KR20030072812A - Lactococcus lactis CBT-19, manufacturing method of the culture broth concentration and the cosmetic preparation - Google Patents
Lactococcus lactis CBT-19, manufacturing method of the culture broth concentration and the cosmetic preparation Download PDFInfo
- Publication number
- KR20030072812A KR20030072812A KR1020020012017A KR20020012017A KR20030072812A KR 20030072812 A KR20030072812 A KR 20030072812A KR 1020020012017 A KR1020020012017 A KR 1020020012017A KR 20020012017 A KR20020012017 A KR 20020012017A KR 20030072812 A KR20030072812 A KR 20030072812A
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- KR
- South Korea
- Prior art keywords
- lactic acid
- concentrate
- acid bacteria
- weight
- cosmetic composition
- Prior art date
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- C—CHEMISTRY; METALLURGY
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
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Abstract
Description
본 발명은 여드름 원인균에 대한 항균기능을 갖는 유산 균주인 락토코쿠스 락티스(Lactococcus lactis) CBT-19, 이를 이용한 항균 배양액 분리농축물의 제조방법 및 상기 농축물을 포함하는 화장료 조성물에 관한 것으로, 좀 더 상세하게는, 여드름의 원인균이 되는 프로피오니박테리움 애크니스(Propionibacterium acnes)의 생육을 특이적으로 저해할 수 있어서, 여드름 원인균에 대한 항균기능을 갖는 락토코쿠스 락티스 CBT-19, 이를 이용한 항균 배양액 분리 농축물의 제조 방법 및 이를 포함하는 화장료 조성물에 관한 것이다.The present invention relates to a lactococcus lactis (Lactococcus lactis) CBT-19, which is a lactic acid strain having an antibacterial function against acne-causing bacteria, a method for preparing an isolated concentrate of an antimicrobial culture solution using the same, and a cosmetic composition comprising the concentrate. More specifically, it is possible to specifically inhibit the growth of propionibacterium acnes that causes acne, Lactococcus lactis CBT-19 having antibacterial function against acne causing bacteria, using the same It relates to a method for producing an antimicrobial culture separation concentrate and a cosmetic composition comprising the same.
종래부터, 유산균의 배양액 등이 화장품에 적용되어 왔는데, 상기 배양액 등을 화장품에 적용함에 있어서는, 균체 및 불용성 성분을 제거한 유산균 배양액이 주로 이용되어 왔다. 이러한 유산균의 배양액에는 주요 성분으로써 젖산, 유당, 아미노산, 인산염 등이 포함되어 있는 바, 이들 성분은 피부의 각질성분에 친화적으로 작용하거나, 피부의 항산화작용, 보습작용, pH 조절 작용 및 피부균총의 조절 작용 등을 할 수 있어서, 화장품에 유용하게 이용되어 왔으며, 이에 따라, 여러 가지 유산균의 배양액이 그 성분에 따라, 여러 종류의 화장품용 소재로서 개발되어 왔다. 특히, 여러 가지 유산균 중에서도 스트렙토코쿠스 써모필러스(Streptococcus thermophilus), 락토바실러스(Lactobacillus) 및 비피도박테리움(Bifidobacterium)속에 속하는 미생물 중에서 보습효과, 활성 산소 제거 효과 및 면역 부활 기능을 가지는 유산균의 배양액이 화장품에 주로 적용되어 왔으며, 다만, 계속적인 연구의 결과로 적용되는 미생물의 범위는 점차로 넓어지고 있다.Conventionally, a culture solution of lactic acid bacteria and the like have been applied to cosmetics. In applying the culture solution and the like to cosmetics, lactic acid bacteria culture medium from which bacteria and insoluble components have been removed has been mainly used. The lactic acid bacteria culture medium contains lactic acid, lactose, amino acid, phosphate, etc. as the main components, these components are friendly to the keratin components of the skin, or the skin's antioxidant activity, moisturizing effect, pH control action and skin flora It is possible to perform a regulating action and the like, and has been usefully used in cosmetics. Accordingly, various lactic acid bacteria culture media have been developed as various kinds of cosmetic materials according to its components. Particularly, among various lactic acid bacteria, a culture medium of lactic acid bacteria having a moisturizing effect, active oxygen elimination effect and immune revival function among microorganisms belonging to the genus Streptococcus thermophilus, Lactobacillus and Bifidobacterium It has been applied mainly to the cosmetics, but as a result of the continuous research, the range of microorganisms applied is gradually widening.
상기 종래 기술에 의한 화장품용 유산균의 배양 방법 및 이를 이용한 화장료 조성물의 제조 방법에 관하여 좀 더 살펴보면 다음과 같다.Looking at the culture method of the lactic acid bacteria for cosmetics according to the prior art and the manufacturing method of the cosmetic composition using the same as follows.
[표 1]TABLE 1
종래 기술에 의한 유산균 배양액을 이용한 화장료 조성물의 제조 공정Manufacturing process of cosmetic composition using lactic acid bacteria culture medium according to the prior art
상기 표에서 볼 수 있는 바와 같이, 유산균 배양액을 이용한 화장료 조성물을 제조하기 위해서는 우선, 적절한 유산균을 선택하여 이를 배양하게 되는 바, 종래 기술에 있어서, 이러한 유산균 배양은 일반적으로 펩톤류, 육엑기스, 효모추출물, 포도당 및 무기이온이 조합된 발효배지를 이용하여 혐기적 발효장치 내에서 이루어지게 되며, 이러한 배양 단계를 진행한 후, 그 배양액으로부터 균체를 분리하게 된다. 상기 분리 단계는 원심분리 또는 한외여과기를 이용하여 이루어지게 되며, 이러한 방법으로 균체가 분리된 유산균 배양액을 건조분말화 시키거나 액체 상태에서 화장품 성분, 증점제, 유화제 등과 혼합하여 화장료 조성물을 제조하게 된다.As can be seen in the above table, in order to prepare a cosmetic composition using a lactic acid bacteria culture medium, first, an appropriate lactic acid bacterium is selected and cultured. In the prior art, such lactic acid bacteria culture is generally peptone, meat extract, yeast. Extraction, glucose and inorganic ions are made in a fermentation broth using a combination of fermentation broth, and after the culturing step, the cells are separated from the culture. The separation step is performed using a centrifugal separation or an ultrafilter, and in this way, the lactic acid bacteria culture medium from which the cells are separated is dried powder or mixed with a cosmetic component, a thickener, and an emulsifier in a liquid state to prepare a cosmetic composition.
