WO2018030748A1 - Method for separating collagen from liposuction effluent using supercritical process - Google Patents

Method for separating collagen from liposuction effluent using supercritical process Download PDF

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WO2018030748A1
WO2018030748A1 PCT/KR2017/008543 KR2017008543W WO2018030748A1 WO 2018030748 A1 WO2018030748 A1 WO 2018030748A1 KR 2017008543 W KR2017008543 W KR 2017008543W WO 2018030748 A1 WO2018030748 A1 WO 2018030748A1
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collagen
extraction
gas
supercritical fluid
supercritical
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PCT/KR2017/008543
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French (fr)
Korean (ko)
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신용우
김형순
박윤국
최용수
김규병
유성식
한갑수
노성래
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주식회사 도프
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Priority to US16/322,894 priority Critical patent/US11655270B2/en
Priority claimed from KR1020170100339A external-priority patent/KR101954905B1/en
Publication of WO2018030748A1 publication Critical patent/WO2018030748A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

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  • the present invention relates to a method for extracting collagen, and more particularly, to a method for extracting collagen from a liposuction effluent, characterized in that the collagen is extracted by treating the liposuction effluent containing collagen in the presence of a supercritical fluid. .
  • Collagen is the major protein that makes up the connective tissue in the body, making up a very large portion of the body composition proteins 25 to 35%.
  • the collagen composition by body part shows that the dentin is 18%, the dermis under the skin epidermis, 70% of the articular cartilage, 80% of the organic matter of the bone, 80% of the tendon that connects the bone and muscle, and the cornea and the conjunctiva In the main ingredient.
  • Collagen found in animals is mostly type 1 collagen, consisting of 300kDa monomer, and has a covalent bond at a specific site. As such, collagen found in mature tissues has low solubility.
  • the amino acids constituting collagen are glutamic acid, hydroxyproline, glycine, proline and alanine, and among them, the content of hydroxyproline, which is specifically present only in collagen, is high.
  • Collagen loses its ability to synthesize in the body as it ages, and collagen production begins to drop rapidly by about 18 years of age, and is known to be less than half of those at age 40. As you age, your metabolism slows down, and if your old collagen doesn't break down and continues to accumulate, you won't have enough materials to synthesize collagen.
  • Korean Patent Registration No. 10-892605 proposes a method for preparing an extract for seasoning having high collagen content using chicken feet and a technique of using the same for seasoning.
  • a technique has been proposed for producing a seasoning extract having high collagen content by bleaching and sterilizing chicken feet with a chlorine disinfectant, extracting a protein containing collagen with hot water or protease, and then filtering, separating and concentrating.
  • Korean Patent Publication No. 2012-120571 the squid endothelium is separated and collected, washed in alkaline electrolytic water, sterilized and disinfected in acidic electrolyzed water to dry and pulverized squid endothelium at a water content of 10-20%, and then the enzyme is purified. It describes the preparation of collagen peptides which are hydrolyzed using, extract collagen peptides, and concentrate and spray dry. A method of extracting a collagen composition has been proposed in which a non-collagen protein is removed, neutralized and washed with water, followed by hydrolysis by addition of water and neutrase enzymes, followed by secondary hydrolysis with a flavozyme enzyme, followed by filtration and drying.
  • Korean Patent No. 10-1105603 a method of treating collagen with high purity through acid dissolution, pepsin treatment, salt precipitation, filtration, secondary acid dissolution, secondary salt precipitation, phase separation and concentration, acid dissolution, etc. in animal tissues Is proposed.
  • the tissues of the animal are softened with hydrochloric acid solution, then using pepsin enzyme and put into phosphate solution to separate collagen from the tissue and precipitated with sodium chloride to recover the collagen isolated, and then dissolved and diluted in phosphate solution.
  • salt precipitation is used to separate collagen, water is removed, collagen precipitate is pressurized, concentrated, redissolved in phosphate solution, and neutralized with sodium hydroxide solution.
  • collagen is isolated through pepsin treatment and fractionation by salt precipitation and carboxymethylation of collagen contained in the placenta and umbilical cord through a four-step process of pepsin again.
  • Purification by chromatography has been reported (GLANVILLE, RW et al., Eur J Biochem, 95: 383, 1979).
  • the collagen extraction method is a typical extraction method using acid and base treatment and enzyme treatment, and the process is complicated and the purity is not good.
  • Supercritical Fluid Extraction is a method of separating a substance using a supercritical fluid having an intermediate characteristic of a gas and a liquid present at a critical point, that is, above a critical temperature and a critical pressure.
