가하게 되면 단백질 표면의 소수성 부분에 분포하고 있는 물분자를 이온들이 끌고 가게 되면서 단백질 표면의 소수성 부분이 겉으로 노출되며 단백질 간의 소수성 결합에 의해 단백질이 응집되게 되는 염석 현상이 발생되기 때문이다. 특히 이 소수성 단백질 형성은 배지 조성 간에 생산되기 때문에 무균상태 배지를 만들기 위한 121℃ 열구간에서 소수성 단백질을 생산하게 되므로 소수성 단백질의 생성 농도구간을 설정하였다. In the process of preparing the culture medium, the lava seawater was used as the culture water to provide the hydrophobic protein necessary for coating, and the optimal lava seawater concentration capable of culturing high concentration lactic acid bacteria in the supernatant was determined. Polymer electrolyte components, such as proteins, have increased solubility in water at normal constants, while the solubility decreases in water with a high salt concentration, such as lava seawater, and precipitates as a hydrophobic protein. When the salt concentration increases, when the concentration of external ions increases, the water molecules distributed in the hydrophobic portion of the protein surface attract ions, and the hydrophobic portion of the protein surface is exposed to the outside, and the protein aggregates due to hydrophobic binding between proteins. This is because the salting out phenomenon occurs. In particular, since the formation of the hydrophobic protein is produced between the medium composition, the hydrophobic protein is produced in the heat section at 121° C. to make a sterile medium, and thus the concentration concentration section of the hydrophobic protein is set.
(b) 상기 (a)단계에서 결정된 농도의 용암해수를 이용하여 유산균을 고농도로 배양하는 단계; 일반적으로 유산균을 포함한 미생물들은 염농도가 높은 100%의 해수조건에서 생장이 어렵지만 유산균의 생장에 영향이 적은 최적의 용암해수 배합 농도 탐색을 통해 적절한 미네랄을 제공함으로써 유산균의 고농도 배양이 가능하다, (b) culturing the lactic acid bacteria at a high concentration using the lava seawater at the concentration determined in the step (a); In general, microorganisms including lactic acid bacteria are difficult to grow under 100% high salt concentration, but it is possible to cultivate high concentrations of lactic acid bacteria by providing appropriate minerals through exploration of the optimal concentration of lava seawater with little effect on the growth of lactic acid bacteria.
(c) (b)단계에서 유산균을 가열처리하고 균체만을 농축시키는 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법;(c) a method for preparing a hydrophobic protein-coated paraprobiotics derived from lava seawater that heat-treats lactic acid bacteria in step (b) and concentrates only the cells;
가열처리 유산균으로부터 균체만을 농축하기 위해 배지성분을 막여과 농축기로 제거하면서 생산된 소수성 단백질을 파라프로바이오틱스에 코팅하는 농축단계로서, 최종적으로 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스를 생산할 수 있다.In order to concentrate only the cells from the heat-treated lactic acid bacteria, it is a concentration step of coating the produced hydrophobic protein on the paraprobiotics while removing the medium component with a membrane filtration concentrator, and finally, the hydrophobic protein-coated paraprobiotics derived from lava seawater can be produced.
보다 더 자세하게, 본 발명은 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법 및 이를 이용한 면역활성이 우수한 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스에 관한 것이다. In more detail, the present invention relates to a method for preparing a hydrophobic protein-coated paraprobiotic derived from lava seawater and a hydrophobic protein-coated paraprobiotic derived from lava seawater having excellent immunological activity using the same.
본 발명을 통해 용암해수를 유산균 배양수로 이용하여 유산균 무균배지 조제 시 가압멸균과정으로 소수성 단백질을 침전 석출시키고 유산균을 고농도로 배양할 수 있는데, 가열처리를 통해 배양된 균체를 농축하면서 배지의 멸균 과정에서 생성된 소수성 염석 단백질이 파라프로바이오틱스에 코팅되게 하여 자가응집반응을 통해 만들어지는 마이쎌(micelle) 구조형성을 방지하여 최종적으로 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스 제조방법인 것이다. When preparing lactic acid bacteria aseptic medium using lava seawater as lactic acid bacteria culture water through the present invention, hydrophobic proteins can be precipitated and precipitated by autoclaving and lactic acid bacteria can be cultured at a high concentration. Sterilization of the medium while concentrating the cultured cells through heat treatment It is a method for preparing a hydrophobic protein-coated paraprobiotics derived from lava seawater by preventing the formation of a micelle structure formed through an auto-aggregation reaction by coating the hydrophobic proteolytic protein produced in the process to a paraprobiotic.
소수성이 증가된 파라프로바이오틱스를 통해 장 점막 세포인 소수성 특징을 갖는 뮤신과의 반응성을 높임으로써 궁극적으로는 종래의 기술이 친수성 파라프로바이오틱스의 장 점막 부착 저하로 면역반응을 극대화시키지 못한 부분을 본 발명의 제조방법을 통해 극복함으로써 면역활성이 우수한 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스를 최초로 제조한 것이다. 이를 통해 제조된 대량생산된 고농도의 파라프로바이오틱스를 사용하여 면역활성효과를 갖는 식품 및 건강기능식품, 의약품의 원료를 만드는 종래의 기술적 한계를 극복할 수 있어 산업적으로 경제적 응용가치가 높다. By increasing the reactivity with mucins having hydrophobic characteristics, which are intestinal mucosal cells through paraprobiotics with increased hydrophobicity, ultimately, the prior art has failed to maximize the immune response due to a decrease in the adhesion of the intestinal mucosa of the hydrophilic paraprobiotics. By overcoming through the manufacturing method, it is the first to produce a hydrophobic protein-coated paraprobiotic derived from lava seawater with excellent immune activity. By using the mass-produced high-concentration para-probiotics produced through this, it is possible to overcome the conventional technical limitations of making foods, health functional foods, and pharmaceutical raw materials having an immune-activating effect, and thus has a high economical industrial value.