한편, 염증성 여드름 질환에 관련된 임상적 치료요법에 있어서는, 주로 테트라사이클린(tetracycline), 신다마이신(cindamycine), 에리스로마이신 (erythromycin)등의 항생제가 주로 이용되어 왔으나, 이러한 항생제를 장기간 투여할 경우, 여드름의 원인균이 내성을 가지게 됨으로써, 치료효과가 떨어지는 문제점이 있었으며, 여드름 유발균인 프로피오니박테리움 애크니스(Propionibacterium acnes)는 그 인위적 배양이 어려워서, 이들 약제에 대한 감수성 실험이 미약할 수 밖에 없으므로, 적절한 약제선정에 어려움이 따랐고, 더구나,. 상기 약제들은 모두 항생제이므로 화장품으로서의 이용이 불가능한 문제가 있었다.On the other hand, in the clinical treatment associated with inflammatory acne disease, antibiotics such as tetracycline, cindamycin, and erythromycin have been mainly used.However, when such antibiotics are administered for a long time, acne As the causative bacteria of the resistant to, there was a problem that the treatment effect is inferior, and propionibacterium acnes, an acne-causing bacterium, is difficult to artificially cultivate, so the sensitivity test for these drugs is inevitably weak. Difficulties in selecting the right medication, Since the drugs are all antibiotics, there is a problem that cannot be used as cosmetics.
이와 같이, 종래 기술에 있어서, 유산균 배양액의 화장품 이용은 그 배양액에 포함된 여러 가지 성분을 통하여 보습 효과, 면역 부활 기능, 활성 산소 제거 기능, 유기산 등에 의한 정균 작용 등의 부수적인 효과를 거두기 위한 것에 국한될 뿐, 특정 균주의 생육을 저해함으로써, 여드름 등의 피부 질환을 치유하기 위한 적용은 없었던 것이 사실이다. 이 때문에, 여드름 등의 피부 질환은, 그 치료를 위하여 주로 항생제가 이용되어 왔으나, 항생제의 과다 투여로 인한 내성의 발생, 화장품에는 적용할 수 없는 한계 등 그 문제점이 많았으며, 이러한 종래 기술의 문제점으로 인하여, 항균 미생물의 배양액이 적용됨으로써, 여드름 등의 피부 질환을 유발하는 원인균의 생육을 저해할 수 있고, 이에 따라, 부작용이 없이 여드름 등의 피부 질환을 근본적으로 치유할 수 있도록 하는 기능성 화장품의 개발이 절실히 요구되어 왔다.As described above, in the prior art, cosmetic use of the lactic acid bacteria culture medium is intended to achieve ancillary effects such as moisturizing effect, immune activating function, reactive oxygen removal function, bacteriostatic action by organic acid, etc. through various components contained in the culture medium. It is true that there is no application for treating skin diseases such as acne by limiting the growth of specific strains. For this reason, skin diseases such as acne have been mainly used for the treatment of the antibiotics, but many problems such as the occurrence of resistance due to the overdose of antibiotics, limitations that cannot be applied to cosmetics, such problems of the prior art Due to the application of the culture medium of the antimicrobial microorganisms, it is possible to inhibit the growth of the causative bacteria causing skin diseases such as acne, and accordingly, the functional cosmetics to fundamentally cure skin diseases such as acne without side effects Development has been urgently needed.
특히, 이러한 항균 미생물의 배양액은 내성 등의 부작용이 없고, 항생제에 비하여 일상 생활에서 사용되는 화장품에 비교적 쉽게 적용될 수 있어서, 여드름 등의 치료에 있어서, 그 효용이 크다고 할 것인 바, 이러한 효능을 가질 수 있는 유산균주, 그 배양액 분리 농축물의 제조 방법 및 이를 이용한 화장료 조성물의 필요성은 더욱 크다고 할 것이다.In particular, the culture medium of these antimicrobial microorganisms has no side effects such as resistance and can be applied relatively easily to cosmetics used in daily life as compared to antibiotics, and thus the utility thereof is great in the treatment of acne. The need for a lactic acid strain that can have, a method for producing a culture concentrate concentrate thereof, and a cosmetic composition using the same is even greater.
본 발명은 상기와 같은 문제점을 해결하고자 안출한 것으로써, 여드름 원인균인 프로피오니박테리움 애크니스(Propionibacterium acnes)를 선택적으로 사멸시킬 수 있어서, 상기 여드름 원인균의 생육을 특이적으로 억제할 수 있는 유산균주 락토코쿠스 락티스(Lactococcus lactis) CBT-19와 상기 본 발명에 의한 균주의 배양액 분리 농축물 및 이러한 분리 농축물을 포함함으로써, 여드름 예방 및 치료 효과를 가지게 되는 화장료 조성물을 제공하는데 그 목적이 있다.The present invention has been made to solve the above problems, it is possible to selectively kill the acne causative bacteria Propionibacterium acnes (Propionibacterium acnes), lactic acid bacteria that can specifically inhibit the growth of the acne causative bacteria The main purpose of the present invention is to provide a cosmetic composition having a preventive and therapeutic effect of acne by including Lactococcus lactis CBT-19 and the culture concentrate separation concentrate of the strain according to the present invention, and such separation concentrate. have.