  • the supercritical fluid extraction technology is based on the principle of solvent extraction and the solute molecules contained in the raw material are low density from the high density condensed phase by the solubility difference between the raw material and the supercritical fluid. It is a combination of the distillation principle, which is an evaporation phenomenon that moves to the supercritical fluid, which is an expanded phase.
  • the supercritical fluid extraction technology as described above separates various substances, including isomers, which are difficult to separate by conventional methods, separation of thermodenatured mixtures, purification of high molecular materials, separation of active ingredients such as medicines and perfumes from natural plants. Made it possible.
  • the supercritical fluid extraction technology is advantageous in that it is harmless to the human body, has a low cost of solvent, and does not require an additional concentration process. Therefore, the supercritical fluid extraction technology has been applied to the pharmaceutical industry, food industry, cosmetics and perfume industry, chemical industry, energy industry and various other fields.
  • Carbon dioxide is generally used as a supercritical fluid. This is because carbon dioxide has a low critical temperature (31 °C) and critical pressure (7.29MPa), which can be easily adjusted to supercritical conditions, and is widely present in nature, colorless, odorless, harmless to humans, and chemically stable. to be.
  • adipose tissue is the largest part of our body, and about one-third to two-thirds of the adipose tissue is fat-containing fat cells and the rest are blood cells, vascular endothelial cells and fat cells progenitor cells ( preadipocyte). It is involved in energy storage and metabolism of the body and exists around the skeleton, nerves, and cardiovascular system to act as a buffer for external shocks and to prevent adhesions outside the protective film of important organs. In addition, it has a very rich micro-vascular network in the tissue and reacts sensitively to the delivery materials, such as hormones and growth factors, and is very important as an auxiliary role of the skeleton to maintain the delicate contour and shape of our body.
  • the fat organization is a huge organ rather than an organization.
  • mesenchymal stem cells in the adipose tissue has been actively studied to induce differentiation from adipose tissue to various mesodermal tissue.
  • Adipose tissue can be obtained by liposuction, which is recently performed for cosmetic surgery in obese patients. Liposuction has been performed safely and easily since the past 30 years. In general, liposuction in clinical practice can yield hundreds to thousands of liposuction effluents at once. In the past, these tissues have just been abandoned, but recently they are used in the clinic for autologous fat transplantation and also by stem cell researchers to obtain stem cells.
  • liposuction effluents contain not only stem cells but also collagen that binds adipose tissue and connects cells, but the extraction method has not been established and is being discarded as it is.
  • the present inventors have made intensive efforts to develop a method of extracting human-derived collagen with high yield and high purity.
  • the human body-derived collagen has a high yield. It confirmed that it could obtain, and completed this invention.
  • the present invention provides a method for extracting collagen from the liposuction effluent characterized in that the collagen is extracted by treating the liposuction effluent containing collagen in the presence of a supercritical fluid.
  • Figure 1 illustrates a process for extracting collagen using supercritical carbon dioxide from a liposuction effluent.
  • Figure 2 shows the results confirmed by SDS-PAGE collagen isolated according to the present invention.
  • FIG. 3 shows a process of post-treatment and concentration of collagen isolated according to the present invention.
  • Figure 4 shows the result of confirming the collagen band by SDS-PAGE after the concentration of the separated collagen.
  • Figure 5 shows the result confirmed by the Western blot analysis of the concentrated collagen sample.
  • collagen was extracted using carbon dioxide as a supercritical fluid.
  • the present invention relates to a method for extracting collagen from a liposuction effluent, wherein the liposuction effluent containing collagen is treated in the presence of a supercritical fluid to extract collagen.
  • the 'supercritical fluid' refers to a fluid in a gaseous state or a critical temperature and a critical pressure or more under general conditions.
  • the supercritical fluid suitable for use in the present invention is not particularly limited, but the supercritical fluid is carbon dioxide gas, ammonia gas, nitrogen gas, nitrogen monoxide (NO) gas, nitrogen dioxide (NO 2 ) gas, nitrous oxide (N 2).
  • a fluid may be used, and preferably carbon dioxide having a critical temperature of 31 ° C. and a critical pressure of 72.8 atm.
  • the extraction method of the present invention comprises the steps of: (a) filling a supercritical fluid extractor with a liposuction effluent containing collagen; (b) extracting the collagen-containing extract by introducing a supercritical fluid into the extractor through a heat exchanger; And (c) extracting collagen from the liposuction effluent comprising separating the supercritical fluid and the collagen-containing extract mixture under reduced pressure in a pressure sensitive separator.