본 발명에서 파라프로바이오틱스 제조용으로 사용가능한 유산균으로는 락토바실러스 속 (Lactobacillus sp.), 비피도박테리움 속 (Bifidobacterium sp.), 스트렙토코커스 속 (Streptococcus sp.), 락토코커스 속 (Lactococcus sp.), 엔테로코커스 속 (Enterococcus sp.), 페디오코커스 속 (Pediococcus sp.), 바이셀라 속 (Weissella sp.), 바실러스 속 (Bacillus sp.)으로 이루어진 군에서 하나 이상의 균이며, 바람직하게는 락토바실러스 아시도필루스(Lactobacillus acidophilus), 락토바실러스 불가리쿠스 (Lactobacillus bulgaricus), 락토바실러스 카세이(Lactobacillus casei), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 가쎄리(Lactobacillus gasseri), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락토바실러스 람노서스(Lactobacillus rhamnosus), 락토바실러스 존소니이(Lactobacillus johnsonii), 락토바실러스 프란타룸(Lactobacillus plantarum), 락토바실러스 루테리(Lactobacillus reuteri), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 인판티스(Bifidobacterium infantis), 비피도박테리움 락티스(Bifidobacterium lactis), 비피도박테리움 롱검(Bifidobacterium longum), 스트렙토코커스 페칼리스(Streptococcus faecalis), 스트렙토코커스 페시움(Streptococcus faecium), 락토코커스 락티스(Lactococcus lactis ssp. lactis), 엔테로코커스 페칼리스(Enterococcus faecalis), 엔테로코커스 페시움(Enterococcus faecium), 페디오코커스 아시도락티시이(Pediococcus acidolacticii), 페디오코커스 펜토사세우스 (Pediococcus pentosaceus), 류코노스톡 카르노숨(Leuconostoc carnosum), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 가시코미타튬(Leuconostoc gasicomitatum), 류코노스톡 겔리둠(Leuconostoc gellidum), 류코노스톡 인해(Leuconostoc inhae), 류코노스톡 김치이(Leuconostoc kimchii), 류코노스톡 락티스(Leuconostoc lactis), 류코노스톡 메센테로이데스 (Leuconostoc mesenteroides subsp. mesenteroides), 류코노스톡 파라메센테로이데스 (Leuconostoc paramesenteroides), 바이쎌라 시바리아(Weissella cibaria), 바이쎌라 콘푸사(Weissella confusa), 바이쎌라 코리엔시스(Weissella koreensis), 바이쎌라 소리(Weissella soli), 바이쎌라 비리데센스(Weissella viridescens), 바실러스 코아귤란스(Bacillus coagulans), 등으로 이루어진 군에서 하나 이상이 선택될 수 있다. Lactobacillus genus ( Lactobacillus sp.), Bifidobacterium sp., Streptococcus sp., Lactococcus sp. Enterococcus sp., Pediococcus sp., Weissella sp., Bacillus sp. Bacillus sp., one or more fungi, preferably Lactobacillus asia dopil Ruth (Lactobacillus acidophilus), Lactobacillus Bulgaria kusu (Lactobacillus bulgaricus), Lactobacillus Kasei (Lactobacillus casei), Lactobacillus momentum spread (Lactobacillus fermentum), Lactobacillus is Serena (Lactobacillus gasseri), Lactobacillus helveticus (Lactobacillus helveticus ), Lactobacillus rhamnosus , Lactobacillus johnsonii , Lactobacillus plantarum , Lactobacillus plantarum , Lactobacillus reuteri , Bifidobacterium bifidum gambling Te Solarium breather bracket (Bifidobacterium breve), Bifidobacterium Infante Tees (Bifidobacterium infantis), Bifidobacterium lactis (Bifidobacterium lactis), Bifidobacterium ronggeom (Bifidobacterium longum), Streptococcus faecalis (Streptococcus faecalis) , Streptococcus faecium , Lactococcus lactis ssp. lactis ), Enterococcus faecalis , Enterococcus faecium , Pediococcus acidolacticii , Pediococcus pentosaceus , Leuconostock carnosum ( Leuconostoc carnosum ), Leuconostoc citreum , Leuconostoc gasicomitatum , Leuconostoc gellidum , Leuconostoc inhae , Leuconostoc inhae , Kimchi ( Leuconostoc inhae ) Leuconostoc kimchii), current Kono Stock lactis (Leuconostoc lactis), current Kono Stock mesen teroyi death (Leuconostoc mesenteroides subsp. mesenteroides), current Kono Stock Farah mesen teroyi death (Leuconostoc paramesenteroides), by cibaria (Weissella cibaria), by One from the group consisting of Weissella confusa , Weissella koreensis , Weissella soli , Weissella viridescens , Bacillus coagulans , etc. The above may be selected.
또한, 본 발명은 소수성 파라프로바이오틱스 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 면역증강용 건강기능식품을 제공한다. 상기 건강기능식품으로는, 예를 들어, 각종 드링크제, 육류, 소세지, 빵, 캔디류, 스넥류, 면류, 아이스크림, 유제품, 스프, 이온음료, 음료수, 알코올 음료, 껌, 차 및 비타민 복합제 등이 있다. In addition, the present invention provides a health functional food for enhancing immunity, including hydrophobic para-probiotics and food-acceptable food supplement additives. Examples of the health functional food include, for example, various drinks, meat, sausage, bread, candy, snacks, noodles, ice cream, dairy products, soups, beverages, drinks, alcoholic beverages, chewing gum, tea, and vitamin complexes.
이하, 비교예와 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 비교예와 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들에 의해 제한되는 것으로 해석되지는 않는다. Hereinafter, the present invention will be described in more detail through comparative examples and examples. Comparative Examples and Examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by them.