본 발명은 상기의 목적을 달성하기 위하여, 여드름 원인균인 프로피오니박테리움 애크니스(Propionibacterium acnes)의 생육을 특이적으로 억제할 수 있는 유산균주인 락토코쿠스 락티스(Lactococcus lactis) CBT-19를 제공한다.The present invention to achieve the above object, Lactococcus lactis (Lactococcus lactis) CBT-19 is a lactic acid strain that can specifically inhibit the growth of acne causing bacteria Propionibacterium acnes (Propionibacterium acnes) do.
또한, 본 발명은 3~5중량%의 포도당, 0.5~1중량%의 효모추출물, 1~1.5중량%의 육엑기스, 0.5~1중량%의 단백가수분해물 및 0.01~0.1중량%의 이온성분을 물에 용해시켜, 혐기적 발효관에서 살균하여 제조한 배양액에 상기 본 발명에 의한 균주를 접종하여 유산균을 배양하여 항균물질을 발생시키는 단계와, 고속원심분리 또는 한외여과(Ultrafiltration)를 통하여 상기 유산균 배양액으로부터 균체를 제거하는 단계와, 상기 균체가 제거된 배양여액을 배제분자량 10,000의 한외여과막(Ultrafiltration membrane)에 통과시키고, 상기 투과액을 다시 배제분자량 1,000의 나노미터막(Nanometer membrane)에 통과시켜, 상기 배양여액을 농축시키는 단계와, 상기 농축액에 무균수를 연속적으로 가하고, 그 결과물을 다시 배제분자량 1,000의 나노미터막(Nanometer membrane)에 통과시키면서 젖산을 제거함으로써, 분자량 1,000~10,000 범위의 중성 pH를 나타내는 항균물질 분리농축액을 제조하는 단계를 포함하는 것을 특징으로 하는 유산균 배양액의 분리농축물 제조방법을 제공한다.In addition, the present invention is 3 to 5% by weight of glucose, 0.5 to 1% by weight of yeast extract, 1 to 1.5% by weight of meat extract, 0.5 to 1% by weight of protein hydrolyzate and 0.01 to 0.1% by weight of ionic components Inoculating the strain according to the present invention in a culture solution prepared by dissolving in water and sterilizing in an anaerobic fermentation tube to cultivate the lactic acid bacteria to generate an antimicrobial material, the lactic acid bacteria through high-speed centrifugation or ultrafiltration (Ultrafiltration) Removing the cells from the culture medium, passing the culture filtrate from which the cells were removed, and passing the ultrafiltration membrane with an exclusion molecular weight of 10,000, and passing the permeate through a nanometer membrane with an exclusion molecular weight of 1,000 again. Concentrating the culture filtrate, and aseptic water is continuously added to the concentrate, and the resultant is passed through the nanometer membrane of the exclusion molecular weight 1,000 to remove the lactic acid By a molecular weight of 1,000 to 10,000 to provide a method for producing separated concentrate of lactic acid bacteria culture comprising the steps of preparing an antimicrobial material separating concentrated liquid showing a neutral pH in the range.
상기 본 발명에 의한 분리 농축물 제조 방법에 있어서, 상기 이온성분으로는, 암모니움 시트레이트, 소디움 아세테이트, 디포타시움 포스테이트, 마그네슘설페이트, 망간 설페이트, 소디움 클로라이드로 이루어진 그룹에서 선택된 물질을 사용함이 바람직하다.In the separation concentrate production method according to the present invention, as the ionic component, it is preferable to use a material selected from the group consisting of ammonium citrate, sodium acetate, dipotassium formate, magnesium sulfate, manganese sulfate, sodium chloride. Do.
또한, 상기 본 발명에 의한 분리 농축물 제조 방법은 상기 항균물질 분리농축액을 제조하는 단계를 진행한 후에, 제조된 분리농축물을 동결건조하거나 분사식 유동층 건조하는 단계를 부가적으로 포함하여 구성될 수도 있다. 이러한 단계를 부가적으로 포함함으로써, 제조된 분리 농축액의 저장 안정성을 향상시킬 수 있게 된다.In addition, the separation concentrate production method according to the present invention may further comprise the step of lyophilizing the prepared concentrate concentrate or sprayed fluid bed drying after the step of preparing the antimicrobial substance concentrate concentrate. have. By additionally including this step, it is possible to improve the storage stability of the prepared separated concentrate.
본 발명은 또한, 상기의 방법에 의하여 제조된 유산균 배양액의 분리 농축물을 0.01중량% 이상의 농도 또는 10Au/G이상의 항균 활성을 가지도록 포함하는 것을 특징으로 하는 화장료 조성물을 제공한다. 이러한 화장료 조성물은 여드름 원인균의 생육을 억제할 수 있으므로, 이러한 조성물을 이용하여 화장품을 제조함으로써, 여드름에 대한 치료 효과를 거둘 수 있게 되는 것이다.The present invention also provides a cosmetic composition comprising the separated concentrate of the lactic acid bacteria culture medium prepared by the above method to have a concentration of 0.01% by weight or more or an antimicrobial activity of 10Au / G or more. Since the cosmetic composition can inhibit the growth of acne-causing bacteria, it is possible to achieve a therapeutic effect on acne by manufacturing a cosmetic using such a composition.