  • the temperature of the extraction step is 10 ⁇ 50 °C
  • the pressure is preferably used 100 ⁇ 500bar conditions.
  • the supercritical fluid separated in step (c) may further include the step of circulating the reservoir and recirculating under reduced pressure with the supercritical fluid supplemented from the outside, and collecting the separated extract. have.
  • the 'co-solvent' refers to a solvent other than the supercritical fluid added during supercritical fluid extraction.
  • the cosolvent may be further added with a cosolvent selected from the group consisting of ethanol, methanol, acetone, hexane, acetylacetate and methylene chloride.
  • the supercritical fluid extraction temperature is 10 ⁇ 50 °C
  • the pressure may be characterized in that the pressure condition 100 ⁇ 500bar.
  • the cosolvent may be used in an amount of 1 to 500% (w / w) based on the extracted raw material.
  • the co-solvent is used in an amount of 50 to 200% (w / w) based on the extract raw material.
  • the manner in which the co-solvent is administered to the extraction tank is not particularly limited, and the supercritical fluid and the co-solvent in the upward direction from the bottom of the extraction tank to extract the extract to the top of the extraction tank or vice versa supercritical
  • Both top-down extraction methods may be used in which the fluid and the cosolvent are administered from the top of the extraction tank to the downward direction so that the extract flows out to the bottom of the extraction tank.
  • the extraction pressure is preferably 72.8 to 500 atm and the extraction temperature is 31 to 100 ° C. More preferably, extraction pressure is 100-400 atmospheres, and extraction temperature is 35-65 degreeC.
  • the present invention comprises the steps of: (a) injecting liposuction effluent into the supercritical extraction device, and supplying the extraction solvent CO 2 to the extraction reactor; (b) removing lipids by extraction by the pressure of the CO 2 fed to the extraction reactor with 100 ⁇ 300bar, 10 ⁇ 50 °C the temperature, via the discharge port is cooler installed; And (c) adjusting the pressure to atmospheric pressure and separating the reactor to obtain collagen.
  • the method relates to extracting collagen from a liposuction effluent using supercritical fluid extraction.
  • a solvent selected from the group consisting of ethanol, hexane, methanol, acetone, hexane, ethyl acetate and methylene chloride may be further supplied as a cosolvent (co-solvent).
  • the lipid is extracted in the oil phase, and the extracted lipid is a lipid derived from a lipid droplet of adipose tissue contained in the liposuction effluent and includes triacylglycerol, sterol ester, and the like.
  • the liposuction effluent is filled in the extractor 1, and supercritical carbon dioxide heated appropriately for extraction through the heat exchanger 9 Supply to the bottom of the extractor 1.
  • the supercritical carbon dioxide supplied in this way is contacted with the packed liposuction effluent to extract collagen, then rises and is released out of the extractor.
  • the mixture of extracted supercritical carbon dioxide and high-purity collagen is decompressed through the pressure reducing valve 2 and reduced to Transferred.
  • Extracted collagen and carbon dioxide are separated in the decompressor 3, the separated carbon dioxide is liquefied through the heat exchanger 4 circulated to the storage tank 5 reused, the collagen separated in the decompressor 3 is collected as a final product.
  • the carbon dioxide storage tank 6 can be supplemented with carbon dioxide from the outside to compensate for some losses incurred in the entire process in addition to the carbon dioxide supplied in circulation.
  • the carbon dioxide stored in the reservoir 5 is pressurized by the pump 7 and supplied to the extractor again through the heat exchanger 9 in a supercritical state.
  • the cosolvent is supplied through the cosolvent inlet 8 as needed.
  • This process can proceed continuously until the targeted collagen extraction yield is reached from the inhaled fat.
  • two or more extractors 1 are installed to control a plurality of supply valves and a plurality of discharge valves alternately. In an extractor not used, the extracted raw material is removed and a new liposuction effluent is removed. Fill and prepare for next extraction.
  • the high content and high purity collagen extraction method using the supercritical fluid according to the present invention basically fills the liposuction effluent into the extractor, and inputs the supercritical fluid to the extractor filled with the liposuction effluent to extract collagen.
  • a mixture of the extracted supercritical fluid and collagen is separated under reduced pressure, and the separated fluid is pressurized by a pump to recycle.
  • the supercritical fluid may be used in various ways, it is most preferred to use carbon dioxide, the pressure is 100 ⁇ 500bar, the temperature is preferably maintained at 10 ⁇ 50 °C, more preferably the temperature is 20 ⁇ It is preferable to maintain the pressure at 50 ° C and 200 to 400 bar.