비교예 1. 유산균 기본배지 제조 및 배양방법Comparative Example 1. Preparation and culture method of lactic acid bacteria basic medium
유산균을 배양하기 위한 기본배지로 포도당 3%(w/v), 효모추출물 2%(w/v), 소이펩톤 2%(w/v) 및 카제인 1%(w/v)에 물을 첨가한 부피가 총 100%(v)가 되도록 발효조에 가하여 용해 후 0.15Mpa, 121℃에서 30분간 가압멸균하였다. 이에 멸균된 Lactobacilli MRS Broth(BD, USA)에서 37℃, 24시간 배양된 유산균 종배양액을 발효조의 멸균배지에 접종하고 100rpm, 37℃ 조건으로 20시간 동안 40% (v/v)의 포도당-수산화나트륨 용해액(40%(w/v) 포도당 및 40%(w/v) 수산화나트륨이 1:1의 부피비로 혼합된 수용액)을 사용하여 pH를 6.0~7.5로 보정하며 fed-batch 배양하였다. * 유산균 종배양액 : 락토바실러스 람노서스(L. rhamnosus) 균주를 Lactobacilli MRS Broth(BD)에서 37℃ 조건에서 24시간 배양한 것, 이하 유산균 종배양액이라 함은 이를 말함. * 또한 가장 대표적인 실험은 상기 락토바실러스 람노서스(Lactobacillus rhamnosus ; L. rhamnosus) 균주를 이용하였으나 각 균주별 실험을 비교하기 위해 동일한 방법으로 엔테로코커스 페칼리스(Enterococcus faecalis ; E. faecalis) 와 비피도박테리움 브레브(Bifidobacterium breve ; B. breve)를 사용하기도 하였다. As a basic medium for culturing lactic acid bacteria, water was added to 3% (w/v) of glucose, 2% of yeast extract (w/v), 2% of soypeptone (w/v), and 1% of casein (w/v). It was added to the fermenter so that the volume was 100% (v) in total, and dissolved and then autoclaved at 0.15 Mpa and 121°C for 30 minutes. To this, the lactic acid bacteria seed culture solution cultured at 37° C. for 24 hours in sterilized Lactobacilli MRS Broth (BD, USA) was inoculated into a sterile medium of a fermenter, and 40% (v/v) glucose-hydroxylation for 20 hours at 100 rpm, 37° C. Sodium lysate (40% (w/v) glucose and 40% (w/v) aqueous solution of sodium hydroxide mixed in a volume ratio of 1:1) was used to adjust the pH to 6.0-7.5 and fed-batch culture. * Lactobacillus seed culture solution: Lactobacillus rhamnosus ( L. rhamnosus ) strain was cultured for 24 hours at 37°C in Lactobacilli MRS Broth (BD), hereinafter referred to as lactic acid bacteria seed culture solution. * In addition, the most representative experiment was the Lactobacillus rhamnosus ( Lactobacillus rhamnosus; L. rhamnosus ) strain, but enterococcus faecalis (E. faecalis ) in the same way to compare the experiment for each strain Wow Bifidobacterium breve (B. breve ) was also used.
비교예 2. 비교 조건의 파라프로바이오틱스 제조방법Comparative Example 2. Paraprobiotic production method under comparative conditions
비교예 1에서 배양된 배양액을 0.15Mpa, 121℃에서 30분간 가압멸균 후 막여과 농축기(여과막 사이즈 0.1㎛, 제조사 DOW separation systems, Serial No.546/57 205-92.)를 통과시켜 배지성분을 모두 제거한 뒤 농축된 균체에 부형제를 혼합한 후 건조하여 파라프로바이오틱스 분말을 회수하였다. The culture medium cultured in Comparative Example 1 was autoclaved at 0.15 Mpa, 121° C. for 30 minutes, and then passed through a membrane filtration concentrator (filtration membrane size 0.1 μm, manufacturer DOW separation systems, Serial No.546/57 205-92.) After removing all of the mixture, the excipient was mixed with the concentrated cells and dried to recover the paraprobiotic powder.
실시예 1. 용암해수를 이용한 유산균 코팅용 소수성 단백질 생산Example 1. Production of hydrophobic protein for lactobacillus coating using lava seawater
용암해수 농도에 따라 배지 내에 침전된 소수성 단백질 생성량을 비교하기 위하여 상수(물)에 용암해수를 10~100%(v/v) 농도로 혼합하여 배양용수를 제조하고, 포도당 3%(w/v), 효모추출물 2%(w/v), 소이펩톤 2%(w/v) 및 카제인 1%(w/v)에 배양용수를 첨가한 부피가 총 100%(v)가 되도록 용해 후 0.15Mpa, 121℃에서 30분간 멸균하여 멸균 배지를 제조하였다. In order to compare the amount of hydrophobic protein precipitated in the medium according to the concentration of lava seawater, lava seawater is mixed with constant (water) at a concentration of 10 to 100% (v/v) to prepare culture water and glucose 3% (w/v) ), 2% of yeast extract (w/v), 2% of soy peptone (w/v) and 1% of casein (w/v), after dissolving so that the volume of culture water is 100% (v), 0.15 Mpa Sterilized medium was prepared by sterilization at 121°C for 30 minutes.
실시예 2. 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조Example 2. Preparation of hydrophobic protein-coated paraprobiotics derived from lava seawater
실시예 1의 멸균배지별로 유산균 종배양액을 접종하여 유산균을 배양한 후 0.15Mpa, 121℃에서 30분간 가열한 뒤, 막여과 농축기를 사용하여 배지성분을 제거함과 동시에 균체와 용암해수 유래 소수성 단백질을 코팅시킨 후, 농축이 완료된 농축물을 건조하여 파라프로바이오틱스 분말을 수득하였다. After inoculating the lactic acid bacteria seed culture solution for each sterilized medium of Example 1, culturing the lactic acid bacteria, heating them at 0.15 Mpa, 121° C. for 30 minutes, removing the medium component using a membrane filtration concentrator, and simultaneously removing the bacteria and lava seawater-derived hydrophobic protein. After coating, the concentrated concentrate was dried to obtain a paraprobiotic powder.
실험예 1. 용암해수를 배양용수로 이용하여 제조한 멸균 배지 내의 소수성 침전 단백질의 확인Experimental Example 1. Identification of hydrophobic precipitated protein in sterile medium prepared using lava seawater as culture water
실시예 1과 비교예 1의 각 멸균배지를 4℃에서 24시간 정치후 6,000rpm, 30분간 원심분리하여 미네랄과 함께 침전된 소수성 단백질을 회수하고 동결건조하여 멸균된 배지 건조물 중의 단백질량을 BCA protein assay로 측정하여 단백질량을 확인하여 도 1에 나타내었다. Each sterilized medium of Example 1 and Comparative Example 1 was left standing at 4° C. for 24 hours, and then centrifuged at 6,000 rpm for 30 minutes to recover the hydrophobic protein precipitated with minerals and lyophilized to determine the amount of protein in the sterilized medium dry matter BCA protein The amount of protein was measured by assay and shown in FIG. 1.
그 결과, 도 1에서 보는 바와 같이, 배양용수로서 용암해수 30%(v/v) 첨가부터 소수성 침전 단백질량이 늘어나 80%(v/v)에서 최고값인 28.5 mg/ml을 나타낸 이후, 감소경향을 나타내었다. As a result, as shown in FIG. 1, since the amount of hydrophobic precipitated protein increased from the addition of 30% of lava seawater (v/v) as culture water, the maximum value at 80% (v/v) was 28.5 mg/ml, and the tendency to decrease. It was shown.