상기 본 발명에 의한 조성물에 있어서, 상기 조성물은 적용되는 화장품의 종류에 적합하도록 W/O, O/W 유화제형, 투명스킨, 현탁스킨, 젤제형 등으로 제조될 수 있으며, 유해세균에 대한 정균작용, 미백, 보습, 항산화 효과를 나타내는 보조 성분으로서 소디움히알루로닉산(Sodium hyaluronic acid), 락토페린(Lactoferrin), 혈청 단백질(Serum protein), 판테틴(Pantethine), 살리실산(Salicylic acid), 트리클로산(Triclosan) 등을 부가적으로 포함할 수 있다.In the composition according to the present invention, the composition can be prepared in W / O, O / W emulsifier type, transparent skin, suspension skin, gel formulation, etc. to suit the type of cosmetics applied, bacteriostatic against harmful bacteria Sodium hyaluronic acid, Lactoferrin, Serum protein, Pantethine, Salicylic acid, Triclosan as an auxiliary ingredient showing action, whitening, moisturizing and antioxidant effect ) May be additionally included.
도 1은 본 발명에 의한 균주인 락토코쿠스 락티스 CBT-19의 배양액 분리 농축물을 제조하고, 상기 농축물을 포함하는 화장료 조성물을 제조하는 공정을 나타낸 순서도이고,1 is a flowchart illustrating a process of preparing a culture concentrate isolated concentrate of Lactococcus lactis CBT-19, which is a strain according to the present invention, and preparing a cosmetic composition comprising the concentrate,
도 2는 락토코쿠스 락티스 CBT-19의 전자현미경 관찰도이며,2 is an electron microscope observation of Lactococcus lactis CBT-19,
도 3은 락토코쿠스 락티스 CBT-19의 항균 물질 발효 경시변화를 나타내는 그래프이고,Figure 3 is a graph showing the changes over time antimicrobial fermentation of Lactococcus lactis CBT-19,
도 4는 락토코쿠스 락티스 CBT-19 배양액 분리 농축물의 여드름 원인균인 프로피오니박테리움 에크니스(Propionibacterium acnes)에 대한 생육저지환 관찰도이다.FIG. 4 is a diagram showing growth hypoglycemia of propionibacterium acnes, which is a cause of acne of Lactococcus lactis CBT-19 culture isolate concentrate.
이하, 첨부한 도면을 참조하여 본 발명에 의한 유산균주, 배양액 분리농축물의 제조 방법 및 이를 포함하는 화장료 조성물에 관해 좀더 상세하게 설명하기로 한다.Hereinafter, with reference to the accompanying drawings will be described in more detail with respect to lactic acid bacteria strain, a method for producing a culture concentrate separation composition and a cosmetic composition comprising the same.
상기한 바와 같이, 본 발명은 여드름 원인균에 특이적 저해활성을 지닌 신규한 유산균주의 개발, 상기 균주에 대한 최적 발효배지 및 발효공정의 구성, 상기 발효를 통해 형성된 배양액으로부터, 항균활성을 가진 배양액 분리농축물을 제조하는 방법, 상기 분리 농축물을 포함하며 화장료 조성에 용이한 액제 또는 수용성 분말 상태의 조성물과, 이를 이용하여 제조되는 다양한 제형의 화장료에 관한 것이다.As described above, the present invention, the development of a novel lactic acid strain with a specific inhibitory activity to the acne causative bacteria, the composition of the optimum fermentation medium and fermentation process for the strain, from the culture medium formed through the fermentation, separation of the culture medium having antibacterial activity The present invention relates to a method of preparing a concentrate, a composition in a liquid or water-soluble powder state containing the separated concentrate and easy to make up a cosmetic composition, and cosmetics of various formulations prepared using the same.
이러한 본 발명에 사용되는 유산균주는 락토코쿠스 락티스(Lactococcus lactis)로 동정되는 락토코쿠스 락티스 CBT-19 유산구균으로서, 여드름 원인균으로 알려진 프로피오니박테리움 애크니스(Propionibacterium acnes)에 대하여 특이적 저해활성을 발휘하는 물질을 생산한다. 상기 균주에 대한 최적 발효배지 및 발효조건은 여드름 원인균에 대한 생육 억제 물질의 대량생산에 적합하도록 구성되었으며, 항균물질의 분리 농축 과정은 원심분리 또는 한외여과를 통한 균체 및 불용성 성분의 제거, 배제분자량을 이용한 항균활성분획의 포집 및 농축, 유기산의 제거 공정으로 구성된다. 건조분체화 공정은 농축액의 살균 후 수용성 포집제를 이용한 분사식 유동층 건조 또는 동결건조를 통하여 이루어 진다.Lactobacillus used in the present invention is Lactococcus lactis (Lactococcus lactis) is identified as Lactococcus lactis CBT-19 lactobacillus, specific for the Propionibacterium acnes known as acne causing bacteria (Propionibacterium acnes) Produces substances with inhibitory activity. The optimum fermentation medium and fermentation conditions for the strain were configured to be suitable for mass production of growth inhibitory substances against acne-causing bacteria, and the separation and concentration process of the antimicrobial material was performed by centrifugation or ultrafiltration to remove the cells and insoluble components, and exclude molecular weight. It consists of the process of capturing and concentrating the antimicrobial active fractions and removing organic acids. The dry powderization process is performed by spraying fluidized bed drying or lyophilization using a water-soluble collecting agent after sterilization of the concentrate.