  • Collagen extracted by the present invention has the advantage that can be extracted in a polymer state as compared to the collagen extracted by a conventional method.
  • the collagen band of the polymer was confirmed by electrophoresis of the collagen isolated by supercritical extraction (FIGS. 2 and 4), and it was confirmed that it was type I collagen by Western blot analysis (FIG. 5).
  • the supercritical fluid extract prepared according to the method of the present invention may be prepared in the form of cosmetics such as lotion, essence and pack according to a known method.
  • the lotion is generally based on purified water, ethanol and humectants. To this is added an acid, alkali or astringent depending on the purpose.
  • Essence refers to a high concentration of a cosmetic ingredient having a skin moisturizing light aging inhibitory effect.
  • the main components of the essence are skin softeners, moisturizers, solvents, emulsifiers and thickeners.
  • the pack is used for the purpose of providing proper tension to the skin and blocking the air from the outside to facilitate the absorption of nutrients.
  • the main components of the pack are film forming agents, humectants, emulsifiers and solvents.
  • the supercritical fluid extract prepared according to the method of the present invention may be prepared in a gel or solid cosmetic form according to a known method.
  • the gel composition can be prepared by further mixing the thickener with the solution composition described above.
  • the composition in solid form consists of emollients, thickeners, emulsifiers and solvents.
  • the separated oily lipids begin to be extracted at the outlet where the chiller is installed.
  • adjust the valve on the outlet to raise the pressure to 300 bar and hold for 15 minutes. If additional oil is removed from the outlet, wait 30 minutes for the oil to be completely removed.
  • the pressure was slowly adjusted to atmospheric pressure, the reactor was separated, and the remaining tissue was extracted to check for the presence of collagen.
  • the agitator shear auxiliary tank was filled with 10 ml of 99.9% EtOH per 100 ml of fat, oil was extracted in the same process as above, and collagen was separated.
  • the collagen separated by supercritical extraction was electrophoresed using SDS-PAGE. As shown in FIG. 2, the collagen band was confirmed.
  • Protein concentration of the entire stock solution before concentration was measured by the Bradford method, collagen was not measured by protein quantification by the Bradford method.
  • the protein concentration of the total stock solution was 0.239 (mg / ml), the protein concentration of the supernatant was 0.179 mg / ml, and no protein was detected in the precipitate (Table 1).
  • the present invention it is possible to extract the collagen of the liposuction effluent, which is conventionally discarded, in high molecular state, and the high purity collagen of the extracted polymer may be widely used in medicine, pharmaceuticals, and cosmetics.
  • the present invention it is possible to extract the collagen of the liposuction effluent, which is conventionally discarded, in high molecular state, and the high purity collagen of the extracted polymer may be widely used in medicine, pharmaceuticals, and cosmetics.

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Abstract

The present invention relates to a method for extracting collagen from liposuction effluent, wherein collagen is extracted by treating collagen-containing liposuction effluent in the presence of a supercritical fluid. According to the present invention, conventionally discarded collagen in liposuction effluent can be extracted at high purity, and the extracted high purity collagen can be widely used in medical, pharmaceutical, and cosmetic products.

Description

지방흡입 유출물로부터 초임계공정을 이용하여 콜라겐을 분리하는 방법 Separation of collagen from liposuction effluent using supercritical process
본 발명은 콜라겐의 추출방법에 관한 것으로, 더욱 자세하게는 콜라겐을 함유하는 지방흡입 유출물을 초임계 유체 존재 하에 처리하여 콜라겐을 추출하는 것을 특징으로 하는 지방흡입 유출물로부터 콜라겐의 추출방법에 관한 것이다.The present invention relates to a method for extracting collagen, and more particularly, to a method for extracting collagen from a liposuction effluent, characterized in that the collagen is extracted by treating the liposuction effluent containing collagen in the presence of a supercritical fluid. .