또한, 상기 소수성 침전 단백질 내에는 용암해수로부터 유래된 천연 미네랄이 형성되어 있는데, 이 천연미네랄이 최종 형성되는 파라프로바이오틱스에 얼마나 이입되는지 알아보기 위해 함량을 확인하였다. 이를 위해 위하여 대표적으로 무기질 (Ca, Mg, Sr) 함량을 측정하였고, 비교예 2와 실시예 2 시료(용암해수 30%(v/v) 배지에서 배양된 시료)를 1 g 취하고, 약 0.01~1,000mg 범위의 표준시약을 취한 후 희석하여 질산 12mL를 가한 후 microwave로 1시간 동안 분해하고 냉각시킨다. 3차 증류수로 50ml fill-up하여 ICPOES(PerkinElmer precisely optical Emission spectrometer, Optima 5300DV)로 분석하였고 해당 결과를 표 1에 나타내었다. 그 결과 실시예 2의 시료에서 소수성 침전 단백질 내에 형성된 칼슘, 마그네슘, 셀레늄의 양이 칼슘 0.41mg/g, 마그네슘 1.19mg/g, 셀레늄 0.000005mg/g으로 확인되고, 물을 배양용수로 이용하여 제조된 균체의 파라프로바이오틱스에 해당되는 비교예 2에서는 이보다 현저히 낮은 미네랄들이 포함되거나 존재하지 않는 것을 알 수 있었다. In addition, in the hydrophobic precipitated protein, natural minerals derived from lava seawater are formed, and the content is checked to see how much the natural minerals are introduced into the final formed paraprobiotics. For this purpose, representatively, inorganic (Ca, Mg, Sr) content was measured, and 1 g of samples of Comparative Example 2 and Example 2 (cultured in
이 결과를 토대로 반복적인 실험을 통해 실시예 2의 방법을 통해 얻을 수 있는 파라프로바이오틱스 내에는 칼슘 0.2~2.0mg/g, 마그네슘 0.5~2.0mg/g, 셀레늄 0.000001~0.00001mg/g이 대략 포함되는 것을 확인하였다.Based on these results, the paraprobiotics that can be obtained through the method of Example 2 through repeated experiments include approximately 0.2 to 2.0 mg/g of calcium, 0.5 to 2.0 mg/g of magnesium, and 0.000001 to 0.00001 mg/g of selenium. Was confirmed.
실험예 2. 용암해수 농도에 따른 파라프로바이오틱스수 비교Experimental Example 2. Comparison of paraprobiotic water according to lava seawater concentration
실시예 2와 비교예 2에서 제조된 파라프로바이오틱스 분말을 haemocytometer을 이용하여 균체수를 측정하여 도 2에 나타내었다. The paraprobiotic powders prepared in Example 2 and Comparative Example 2 are shown in FIG. 2 by measuring the number of cells using a haemocytometer.
용암해수 첨가 농도에 따른 파라프로바이오틱스수는 생균수에 비례하기에, 도 2에서 보는 바와 같이 용암해수 30% (v/v) 첨가하여 배양 진행할 때 최종적으로 유산균이 가장 잘 배양됨을 알 수 있다. 따라서 유산균 배양에 최적인 용암해수의 농도는 30% (v/v)로 최적화하였다. Since the paraprobiotic water according to the concentration of the lava seawater is proportional to the number of live bacteria, as shown in FIG. 2, it can be seen that lactic acid bacteria are best cultured finally when the culture is performed by adding 30% of lava seawater (v/v). Therefore, the concentration of lava seawater optimal for lactic acid bacteria culture was optimized to 30% (v/v).
이에 하기 표 2와 같이, 실시예 1의 배지 중 배양용수 중 30% (v/v) 농도의 용암해수로 제조한 배지와 비교예 1의 배지만 500L 단위로 실시예 2와 비교예 2의 배양, 파라프로바이오틱스화 과정을 수행하여 유산균의 파라프로바이오틱스개수를 비교하였으며 예상한 바와 같은 결과가 나와 대량의 파라프로바이오틱스 제조 시스템을 갖춤을 입증하였다. Accordingly, as shown in Table 2 below, only the medium prepared in lava seawater at a concentration of 30% (v/v) in culture water in the medium of Example 1 and the medium of Comparative Example 1 were cultured in Example 2 and Comparative Example 2 in 500 L units, The paraprobiotic process was performed to compare the number of paraprobiotics of lactic acid bacteria, and the results as expected were shown and proved to be equipped with a mass production system of paraprobiotics.
실험예 3. 용암해수 농도에 따른 파라프로바이오틱스의 소수성 비교Experimental Example 3. Comparison of hydrophobicity of paraprobiotics according to lava seawater concentration
용암해수 농도별로 생산한 파라프로바이오틱스 분말을 0.1g PBS buffer 10 mL에 현탁한 후, 원심분리 (12,000 rpm, 10 min, 4℃)하여 PBS buffer로 2회 washing하였다. PBS buffer에 OD600=0.5±0.02로 맞춰 pellet을 희석한다. 바로 초기 OD값을 측정한 후, 희석액 3 mL과 용매(Xylene) 1 mL을 섞은 후 (2 min간 vortexing), room temperature에서 30 min 정치시켰다. 정치 후, 정치액의 water 부분을 채취하여 30분간 둔 상태의 OD값을 측정하여 소수성을 계산하여 표 3에 나타내었다.The paraprobiotic powder produced by lava seawater concentration was suspended in 10 mL of 0.1 g PBS buffer, and centrifuged (12,000 rpm, 10 min, 4°C) to wash twice with PBS buffer. Dilute the pellet in PBS buffer with OD 600 =0.5±0.02. Immediately after measuring the initial OD value, 3 mL of diluent and 1 mL of solvent (Xylene) were mixed (vortexing for 2 min) and left at room temperature for 30 min. After standing, the water portion of the stationary liquid was collected and the OD value in a state left for 30 minutes was measured to calculate the hydrophobicity and shown in Table 3.
세포 표면 소수성 계산식Cell surface hydrophobicity formula
세포 표면 소수성 (%) = (1-(30분 후 흡광도값 / 초기 흡광도값)) x 100Cell surface hydrophobicity (%) = (1-(absorbance value after 30 minutes / initial absorbance value)) x 100
용암해수 첨가 농도에 따른 유산균 균체 표면의 소수성(hydrophobicity, %)을 백분율로 표기하였을 때, 용암해수 30% (v/v) 첨가시, 대부분의 유산균에서 가장 높은 소수성을 나타내었다. 이는 일반 파라프로바이오틱스 분말 대비, 실시예 2의 용암해수로 제조한 파라프로바이오틱스 엔테로코커스 페칼리스가 44.7%, 락토바실러스 람노서스가 47.7%, 비피도박테리움 브레비가 43.4% 증가함으로써 용암해수 염석으로 생성된 단백질이 각 유산균에 코팅되어 분말상태에서도 높은 소수성을 나타내었다. When the hydrophobicity (%) of the surface of lactic acid bacteria according to the concentration of lava seawater was expressed as a percentage, when 30% (v/v) of lava seawater was added, it showed the highest hydrophobicity in most lactic acid bacteria. This is produced by the lava seawater salt by increasing the paraprobiotics Enterococcus pecalis prepared by the lava seawater of Example 2 to 44.7%, Lactobacillus rhamnosus 47.7%, and Bifidobacterium brevi 43.4% compared to the general paraprobiotic powder. The protein was coated on each lactic acid bacterium and showed high hydrophobicity even in powder form.