이와같은 공정으로 획득되어진 유산균 배양 분획건조물은 유화제 및 기타 화장품 원료 등과 혼합하여 유연화장수, 영양화장수, 에센스, 크림, 로션, 메이크업, 색조 화장품 등의 모든 화장품 제형 등의 화장료로 제조되어질 수 있다.The lactic acid bacteria culture fraction dried obtained by such a process may be mixed with an emulsifier and other cosmetic raw materials, and may be prepared as a cosmetic such as all cosmetic formulations such as soft cosmetics, nutrient cosmetics, essences, creams, lotions, makeup and color cosmetics.
상기와 같은, 본 발명에 의한 균주, 분리 농축물의 제조방법 및 이를 포함하는 화장료 조성물에 관해 상세하게 설명하면 다음과 같다.As described above in detail with respect to the strain according to the present invention, a method for producing a concentrate and a cosmetic composition comprising the same.
1) 균주선발(101)1) Strain Selection (101)
락토코쿠스 락티스의 상기 배양액으로부터 균체를 분리한 유산균 배양 여액에 대해, 지시균으로서 프로피오니박테리움 애크니스(Propionibacterium acnes)를 반유동 배지에서 배양하여가며, 저지환 시험을 진행한다. 위와 같은 시험 결과 800Au/ml이상의 항균활성을 발휘하는 유산종균을 선별하여, 선별된 균주를 락토코쿠스 락티스 CBT-19 (Lactococcus lactis CBT-19)라고 명명하고, 이를 부다페스트 조약에 의한 국제 기탁기관(KCTC: 한국생명공학연구원 유전자원센터 유전자은행)에 기탁하여, 기탁 번호 (KCTC10180BP)를 부여받았다.The lactic acid bacteria culture filtrate from which the cells were isolated from the culture medium of Lactococcus lactis was treated with propionibacterium acnes in a semi-fluid medium as an indicator bacterium, and a hypotonic test was conducted. As a result of the above test, lactic acid spawn with an antimicrobial activity of 800 Au / ml or more was selected, and the selected strain was named Lactococcus lactis CBT-19, which is an international depository institution under the Budapest Treaty. (KCTC: Korea Institute of Biotechnology and Genetic Resources Gene Bank) was given a deposit number (KCTC10180BP).
2) 균주의 특성 조사2) Characterization of the strain
상기와 같은 방법으로 제조된 유산균주 락토코쿠스 락티스 CBT-19에 관련한 생리학적, 생화학적 특성 등을 조사하여, 이를 하기의 표 1, 2에 나타내었으며, 상기 균주의 전자 현미경 사진을 도 2에 표시하였다.The physiological and biochemical properties related to the lactobacillus lactococcus lactis CBT-19 prepared by the above method were investigated, and the results are shown in Tables 1 and 2 below, and the electron micrographs of the strains were shown in FIG. 2. Marked on.
표 1. 락토코쿠스 락티스 CBT-19균의 효소활성 시험Table 1. Enzyme activity test of Lactococcus lactis CBT-19
표 2. 락토코쿠스 락티스(Lactococcus lactis) CBT-19균의 당자화능 시험Table 2. Glycosylation of Lactococcus lactis CBT-19
3) 항균활성 물질의 발효(102)3) Fermentation of Antimicrobial Active Materials (102)
상기한 바와 같이, 본 발명에 사용된 유산균주는 락토코쿠스 락티스(Lactococcus lactis) CBT-19로서, 이는 락토코쿠스 락티스를 혐기적으로 배양한 후, 이를 통해 제조된 유산균 배양여액에 대해 지시균으로 프로피오니박테리움 애크니스(Propionibacterium acnes)를 사용하여, 반유동배지에서 저지환 시험을 진행함으로써, 800Au/ml이상의 항균활성을 발휘하는 유산균을 선별하여 제조된 것이다. 상기 균주에 의한 항균물질 생산은 3~5중량%의 포도당, 0.5~1중량%의 효모추출물, 1~1.5중량%의 육엑기스, 0.5~1중량%의 단백가수분해물 및 0.01~0.1중량%의 암모니움 시트레이트, 소디움 아세테이트, 디포타시움 포스테이트, 마그네슘 설페이트, 망간 설페이트 또는 소디움 클로라이드 등의 이온성분을 물에 용해시켜 제조된 배양용 배지에, 상기 본 발명에 의한 균주를 접종하고 산소가 제거된 혐기적 조건으로 16시간 발효함으로써 이루어진다. 항균물질 발효공정의 경시변화는 도 3에 나타내었는데 16시간 경과시 여드름 원인균인 프로피오니박테리움 애크니스(Propionibacterium acnes)에 대하여 최대 1600 Au/ml의 저해활성을 나타내는 항균활성 물질이 생산되었다.As described above, the lactobacillus used in the present invention is Lactococcus lactis CBT-19, which is incubated anaerobicly with Lactococcus lactis, and then directed to the lactic acid bacteria culture filtrate prepared therefrom. As a bacterium, by using a propionibacterium acnes (Propionibacterium acnes), by carrying out a low-ring test in a semi-fluid medium, lactic acid bacteria exhibiting an antimicrobial activity of more than 800 Au / ml was selected. Production of the antimicrobial material by the strain is 3 to 5% by weight of glucose, 0.5 to 1% by weight of yeast extract, 1 to 1.5% by weight of meat extract, 0.5 to 1% by weight of protein hydrolyzate and 0.01 to 0.1% by weight of The culture medium prepared by dissolving ionic components such as ammonium citrate, sodium acetate, dipotassium formate, magnesium sulfate, manganese sulfate, or sodium chloride in water was inoculated with the strain according to the present invention and oxygen was removed. By fermentation for 16 hours under anaerobic conditions. The change in the fermentation process of the antimicrobial agent is shown in FIG. 3, wherein an antimicrobial active agent exhibiting an inhibitory activity of up to 1600 Au / ml was produced against the acne causing bacterium Propionibacterium acnes.