콜라겐(collagen)은 몸속에서 결합조직을 이루는 주요한 단백질로, 신체구성 단백질 중 25 ~ 35 %의 매우 많은 부분을 차지한다. 인체 부분별 콜라겐 구성비를 보면, 이의 상아질 18%, 피부표피 아래 진피의 70%, 관절 연골의 50%, 뼈의 유기물 중 80%, 뼈와 근육을 이어주는 힘줄의 80% 그리고, 눈의 각막과 결막에서 주성분을 이루고 있다. 동물에서 발견되는 콜라겐은 대부분 제 1형 콜라겐으로, 300kDa의 단량체로 이루어져 있으며, 특정부위에 공유결합을 가지고 있다. 그렇기 때문에 성숙한 조직에서 발견되는 콜라겐은 낮은 용해성을 가진다. 또한 콜라겐을 구성하는 아미노산은 글루탐산, 하이드록시프롤린, 글리신, 프롤린 및 알라닌 등이며, 그 중 콜라겐에만 특이적으로 존재하는 하이드록시프롤린의 함량이 높은 것이 특징이다.Collagen (collagen) is the major protein that makes up the connective tissue in the body, making up a very large portion of the body composition proteins 25 to 35%. The collagen composition by body part shows that the dentin is 18%, the dermis under the skin epidermis, 70% of the articular cartilage, 80% of the organic matter of the bone, 80% of the tendon that connects the bone and muscle, and the cornea and the conjunctiva In the main ingredient. Collagen found in animals is mostly type 1 collagen, consisting of 300kDa monomer, and has a covalent bond at a specific site. As such, collagen found in mature tissues has low solubility. In addition, the amino acids constituting collagen are glutamic acid, hydroxyproline, glycine, proline and alanine, and among them, the content of hydroxyproline, which is specifically present only in collagen, is high.
콜라겐은 나이가 들수록 체내에서 합성하는 능력을 잃게 되는데, 18세 정도가 되면 콜라겐 생성 속도는 급속히 떨어지기 시작하여 40세에는 18세에 비해 절반 이하가 되는 것으로 알려져 있다. 또한 나이가 들게 되면 신진대사가 둔해져, 오래된 콜라겐이 분해되지 않고 계속 축적되면 콜라겐을 합성하는 재료도 부족해짐으로써 노화가 촉진된다.Collagen loses its ability to synthesize in the body as it ages, and collagen production begins to drop rapidly by about 18 years of age, and is known to be less than half of those at age 40. As you age, your metabolism slows down, and if your old collagen doesn't break down and continues to accumulate, you won't have enough materials to synthesize collagen.
이러한 이유로 인해, 식품이나 화장품, 의약 관련 분야 등에서는 콜라겐 개발과 이를 이용한 화장품, 식용 및 생체 소재 개발 등에 대해 지속적으로 연구되고 있다. 지금까지는 소나 돼지에서 얻어진 콜라겐이 주로 이용되어 왔지만, 소에서 발생되는 광우병의 발병 이후로 소나 돼지 이외의 어류나 기타 축산물에서 유래하는 콜라겐이 주목을 받아 화장품이나 건강식품, 의약품 등의 원료로서 이용이 검토되고 있다. 종래에 일반적인 콜라겐 소 돼지와 같은 축산물 외에 오징어 등의 어류나, 녹용, 닭발 등과 같은 동물의 특수부위에서 콜라겐을 추출하는 방법이 알려지고 있다.For this reason, in the fields of food, cosmetics, medicine, etc., research on the development of collagen and the development of cosmetics, edible and biological materials using the same have been continuously conducted. Until now, collagen obtained from cows and pigs has been mainly used, but since the development of mad cow disease in cows, collagen derived from fish and other livestock products other than cows and pigs has attracted attention, and has been used as a raw material for cosmetics, health foods and medicines. It is considered. BACKGROUND ART Conventionally, a method of extracting collagen from special parts of fish such as squid, antler, chicken feet, etc. in addition to livestock products such as general collagen bovine pig is known.
한국특허등록 제10-892605호에서는 닭발을 이용한 콜라겐 함유량이 높은 조미료용 추출물 제조방법과 이를 조미료에 활용하는 기술이 제안되어 있다. 여기서는 닭발을 염소계 소독제로 표백 및 살균하고, 열수 또는 단백질분해효소로 콜라겐을 포함하는 단백질을 추출한 다음, 여과, 분리 및 농축하여 콜라겐 함유량이 높은 조미료용 추출물을 제조하는 기술이 제안되어 있다.Korean Patent Registration No. 10-892605 proposes a method for preparing an extract for seasoning having high collagen content using chicken feet and a technique of using the same for seasoning. Here, a technique has been proposed for producing a seasoning extract having high collagen content by bleaching and sterilizing chicken feet with a chlorine disinfectant, extracting a protein containing collagen with hot water or protease, and then filtering, separating and concentrating.