또한 도 11의 다양한 균주로부터 얻은 결과를 통해 본 발명의 소수성을 갖는 파라프로바이오틱스 제조방법이 특정 균주들에 제한되지 않음을 보여준다. In addition, the results obtained from the various strains of FIG. 11 show that the method for producing paraprobiotics having hydrophobicity of the present invention is not limited to specific strains.
실험예 4. 용암해수 농도에 따른 파라프로바이오틱스의 뮤신 부착성 비교Experimental Example 4. Comparison of mucin adhesion of paraprobiotics according to lava seawater concentration
Immunoplate(Nunc)의 well에 10 mg/mL mucin solution을 200 ㎕씩 분주하고 4℃, overnight(18시간 이상 반응) 하여 mucin을 well에 부착시켰다. 각 well의 mucin solution을 제거하고, PBS (1X,pH7.4) 200 ㎕씩 분주하여 1 회 washing 하였다. 배지 제조용 용암해수 농도별로 생산된 파라프로바이오틱스 분말을 0.85% NaCl에 혼합하여 약 108 CFU/mL으로 희석하였다. 파라프로바이오틱스 희석액 100 ㎕를 미리 제조한 각 mucin well에 분주하고 37℃, 2 시간 동안 정치시켰다. Well의 상등액을 제거하고, mucin에 부착하지 않은 균체를 PBS (1X, pH7.4) 200 ㎕로 5 회 washing 하여 제거하였다. 0.1% Triton X-100를 100 ㎕씩 분주하고 뚜껑을 덮어 1,000 rpm, 20 분간 자동 교반하였다. 교반이 완료된 well로부터 pipetting하여 mucin에 부착된 균체를 회수하였다. 회수액을 0.85%(w/v) NaCl 수용액으로 serial dilution하여 hemocytometer로 부착된 균수를 측정하였으며, 이를 표 4에 나타내었다.200 μl of 10 mg/mL mucin solution was dispensed into wells of an immunoplate (Nunc), and mucin was attached to the wells at 4° C. overnight (over 18 hours reaction). The mucin solution of each well was removed, and 200 µl of PBS (1X, pH7.4) was dispensed and washed once. The paraprobiotic powder produced by lava seawater concentration for medium production was mixed with 0.85% NaCl and diluted to about 10 8 CFU/mL. 100 μl of a paraprobiotic dilution was dispensed into each mucin well prepared in advance and left at 37° C. for 2 hours. The supernatant of the well was removed, and the cells not attached to mucin were removed by washing 5 times with 200 μl of PBS (1X, pH7.4). 0.1% Triton X-100 was dispensed in 100 µl increments and covered with a lid to automatically stir at 1,000 rpm for 20 minutes. The cells adhered to the mucin were recovered by pipetting from the wells that had been stirred. The recovered solution was serially diluted with 0.85% (w/v) NaCl aqueous solution to measure the number of bacteria attached with a hemocytometer, which is shown in Table 4.
부착성 계산식Adhesion calculation formula
부착성 (%) = (부착균수)/(접종균수) x 100Adhesion (%) = (Number of bacteria attached)/(Number of bacteria inoculated) x 100
상기 계산식에서 균수는 cells/g으로 확인한다. In the above calculation formula, the number of bacteria is confirmed as cells/g.
표 4의 결과를 참고할 때, 소수성이 증가된 용암해수 농도별 소수성 단백질 코팅 파라프로바이오틱스와 표면 소수성을 가진 면역세포와의 부착능을 균주별로 살펴보았을 때, 용암해수 30% (v/v) 첨가시, 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 뮤신 부착능이 가장 높은 것으로 나타났다. 이는 일반적인 파라프로바이오틱스 제조방법(비교예 1의 배지에서 배양된 것)과 비교하여 엔테로코커스 페칼리스가 56%, 락토바실러스 람노서스가 44%, 비피도박테리움 브레비가 51% 증가함으로써 실제 면역활성을 일으키기 위한 전제조건인 뮤신 부착능이 용암해수를 배양용수로 사용한 파라프로바이오틱스 제조방법에서 높게 나타나는 것으로 확인되었다. 즉, 본 발명에서 제조한 파라프로바이오틱스가 장내 면역세포의 점액질을 구성하는 상기 뮤신 부착능 향상을 통해 체내의 면역반응을 증강시킬 수 있는 것을 확인할 수 있다. When referring to the results of Table 4, when the adhesion ability of hydrophobic protein-coated paraprobiotics by surface concentration with increased hydrophobicity and immune cells with surface hydrophobicity was examined by strain, when
또한 도 12의 다양한 균주로부터 얻은 결과를 통해 본 발명의 뮤신 부착능이 증가된 파라프로바이오틱스 제조방법이 특정 균주들에 제한되지 않음을 보여준다. In addition, the results obtained from the various strains of FIG. 12 show that the method for manufacturing paraprobiotics having increased mucin adhesion ability of the present invention is not limited to specific strains.
실험예 5. 용암해수로 배양한 가열처리 유산균와 용암해수로 배양하지 않은 파라프로바이오틱스의 유전자 발현 증가를 통한 면역활성 비교Experimental Example 5. Comparison of immune activity through increased gene expression of heat-treated lactic acid bacteria cultured with lava seawater and paraprobiotics not cultured with lava seawater
생쥐의 대식세포인 Raw264.7 세포에 리포폴리사카라이드(LPS) 0.1㎍/㎖을 처리하여 양성대조군으로 사용하며(염증 반응을 일으키는 소재로 사용한 것이 아님) 비교예 2와 실시예 2의 시료의 면역증진 관련 유전자 발현 증가율을 Real-Time PCR 방법을 이용하여 측정하였다. 10% FBS-DMEM 배지에 2x106 개의 Raw264.7 세포를 현탁시켜 60 mm 디쉬에 접종하여 부착시켰다. The macrophages of mice were treated with 0.1 μg/ml of lipopolysaccharide (LPS) in Raw264.7 cells and used as a positive control (not used as a material for causing an inflammatory reaction) of the samples of Comparative Examples 2 and 2 The rate of increase in gene expression related to immune enhancement was measured using a Real-Time PCR method. 2x10 6 Raw264.7 cells were suspended in 10% FBS-DMEM medium and inoculated in a 60 mm dish.