4)발효액 분리농축 및 젖산제거(103-106)4) Separated concentration of fermentation solution and removal of lactic acid (103-106)
상기와 같은 방법으로 발효 단계를 진행한 후, 상기 발효액은 15,000 RPM 이상의 고속원심분리기 또는 균체분리용 한외여과기에 의해 균체와 여액으로 분리된다. 균체가 분리된 발효여액은 배제분자량 10,000의 한외여과막(Ultrafiltrationmembrane)으로 1차 여과하고, 1차 여과한 투과액에 대하여 배제분자량 1,000을 지닌 나노미터막(Nanometer membrane)으로 분리, 농축시켜 분자량 1000~10,000사이의 활성분획만을 포집하여 농축하고, 상기 농축액에 대해서는 5배량의 무균수를 연속적으로 가하여, pH가 6.0이상이 될 때까지 Dia-filtration함으로써, 젖산 성분을 완전 제거 한 후, 유기산이 제거된 최종 농축액의 부피를 발효원액에 대비하여 1/10의 부피로 조절한다. 발효액으로부터 항균물질을 분리 포집하고 젖산을 제거하는 공정에서의 항균활성 물질의 경시변화는 표 3에, 최종 농축액의 항균활성 관찰도는 도 4에 나타내었다.After proceeding the fermentation step in the same manner as above, the fermentation broth is separated into cells and filtrate by a high-speed centrifuge or cell separation ultrafiltration for 15,000 RPM or more. The fermented filtrate from which the cells were separated was first filtered through an ultrafiltration membrane with an exclusion molecular weight of 10,000, and then separated and concentrated with a nanometer membrane having an exclusion molecular weight of 1,000 with respect to the filtered permeate. Only 10,000 active fractions were collected and concentrated, and 5 times aseptic water was added to the concentrated solution continuously, and dia-filtration was carried out until the pH was 6.0 or more, to completely remove the lactic acid component, and then remove the organic acid. The volume of the final concentrate is adjusted to 1/10 the volume of the fermentation stock solution. The change over time of the antimicrobial active material in the process of separating and collecting the antimicrobial material from the fermentation broth and removing lactic acid is shown in Table 3, and the degree of antimicrobial activity of the final concentrate is shown in FIG. 4.
표 3. 락토코쿠스 락티스 CBT-19의 발효에 의한 항균물질 농축액 제조시 공정별 항균활성의 경시도Table 3. Severity of Antimicrobial Activity by Process in the Preparation of Antimicrobial Concentrates by Fermentation of Lactococcus Lactis CBT-19
5)건조분체화(107)5) Dry powdering (107)
상기한 농축액에 대비하여 중량비 1~10%의 유당, 말토덱스트린, 만니톨, 탈지분유를 혼합하여 용해시키고 80℃ 온도조건에서 교반하여 가면서, 2회 30분간 열처리 하여 기타 잡균 및 유산균 등을 살균하고 동결건조기에 투입하여 건조시키거나 또는 분사식 유동층건조기에서 건조하고 분쇄하여 분말을 제조한다. 경우에 따라 항균활성 물질의 농축액을 화장료 원료로 직접사용하는 경우에는 건조분체화 공정을 생략할 수 있다.Lactose, maltodextrin, mannitol, and skim milk powder in a weight ratio of 1 to 10% of the above-mentioned concentrates are mixed and dissolved, and stirred at 80 ° C. under a 30-minute heat treatment to sterilize and freeze other miscellaneous bacteria and lactic acid bacteria. Powder is prepared by drying in a drier or by drying and pulverizing in a spray fluidized bed dryer. In some cases, when the concentrate of the antimicrobial active substance is directly used as a cosmetic raw material, the dry powdering process may be omitted.
8)화장료 제조(108)8) Cosmetics Manufacturing (108)
상기 항균물질 농축물을 원료로 포함한 화장료 제형에 있어서 특별히 한정되는 것은 없다. 본 발명에 의한 화장료의 제형에는 구체적으로 유연화장수, 영양화장수, 에센스, 크림, 로션, 메이크업, 색조 화장품 등의 모든 화장품 제형이 가능하며, 각 제형의 화장료 제조에 있어서 점증제, 방부제, 향료, 착색료 등의 일반적인 원료성분들은 화장료의 제조방법, 제형, 사용목적 등에 따라 적절하게 선택하여 배합할 수 있다. 화장료의 제조시 본 발명의 핵심성분인 항균물질 농축물은 전체 조성물에 대하여 0.01% 이상의 농도 또는 10Au/g 이상의 항균활성을 유지하도록 배합하며, 추가적으로 기타 유해세균에 대한 정균작용, 미백, 보습, 항산화 효과를 나타내는 보조 성분으로서 소디움히알루로닉산(Sodium hyaluronic acid), 락토페린(Lactoferrin), 혈청 단백질(Serum protein), 판테틴(Pantethine), 살리실산(Salicylic acid), 트리클로산(Triclosan) 등을 단독 또는 혼합하여 화장료를 제조한다.There is no particular limitation in the cosmetic formulation containing the antimicrobial concentrate as a raw material. In the formulation of the cosmetics according to the present invention, all cosmetic formulations such as soft cosmetics, nourishing cosmetics, essences, creams, lotions, makeups, and color cosmetics can be used, and thickeners, preservatives, fragrances, and coloring agents in the preparation of cosmetics of each formulation can be used. General raw materials such as may be appropriately selected and blended according to the manufacturing method, formulation, purpose of use, and the like. The antimicrobial concentrate, which is a key ingredient of the present invention, is formulated to maintain a concentration of at least 0.01% or at least 10 Au / g of antimicrobial activity in the preparation of cosmetics, and additionally, bacteriostatic action, whitening, moisturizing, and antioxidant activity against other harmful bacteria. Sodium hyaluronic acid, Lactoferrin, Serum protein, Panthine, Salicylic acid, Triclosan, etc. Prepare cosmetics.