한국특허공개 제2012-120571호에서는 오징어 내피 부분을 분리 선별 취합하고 알칼리 전해수에 세척한후 산성전해수에 살균 소독하는 전처리 공정을 거친 오징어 내피를 10-20%의 수분율로 건조하고 분쇄한 다음 효소를 이용하여 가수분해하여 콜라겐 펩타이드를 추출하고 농축하여 스프레이 건조하는 콜라겐 펩타이드 제조에 관해 기술하고 있으며, 또 한국특허공개 제2012-134935호에서는 오징어 껍질을 건조 및 분쇄한 오징어 껍질분말에 수산화나트륨 수용액을 가하여 비콜라겐 단백질을 제거하고, 중화 및 수세한 다음 물과 뉴트라제 효소를 첨가하여 가수분해하고, 다시 플라보자임 효소로 2차 가수분해하여 여과, 건조하는 콜라겐 조성물의 추출방법이 제안되어 있다. In Korean Patent Publication No. 2012-120571, the squid endothelium is separated and collected, washed in alkaline electrolytic water, sterilized and disinfected in acidic electrolyzed water to dry and pulverized squid endothelium at a water content of 10-20%, and then the enzyme is purified. It describes the preparation of collagen peptides which are hydrolyzed using, extract collagen peptides, and concentrate and spray dry. A method of extracting a collagen composition has been proposed in which a non-collagen protein is removed, neutralized and washed with water, followed by hydrolysis by addition of water and neutrase enzymes, followed by secondary hydrolysis with a flavozyme enzyme, followed by filtration and drying.
한국특허등록 제10-1105603호에서는 동물 조직에서 산 용해, 펩신처리, 염 침전, 여과, 2차 산 용해, 2차 염 침전, 상 분리 및 농축, 산 용해 등을 거쳐서 콜라겐을 고순도로 처리하는 방법이 제안되어 있다. 여기서는 동물의 조직을 염산용액으로 연화시킨 후 펩신효소를 이용하고 인산용액에 넣어 콜라겐을 조직으로부터 분리해내고 염화나트륨으로 침전시킴으로써 효소분리된 콜라겐을 회수하는 방법과, 이를 다시 인산용액에 용해하고 희석한 후 제균 여과한 다음 염 침전시켜서 콜라겐을 분리하고 수분을 제거한 뒤 콜라겐 침전물을 가압, 농축하고 인산용액에 재용해한 후 수산화나트륨용액으로 중화하여 콜라겐 용액을 제조방법을 제안하고 있다.In Korean Patent No. 10-1105603, a method of treating collagen with high purity through acid dissolution, pepsin treatment, salt precipitation, filtration, secondary acid dissolution, secondary salt precipitation, phase separation and concentration, acid dissolution, etc. in animal tissues Is proposed. Here, the tissues of the animal are softened with hydrochloric acid solution, then using pepsin enzyme and put into phosphate solution to separate collagen from the tissue and precipitated with sodium chloride to recover the collagen isolated, and then dissolved and diluted in phosphate solution. After sterilization and filtration, salt precipitation is used to separate collagen, water is removed, collagen precipitate is pressurized, concentrated, redissolved in phosphate solution, and neutralized with sodium hydroxide solution.
또한, 인체 유래 콜라겐을 수득하는 방법으로, 태반과 탯줄에 함유되어 있는 콜라겐을 펩신 처리 및 염 침전에 의한 분획법 및 카르복시 메틸레이션을 거쳐 다시 펩신을 처리하는 4 단계 과정을 통하여 콜라겐을 분리한 후, 크로마토그래피로 정제하는 방법이 보고되어 있다(GLANVILLE, RW et al., Eur J Biochem, 95:383, 1979).In addition, as a method of obtaining collagen derived from the human body, collagen is isolated through pepsin treatment and fractionation by salt precipitation and carboxymethylation of collagen contained in the placenta and umbilical cord through a four-step process of pepsin again. , Purification by chromatography has been reported (GLANVILLE, RW et al., Eur J Biochem, 95: 383, 1979).
그러나 이러한 콜라겐 추출방법은 산과 염기 처리 및 효소처리 등을 이용하는 전형적인 추출방법으로서 공정이 복잡하고 순도가 좋지 못하였다.However, the collagen extraction method is a typical extraction method using acid and base treatment and enzyme treatment, and the process is complicated and the purity is not good.
이러한 종래의 방법들은 고순도의 콜라겐을 얻기 힘들며, 원료에 있어서 동물유래이어서 인체에 대한 의료용으로 사용하기 부적합한 면이 있었다.These conventional methods are difficult to obtain high-purity collagen, and since the raw materials are animal-derived, they are not suitable for medical use for the human body.