하루 후, 10% FBS-DMEM 배지로 교체하고 1.0x107 cells/mL로 희석된 파라프로바이오틱스 시료를 처리하여 24시간 동안 배양했다. 세포의 배지를 제거하고 퀴아졸 (QIAzol, QIAGEN 社) 1 ml를 첨가하여 퀴아젠 사의 RNA 분리법에 따라 RNA를 분리하고 정량한 뒤 1 ㎍의 RNA로 cDNA를 합성하여 Real-Time PCR을 실시하였다. PCR에 사용된 프라이머는 코스모진텍 社에서 합성하여 사용하였고, 프라이머 서열은 하기 표 5에 나타내었다. One day later, the sample was replaced with 10% FBS-DMEM medium and treated with a paraprobiotic sample diluted with 1.0x10 7 cells/mL to incubate for 24 hours. After removing the cell medium and adding 1 ml of QIAzol (QIAGEN), RNA was isolated and quantified according to the RNA separation method of Qiagen, and cDNA was synthesized with 1 μg of RNA to perform Real-Time PCR. Primers used for PCR were synthesized and used by Cosmogenetech, and the primer sequences are shown in Table 5 below.
도 3을 참고하면 면역 대식세포인 Raw264.7 세포를 자극함으로써 활성화되는 초기 면역반응 사이토카인인 TNF-α의 유전자 발현 수준이 비처리군(Control) 대비 실시예 2에서 크게 증가하였다. 또한 비교예 2의 일반적인 파라프로바이오틱스와 비교하여 실시예 2의 용암해수로 제조한 파라프로바이오틱스 엔테로코커스 페칼리스가 307%, 비피도박테리움 브레비가 382%, 락토바실러스 람노서스가 933% 증가한 발현 수준을 보이며 면역세포를 자극함을 알 수 있다. Referring to FIG. 3, the gene expression level of TNF-α, an initial immune response cytokine activated by stimulating the immune macrophages Raw264.7 cells, was significantly increased in Example 2 compared to the untreated group (Control). In addition, compared to the general paraprobiotics of Comparative Example 2, Paraprobiotics Enterococcus pecalis prepared from the lava seawater of Example 2 increased 307%, Bifidobacterium brevi 382%, and Lactobacillus rhamnosus increased by 933%. It can be seen that it stimulates immune cells.
도 4에서 면역 대식세포인 Raw264.7 세포를 자극함으로써 활성화되는 초기 면역 반응 사이토카인인 IL-6의 유전자 발현 수준이 비처리군(Control) 대비 실시예 2에서 증가함을 알 수 있다. 또한 비교예 2의 일반적인 파라프로바이오틱스와 비교하여 실시예 2의 용암해수로 제조한 파라프로바이오틱스 엔테로코커스 페칼리스가 987%, 비피도박테리움 브레비가 459%, 락토바실러스 람노서스가 332% 증가한 것을 확인할 수 있다. In FIG. 4, it can be seen that the level of gene expression of IL-6, an initial immune response cytokine activated by stimulating the immune macrophages Raw264.7 cells, is increased in Example 2 compared to the untreated group (Control). In addition, compared to the general paraprobiotics of Comparative Example 2, it can be seen that Paraprobiotics Enterococcus pecalis prepared from the lava seawater of Example 2 increased 987%, Bifidobacterium brevi 459%, and Lactobacillus rhamnosus 332%. have.
도 5의 결과에서는 면역 대식세포인 Raw264.7 세포를 자극함으로써 면역 반응 개시를 위해 활성화되는 사이토카인인 IL-1β의 유전자 발현 수준이 비처리군(Control) 대비 실시예 2에서 증가하였다. IL-1β는 초기 면역반응에서 매우 중요한 개시 인자인데, 비교예 2의 일반적인 파라프로바이오틱스와 비교하여 실시예 2의 용암해수로 제조한 파라프로바이오틱스 엔테로코커스 페칼리스가 165%, 비피도박테리움 브레비가 93%, 락토바실러스 람노서스가 180% 증가한다. In the results of FIG. 5, the gene expression level of IL-1β, a cytokine activated for initiating an immune response by stimulating the immune macrophages Raw264.7 cells, was increased in Example 2 compared to the untreated group (Control). IL-1β is a very important initiation factor in the initial immune response, compared to the general paraprobiotics of Comparative Example 2, Paraprobiotics Enterococcus pecalis prepared from lava seawater of Example 2 is 165%, Bifidobacterium brevi is 93 %, Lactobacillus rhamnosus increased by 180%.
도 6을 참고하면 면역 대식세포인 Raw264.7 세포를 자극함으로써 면역 반응 개시 단계에서 활성화되는 효소인 NOS의 활성 수준이 비처리군(Control) 대비 실시예 2에서 증가하였다. 비교예 2의 일반적인 파라프로바이오틱스와 비교하여 실시예 2의 용암해수로 제조한 파라프로바이오틱스 엔테로코커스 페칼리스가 175%, 비피도박테리움 브레비가 221%, 락토바실러스 람노서스가 674% 증가됨을 알 수 있다. Referring to FIG. 6, by stimulating the immune macrophages Raw264.7 cells, the activity level of NOS, an enzyme activated in the immune response initiation step, was increased in Example 2 compared to the untreated group (Control). Compared to the general paraprobiotics of Comparative Example 2, it can be seen that Paraprobiotics Enterococcus pecalis prepared from the lava seawater of Example 2 increased 175%, Bifidobacterium brevi 221%, and Lactobacillus rhamnosus 674%. .
도 7에서는 면역 대식세포인 Raw264.7 세포를 자극함으로써 면역 반응 개시 단계에서 IL-1β에 의해 활성화 되는 주요 효소인 COX-2의 유전자 발현 수준이 비처리군(Control) 대비 실시예 2에서 증가함을 확인할 수 있다. 비교예 2의 일반적인 파라프로바이오틱스와 비교하여 실시예 2의 용암해수로 제조한 파라프로바이오틱스 엔테로코커스 페칼리스가 1,482%, 비피도박테리움 브레비가 693%, 락토바실러스 람노서스가 1,242% 증가한 발현 수준을 보인다. In FIG. 7, the gene expression level of COX-2, a major enzyme activated by IL-1β at the initiation stage of the immune response, is increased in Example 2 compared to the untreated group by stimulating the immune macrophages Raw264.7 cells. can confirm. Compared to the general paraprobiotics of Comparative Example 2, Paraprobiotics Enterococcus pecalis prepared from the lava seawater of Example 2 shows an increased expression level of 1,482%, Bifidobacterium brevi 693%, and Lactobacillus rhamnosus 1,242%. .