실시예 1) 락토코쿠스 락티스 CBT-19 균에 의한 여드름 원인균을 저해하는 농축 발효액 및 농축건조물의 제조Example 1 Preparation of a concentrated fermentation broth and a concentrated dried product inhibiting acne causing bacteria by Lactococcus lactis CBT-19
포도당 25Kg, 육엑기스 5Kg, 효모추출물 2.5Kg, 카제이펩톤 2.5kg, 소이펩톤2.5kg, 디포타슘 포스페이트 0.25kg, 암모니움 시트레이트 0.25kg, 소디움 아세테이트 0.25kg, 마그네슘 설페이트 0.05kg, 망간 설페이트 0.05kg을 물 500L에 첨가하여 용해시키고 800L 용량의 혐기적 발효관에 이송하여 121℃에서 15분간 살균하고 미리 배양한 종균 10L를 접종하여 20시간 발효하였다. 발효액을 60L/Hr의 유속으로 연속식 원심분리기에 이송시키면서 균체를 제거하고 배양여액을 배제분자량 10,000의 한외여과기 필터에 통과시키고 투과액을 회수하여 다시 배제분자량 1,000의 나노미터 필터에 통과시켜 분자량 1000~10,000사이의 항균물질 농축액을 50L를 획득하였다. 농축액에 대하여 다시 5배량의 무균수를 연속적으로 가하며 배제분자량 1,000의 나노미터 필터에 통과시켜 투과액과 농축액을 재분리하였다. 이 공정에서 잔존유기산, 색소, 불용성 성분 등이 완전히 제거되었고 여드름 원인균에 대하여 항균활성을 지닌 분자량 1000~10,000사이의 항균물질분획이 선택적으로 분리농축 되었다. 분리농축된 항균물질 포집액은 최종 부피를 발효원액의 1/10수준으로 농축하였고 여드름 원인균에 대한 항균활성 또한 10배로 농축되었다. 항균물질 분리 농축액에 대하여 중량비 5~50% 범위의 만니톨, 유당 등의 수용성 부형제를 사용하여 동결건조 또는 분사식 유동층 건조하여 수용성 분말화 하였다. 이때 항균활성은 최종 분말의 경우 1,600~16,000Au/g이 되도록 희석비를 조정하였다.Glucose 25Kg, Meat Extract 5Kg, Yeast Extract 2.5Kg, Casei Peptone 2.5kg, Soypeptone 2.5kg, Dipotassium Phosphate 0.25kg, Ammonium Citrate 0.25kg, Sodium Acetate 0.25kg, Magnesium Sulfate 0.05kg, Manganese Sulfate 0.05kg The solution was added to 500 L of water to dissolve, transferred to an 800 L anaerobic fermentation tube, sterilized at 121 ° C. for 15 minutes, and inoculated with 10 L of pre-cultivated spawn and fermented for 20 hours. The cells were removed while the fermentation broth was transferred to a continuous centrifuge at a flow rate of 60 L / Hr, the culture filtrate was passed through an ultrafilter filter with a exclusion molecular weight of 10,000, the permeate was recovered, and passed through a nanometer filter with an exclusion molecular weight of 1,000. 50 L of an antimicrobial concentrate between -10,000 was obtained. 5 times of sterile water was continuously added to the concentrate, and the permeate and the concentrate were re-separated by passing through a nanometer filter with an exclusion molecular weight of 1,000. In this process, residual organic acids, pigments, and insoluble components were completely removed, and an antimicrobial fraction with an antimicrobial activity of 1,000 to 10,000 was selectively isolated and concentrated. The concentrated concentration of the antimicrobial agent was concentrated to 1/10 the final volume of the fermentation broth and the antimicrobial activity against acne-causing bacteria was also increased 10-fold. The water-soluble powder was lyophilized or spray-liquid dried using an aqueous excipient such as mannitol and lactose in a weight ratio of 5 to 50% with respect to the separated concentrate of the antimicrobial substance. At this time, the antimicrobial activity was adjusted to a dilution ratio of 1,600 ~ 16,000Au / g for the final powder.
실시예 2)-5) 유산균 배양액 농축건조물을 함유하는 일반 화장수의 제조Example 2) -5) Preparation of general lotion containing lactic acid bacterium concentrate concentrated dry matter
표 4. 실시예2)-5)에 따른 조성비Table 4. Composition ratio according to Example 2) -5)
원료 1)~5)을 혼합하여 균질하게 용해시켰다. 별도의 용기에서 원료 6)~11)을 40℃ 이하의 온도로 가열하면서 혼합하여 용해시킨 후, 먼저 제조된 1)~5)원료 용액에 서서히 투입하고 Agi-mixer에서 균질화 시켜 화장수를 제조하였다. 이때, 여드름 원인균인Propionibacterium acnes를 포함한 피부 유해세균에 대한 항균작용에 관련한 활성성분으로서 주 성분인 5)Lactopad에 대하여 보조성분으로서 7)Salicylic acid, 8)Triclosan을 단독 또는 동시에 일정한 농도로 혼합하여 화장수를 제조하였다.Raw materials 1) to 5) were mixed and dissolved homogeneously. In a separate container, raw materials 6) to 11) were mixed and dissolved while heating to a temperature of 40 ° C. or lower, and then slowly added to the prepared 1) to 5) raw material solution, and homogenized in an Agi-mixer to prepare a lotion. At this time, the active ingredient related to the antibacterial action against the harmful bacteria of the skin including acne cause Propionibacterium acnes 5) Lactopad as the main ingredient 7) Salicylic acid, 8) Triclosan as a secondary ingredient mixed with a constant concentration at the same time Was prepared.