초임계유체 추출기술(Supercritical Fluid Extraction)은 임계점 즉, 임계 온도 및 임계 압력 이상에 존재하는 기체와 액체의 중간 성격을 갖는 초임계유체를 사용하여 물질을 분리하는 방법이다. 상기 초임계유체 추출기술은 추출 원료와 초임계유체의 용해도 차이에 의해 원료 중에 함유된 가용 성분이 초임계유체로 용해되는 용매추출(extraction) 원리와 원료 중에 함유된 용질분자가 고밀도 응축상으로부터 저밀도 팽창상인 초임계유체로 이행하는 증발현상인 증류원리를 복합적으로 이용한 것이다. 상기와 같은 초임계유체 추출기술은 기존의 방법으로는 분리가 어려웠던 이성질체, 열변성 혼합물의 분리, 고분자 물질의 정제, 천연식물로부터 의약, 향료와 같은 유효성분의 분리 등을 비롯한 다양한 물질의 분리를 가능하게 하였다. 또한, 초임계유체 추출기술은 인체에 무해하며 용매의 값이 저렴하고 추가적인 농축공정이 필요하지 않다는 장점이 있다. 따라서, 상기 초임계유체 추출기술은 의약품 공업, 식품공업, 화장품·향료 공업, 화학 공업, 에너지 공업 및 기타 다양한 분야에 응용되고 있다.Supercritical Fluid Extraction is a method of separating a substance using a supercritical fluid having an intermediate characteristic of a gas and a liquid present at a critical point, that is, above a critical temperature and a critical pressure. The supercritical fluid extraction technology is based on the principle of solvent extraction and the solute molecules contained in the raw material are low density from the high density condensed phase by the solubility difference between the raw material and the supercritical fluid. It is a combination of the distillation principle, which is an evaporation phenomenon that moves to the supercritical fluid, which is an expanded phase. The supercritical fluid extraction technology as described above separates various substances, including isomers, which are difficult to separate by conventional methods, separation of thermodenatured mixtures, purification of high molecular materials, separation of active ingredients such as medicines and perfumes from natural plants. Made it possible. In addition, the supercritical fluid extraction technology is advantageous in that it is harmless to the human body, has a low cost of solvent, and does not require an additional concentration process. Therefore, the supercritical fluid extraction technology has been applied to the pharmaceutical industry, food industry, cosmetics and perfume industry, chemical industry, energy industry and various other fields.
초임계유체로는 일반적으로 이산화탄소가 사용되고 있다. 이는 이산화탄소의 경우 낮은 임계 온도(31℃)와 임계 압력(7.29MPa)을 가지고 있어 쉽게 초임계 조건으로 조정이 가능하며 자연계에 널리 존재하고 무색, 무취하며 인체에 무해하고 화학적으로 안정한 장점이 있기 때문이다.Carbon dioxide is generally used as a supercritical fluid. This is because carbon dioxide has a low critical temperature (31 ℃) and critical pressure (7.29MPa), which can be easily adjusted to supercritical conditions, and is widely present in nature, colorless, odorless, harmless to humans, and chemically stable. to be.
한편, 지방조직은 우리 몸에서 가장 많은 부분을 차지하고 있으며, 지방조직의 1/3에서 2/3 정도는 지질을 함유하는 지방세포와 나머지는 혈구세포, 혈관내피세포와 지방세포의 전 구세포(preadipocyte)로 구성되어 있다. 우리 몸의 에너지 저장과 대사에 관여하며 골격이나 신경, 심혈관계 주위에 존재하여 외부의 충격에 완충작용을 하는 역할과, 중요기관의 보호막 밖에서 서로 유착을 막아주는 역할을 담당한다. 또한, 조직 내에 매우 풍부한 미세혈관 망을 가지고 있어 호르몬과 성장인자 등의 전달물질에 민감하게 반응하며, 외모적으로 우리 몸의 섬세한 윤곽과 형상을 유지하는 골격의 보조역할로서 매우 중요하다. 이와 같이 지방조직은 그 역할로 가지는 비중이 조직이기 보다는 하나의 거대한 기관이라 할 수 있다. 또한 최근에는 골수와 마찬가지로 지방조직 내에도 간엽줄기세포(mesenchymal stem cell)들을 가지고 있어 지방조직에서 다양한 중배엽성 조직으로 분화 유도시키는 연구가 활발하다.On the other hand, adipose tissue is the largest part of our body, and about one-third to two-thirds of the adipose tissue is fat-containing fat cells and the rest are blood cells, vascular endothelial cells and fat cells progenitor cells ( preadipocyte). It is involved in energy storage and metabolism of the body and exists around the skeleton, nerves, and cardiovascular system to act as a buffer for external shocks and to prevent adhesions outside the protective film of important organs. In addition, it has a very rich micro-vascular network in the tissue and reacts sensitively to the delivery materials, such as hormones and growth factors, and is very important as an auxiliary role of the skeleton to maintain the delicate contour and shape of our body. As such, the fat organization is a huge organ rather than an organization. In addition, recently, as in the bone marrow has mesenchymal stem cells in the adipose tissue has been actively studied to induce differentiation from adipose tissue to various mesodermal tissue.