이상과 같이 도 3 내지 도 7의 결과를 통해 본 발명의 방법으로 제조한 파라프로바이오틱스가 면역세포를 자극함으로써 면역 증진 효과를 갖는 것을 알 수 있다.As described above, it can be seen from the results of FIGS. 3 to 7 that the paraprobiotics produced by the method of the present invention stimulate the immune cells to have an immune enhancing effect.
실험예 6. 용암해수로 제조한 파라프로바이오틱스와 일반 파라프로바이오틱스의 면역증진 관련 단백질 분비 증가를 통한 면역활성 비교Experimental Example 6. Comparison of immune activity by increasing the secretion of proteins related to immunoproliferation of paraprobiotics and normal paraprobiotics prepared from lava seawater
생쥐의 대식세포인 Raw264.7 세포에 리포폴리사카라이드 0.1㎍/㎖ 처리하여 양성 대조군으로 사용하며, 비교예 2와 실시예 2의 시료에 의한 단백질 분비 증가율을 ELISA을 이용하여 측정하였다. 10% FBS-DMEM 배지에 2x106 개의 Raw264.7 세포를 현탁시켜 24웰 디시에 접종하여 부착시켰다. 하루 후, 10% FBS-DMEM 배지로 교체하고 1.0x107 cells/mL로 희석된 파라프로바이오틱스 시료를 처리하여 24시간 동안 배양했다. ELISA 플레이트를 수세 용액으로 2회 수세하였다. 희석제(assay buffer)로 농도의존적으로 희석된 표준용액과 1:1로 희석된 배양 배지 시료를 플레이트에 첨가하였다. 플레이트에 biotin이 접합된 항 마우스 각 항체를 첨가하고 2시간 동안 37℃에서 반응시켰다. 플레이트를 수세 용액으로 5회 수세하였다. Streptavidin-HRP 용액을 플레이트에 첨가하였다. 플레이트를 1시간 동안 37℃에서 반응시켰고, 수세 용액으로 5회 수세하였다. 플레이트에 테트라메틸 벤지딘(tetramethyl benzidine, TMB) 페록시다제 기질을 첨가하였다. 이 후 플레이트를 실온에서 10분 동안 반응시켰고, 규칙적으로 흔들었다. 정지 용액을 넣어 반응을 종료시킨 후 450nm에서 흡광도를 측정하였다. The macrophages of mice were treated with 0.1 μg/ml of lipopolysaccharide in Raw264.7 cells and used as a positive control, and the increase rate of protein secretion by the samples of Comparative Examples 2 and 2 was measured by ELISA. 2x10 6 Raw264.7 cells were suspended in 10% FBS-DMEM medium and attached by inoculation in a 24-well dish. One day later, the sample was replaced with 10% FBS-DMEM medium and treated with a paraprobiotic sample diluted with 1.0x10 7 cells/mL to incubate for 24 hours. The ELISA plate was washed twice with a washing solution. A standard solution diluted concentration-dependently with a dilution agent (assay buffer) and a culture medium sample diluted 1:1 were added to the plate. Each anti-mouse antibody conjugated to biotin was added to the plate and reacted at 37°C for 2 hours. The plate was washed 5 times with a washing solution. Streptavidin-HRP solution was added to the plate. The plate was reacted at 37° C. for 1 hour, and washed 5 times with a washing solution. Tetramethyl benzidine (TMB) peroxidase substrate was added to the plate. The plate was then reacted for 10 minutes at room temperature and shaken regularly. After stopping the reaction by adding a stop solution, the absorbance was measured at 450 nm.
도 8을 참고하면 면역 대식세포인 Raw264.7 세포를 자극함으로써 활성화되는 초기 면역 반응 사이토카인인 TNF-α의 분비 수준이 비처리군(Control) 대비 실시예 2에서 크게 증가하였다. 또한 비교예 2의 일반적인 파라프로바이오틱스와 비교하여 실시예 2의 용암해수로 제조한 가열처리 파라프로바이오틱스 엔테로코커스 페칼리스가 60%(2.83 pg/μg), 비피도박테리움 브레비가 52%(2.66 pg/μg), 락토바실러스 람노서스가 48%(2.37 pg/μg) 증가한 발현 수준을 보인다.Referring to FIG. 8, the secretion level of TNF-α, an initial immune response cytokine activated by stimulating the immune macrophages Raw264.7 cells, was significantly increased in Example 2 compared to the untreated group (Control). In addition, compared to the general paraprobiotics of Comparative Example 2, the heat-treated Paraprobiotics Enterococcus pecalis prepared from the lava seawater of Example 2 was 60% (2.83 pg/μg), and Bifidobacterium brevi was 52% (2.66 pg/ μg), Lactobacillus rhamnosus showed 48% (2.37 pg/μg) increased expression level.
마찬가지로, 도 9의 결과에서도 면역 대식세포인 Raw264.7 세포를 자극함으로써 활성화되는 초기 면역 반응 사이토카인인 IL-6의 분비 수준이 비처리군(Control) 대비 실시예 2에서 크게 증가하였다. 또한 비교예 2의 일반적인 파라프로바이오틱스와 비교하여 실시예 2의 용암해수로 제조한 가열처리 파라프로바이오틱스 엔테로코커스 페칼리스가 64%(0.267 pg/μg), 비피도박테리움 브레비가 380%(0.019 pg/μg), 락토바실러스 람노서스가 164%(0.089 pg/μg) 증가한다. Likewise, in the results of FIG. 9, the secretion level of IL-6, an initial immune response cytokine activated by stimulating the immune macrophages Raw264.7 cells, was significantly increased in Example 2 compared to the untreated group (Control). In addition, compared to the general paraprobiotics of Comparative Example 2, heat-treated Paraprobiotics Enterococcus pecalis prepared from lava seawater of Example 2 was 64% (0.267 pg/μg), Bifidobacterium brevi was 380% (0.019 pg/ μg), Lactobacillus rhamnosus increased by 164% (0.089 pg/μg).