실시예 6)-9) 항균물질 농축건조물을 함유하는 영양화장수의 제조Example 6) -9) Preparation of Nutrients Longevity Containing Antimicrobial Concentrates
표 5. 실시예 6)-9)에 따른 조성비Table 5. Composition ratios according to Examples 6) -9)
원료 1)~9)를 혼합하고 가열하여 65~70℃까지 온도를 상승시켜 용해시켰다. 별도의 용기에서 원료 10)~11)를 혼합하여 용해시키고 가열하여 65~70℃까지 온도를 상승시키고 앞서 제조한 용액에 서서히 투입하면서 homo-mixer에서 균질화 시켜 에멀젼을 형성시켰다. 이 형성된 에멀젼 용액에 대하여 원료 12)~16)을 순서대로 혼합하여 점증있는 에멀젼을 제조하였다. 이때 일반화장수의 제조와 마찬가지로 여드름 원인균인 Propionibacterium acnes를 포함한 피부 유해세균에 대한 항균작용에 관련한 활성성분으로서 주 성분인 5)Lactopad에 대하여 보조성분으로서 7)Salicylic acid, 8)Triclosan을 단독 또는 동시에 일정한 농도로 혼합하여 영양화장수를 제조하였다.Raw materials 1) -9) were mixed, heated, and dissolved by raising the temperature to 65-70 ° C. In a separate container, raw materials 10) to 11) were mixed, dissolved, heated, and heated to 65-70 ° C., and gradually added to the previously prepared solution, and homogenized in a homo-mixer to form an emulsion. To the formed emulsion solution, raw materials 12) to 16) were mixed in order to prepare an increasing emulsion. At this time, as in the manufacture of normal cosmetics, 7) Salicylic acid, 8) Triclosan as an active ingredient related to antibacterial action against harmful bacteria of the skin including acne-causing bacteria Propionibacterium acnes as the main ingredient, 5) Lactopad The nutrient cosmetics were prepared by mixing with concentration.
이상 실시예 2)~9)에 의하여 만들어진 화장료 조성물들은 각각 실온, 5℃, 37℃, 그리고 45℃에서 한달 이상 보관하면서 제품 중의 층분리 현상을 비교 측정하였는데 모두 안정하여 층분리 등의 현상은 없었으며 항균활성도 그대로 유지되었다. 락토패드(LACTOPAD)를 사용한 화장료 조성물의 제형안정성 및 임상자료는 표6-7에 나타내었다.The cosmetic compositions prepared according to the above Examples 2) to 9) were measured at room temperature, 5 ° C., 37 ° C., and 45 ° C. for at least one month. The antimicrobial activity was also maintained. Formulation stability and clinical data of the cosmetic composition using lactopad (LACTOPAD) are shown in Table 6-7.
표 6. 유산균 배양 농축물을 함유한 화장료 조성물의 제형 안정성Table 6. Formulation Stability of Cosmetic Compositions Containing Lactobacillus Culture Concentrates
표 7. 유산균 배양농축물을 함유한 화장료 조성물의 임상시험Table 7. Clinical Trials of Cosmetic Compositions Containing Lactic Acid Bacteria Culture Concentrates
(성인여성 20명에 대하여 이틀 간격으로 15일간 처리)(20 adult women treated 15 days every two days)
이와같이, 본 발명인 여드름원인균에 대한 항균기능을 갖는 유산균 배양액의 분리농축물 제조방법 및 그 조성물은 여드름 예방 및 치료효과를 가진 기능성 제품의 사용상 효율성을 극대화 한다.As described above, the method for preparing an isolated concentrate of the lactic acid bacteria culture medium having an antibacterial function against the cause of acne of the present invention and its composition maximize the use efficiency of the functional product having the acne prevention and treatment effect.
본 발명인 여드름원인균에 대한 항균기능을 갖는 신규한 유산 균주, 상기 균주의 배양액 분리농축물의 제조방법 및 이를 포함하는 화장료 조성물은 여드름 원인균인 프로피오니박테리움 에크네스(Propionibacterium acnes)에 대하여 특이적 저해활성 기능을 가지고 있어 피부에 서식하는 여드름 원인균 및 유해세균에 대하여 항균작용을 발휘하여 여드름 질환을 치료하고 예방하며, 본 수용성 항균성분의 분리농축물은 화장료 조성에 적합한 물성이므로 유연화장수, 영양화장수, 에센스, 크림, 로션, 메이크업, 색조 화장품 등의 모든 화장품류로 제조될 수 있어 사용자의 사용상 효율성을 극대화 하는 매우 훌륭한 발명이다.The novel lactic acid strain having an antibacterial function against the causative agent of the present invention, a method for producing a culture concentrate isolated from the strain, and a cosmetic composition comprising the same have specific inhibitory activity against the acne causing bacterium Propionibacterium acnes. It has the function to treat and prevent acne disease by exerting antibacterial action against acne causing bacteria and harmful bacteria that inhabit the skin, and the separated concentrate of this water-soluble antimicrobial ingredient is suitable for cosmetic composition, so it is a flexible cosmetic, nutrient cosmetic, essence It can be manufactured with all cosmetics such as creams, lotions, makeup, color cosmetics, etc. It is a very excellent invention that maximizes the efficiency of user use.
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