지금까지의 실험들을 통해 용암해수로 배양한 파라프로바이오틱스가 면역세포의 면역 개시 인자로서 pro-inflammatory cytokines(TNF-α, IL-6, IL-1β)와 면역 활성화 효소(NOS, COX-2)의 발현 및 분비를 촉진시킴으로써 면역 증진 효과가 있음을 입증한다.Through the experiments so far, paraprobiotics cultured with lava seawater are the immune initiating factors of immune cells, pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and immune-activating enzymes (NOS, COX-2). It demonstrates the effect of enhancing immunity by promoting expression and secretion.
실험예 7. 용암해수로 제조한 파라프로바이오틱스와 일반 파라프로바이오틱스의 TEM 사진 비교 Experimental Example 7. Comparison of TEM pictures of paraprobiotics and normal paraprobiotics prepared from lava seawater
실시예 2와 비교예 2의 파라프로바이오틱스 분말을 투과전자현미경(Transmission electron microscope, TEM) 사진을 찍어 비교하였다. 도 10에서 보는 바와 같이, 용암해수로 제조한 파라프로바이오틱스(실시예 2, 좌측 사진)는 전자현미경 사진에서 용암해수 배지에서 생성된 소수성 염석 단백질이 세포 표면이 코팅된 울퉁불퉁한 형상을 볼 수 있다. 반면, 상수로 배양하여 제조한 파라프로바이오틱스(비교예 2, 우측 사진)는 어떠한 코팅도 없었기에 일반 간균 형태를 그대로 나타내어 표면이 매끄러운 상태임을 확인할 수 있었다. The paraprobiotic powders of Example 2 and Comparative Example 2 were compared by taking a transmission electron microscope (TEM) picture. As shown in FIG. 10, paraprobiotics (Example 2, left photo) prepared from lava seawater can be seen in an electron microscope photograph of a lumpy shape coated with a hydrophobic salt salt protein produced in the lava seawater medium on the cell surface. On the other hand, paraprobiotics (Comparative Example 2, photo on the right) prepared by culturing with constant water did not have any coating, so it was confirmed that the surface was smooth by showing the general bacterium form as it is.
Claims (10)
(제2단계) 상기 유산균 배양용 배지에 유산균을 접종하고 배양하여 배양액을 얻고 가열처리하는 단계; 및,
(제3단계) 가열처리된 배양액을 농축하여 배지성분을 제거하면서 파라프로바이오틱스 외부에 용암해수 유래 소수성 단백질을 코팅하고, 농축이 완료된 파라프로바이오틱스 농축물을 건조하여 분말을 수득하는 단계;
를 포함하는 것을 특징으로 하는 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법.(First step) preparing a culture medium for lactic acid bacteria in which hydrophobic proteins are formed using lava seawater as culture water;
(Second step) Inoculating and culturing the lactic acid bacteria in the culture medium for lactic acid bacteria to obtain a culture solution and heat treatment; And,
(Step 3) Concentrating the heat-treated culture medium to remove the medium component, coating the hydrophobic protein derived from lava seawater outside the paraprobiotics, and drying the concentrated paraprobiotics concentrate to obtain a powder;
Method for producing a hydrophobic protein-coated paraprobiotics derived from lava seawater comprising a.
상기 제1단계의 배양용수 내의 용암해수 농도는 30~70%(v/v) 인 것을 특징으로 하는 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법.According to claim 1,
Method for producing a hydrophobic protein-coated paraprobiotics derived from lava seawater, characterized in that the concentration of lava seawater in the culture water of the first step is 30-70% (v/v).
상기 제1단계의 배지는 포도당, 효모추출물, 소이펩톤 및 카제인이 포함되는 것을 특징으로 하는 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법.According to claim 1,
The medium of the first step is a method for producing a hydrophobic protein-coated paraprobiotics derived from lava seawater, which comprises glucose, yeast extract, soypeptone, and casein.
상기 제2단계의 유산균은 락토바실러스 속 (Lactobacillus sp.), 비피도박테리움 속 (Bifidobacterium sp.), 스트렙토코커스 속 (Streptococcus sp.), 락토코커스 속 (Lactococcus sp.), 엔테로코커스 속 (Enterococcus sp.), 페디오코커스 속 (Pediococcus sp.), 바이셀라 속 (Weissella sp.),바실러스 속 (Bacillus sp.)으로 이루어진 군 중에서 1종 이상 선택되는 것을 특징으로 하는 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법.According to claim 1,
Lactobacillus sp. ( Lactobacillus sp.), Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp.), Pediococcus sp., Weissella sp., Bacillus sp. selected from the group consisting of one or more selected from the group consisting of lava seawater-derived hydrophobic protein coating para Method of manufacturing probiotics.
상기 제3단계에서 파라프로바이오틱스가 포함된 배양액을 농축함으로써 파라프로바이오틱스에 소수성을 갖는 염석 단백질이 코팅되는 것을 특징으로 하는 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법.According to claim 1,
Method for producing a hydrophobic protein-coated paraprobiotics derived from lava seawater, characterized in that a hydrochloric acid protein having hydrophobicity is coated on the paraprobiotics by concentrating the culture solution containing paraprobiotics in the third step.
상기 제3단계의 건조는 동결건조, 분무건조 또는 진공건조를 통해 수행가능한 것을 특징으로 하는 용암해수 유래 소수성 단백질 코팅 파라프로바이오틱스의 제조방법.According to claim 1,
The third step of drying can be performed through lyophilization, spray drying, or vacuum drying. The method for preparing hydrophobic protein coated paraprobiotics derived from lava seawater.
상기 파라프로바이오틱스는 뮤신에 대한 부착성이 있는 것을 특징으로 하는 면역증강용 건강기능식품.The method of claim 8,
The para-probiotic is a health functional food for immunity enhancement, characterized in that it has adhesion to mucin.
상기 파라프로바이오틱스는 뮤신에 부착되어 TNF-α(tumor necrosis factor-α), IL-6(interleukin-6), IL-1β(interleukin-1β), NOS(nitric oxide synthase) 및 COX-2(cyclooxygenase-2)로 이루어진 군 중에서 1종 이상 선택되는 면역 개시 인자 또는 면역 활성화 효소의 유전자 발현 또는 단백질 발현을 증가시키는 것을 특징으로 하는 면역증강용 건강기능식품.The method of claim 9,
The paraprobiotics are attached to mucin, TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), IL-1β (interleukin-1β), NOS (nitric oxide synthase) and COX-2 (cyclooxygenase- 2) Immune-initiating health function food for enhancing immunity, characterized in that it increases the gene expression or protein expression of one or more immune-initiating factors selected from the group consisting of.
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