지방조직은 최근 비만환자에서 미용성형 목적으로 시행되고 있는 지방흡입술에 의해 얻을 수 있으며 지방흡입술은 과거 30년 전부터 안전하고 손쉬운 시술로 행하여져 왔다. 일반적으로 임상에서 이루어지는 지방흡입술은 수백~수천 ml의 지방흡입 유출물을 한번에 얻을 수 있다. 과거에는 이들 조직들은 그냥 버려져 왔으나 최근에는 자가지방 이식용으로 임상에서 사용되며 또한 지방세포 연구자들에 의해 줄기세포를 얻는 데 활용되고 있다.Adipose tissue can be obtained by liposuction, which is recently performed for cosmetic surgery in obese patients. Liposuction has been performed safely and easily since the past 30 years. In general, liposuction in clinical practice can yield hundreds to thousands of liposuction effluents at once. In the past, these tissues have just been abandoned, but recently they are used in the clinic for autologous fat transplantation and also by stem cell researchers to obtain stem cells.
이러한 지방흡입 유출물에는 줄기세포뿐만아니라 지방조직을 결착하고 세포간을 연결하는 콜라겐이 다량 함유되어 있으나, 이에 대한 추출법이 확립되어 있지 않아, 그대로 버려지고 있는 실정이다. These liposuction effluents contain not only stem cells but also collagen that binds adipose tissue and connects cells, but the extraction method has not been established and is being discarded as it is.
이에, 본 발명자들은 인체유래 콜라겐을 높은 수율과 고순도로 추출하는 방법을 개발하고자 예의 노력한 결과, 인체유래 지방흡입유출물을 이용하여 초임계 추출방법을 사용하는 경우 고순도의 인체 유래 콜라겐을 높은 수율로 얻을 수 있는 것을 확인하고, 본 발명을 완성하게 되었다.Thus, the present inventors have made intensive efforts to develop a method of extracting human-derived collagen with high yield and high purity. When using supercritical extraction method using human-derived liposuction effluent, the human body-derived collagen has a high yield. It confirmed that it could obtain, and completed this invention.
발명의 요약Summary of the Invention
본 발명의 목적은 지방흡입유출물로부터 초임계 추출방법을 이용하여 콜라겐을 추출하는 방법을 제공하는데 있다. It is an object of the present invention to provide a method for extracting collagen from a liposuction effluent using a supercritical extraction method.
상기 목적을 달성하기 위하여, 본 발명은 콜라겐을 함유하는 지방흡입 유출물을 초임계 유체 존재 하에 처리하여 콜라겐을 추출하는 것을 특징으로 하는 지방흡입 유출물로부터 콜라겐의 추출방법을 제공한다.In order to achieve the above object, the present invention provides a method for extracting collagen from the liposuction effluent characterized in that the collagen is extracted by treating the liposuction effluent containing collagen in the presence of a supercritical fluid.
도 1은 지방흡입 유출물로부터 초임계 이산화탄소를 이용하여 콜라겐을 추출하는 공정을 도시한 것이다.Figure 1 illustrates a process for extracting collagen using supercritical carbon dioxide from a liposuction effluent.
도 2는 본 발명에 따라 분리한 콜라겐을 SDS-PAGE로 확인한 결과를 나타낸 것이다.Figure 2 shows the results confirmed by SDS-PAGE collagen isolated according to the present invention.
도 3은 본 발명에 따라 분리한 콜라겐을 후처리하여 농축하는 과정을 나타낸 것이다.3 shows a process of post-treatment and concentration of collagen isolated according to the present invention.
도 4는 분리된 콜라겐을 농축한 후 SDS-PAGE로 콜라겐 밴드를 확인한 결과를 나타낸 것이다.Figure 4 shows the result of confirming the collagen band by SDS-PAGE after the concentration of the separated collagen.
도 5는 농축한 콜라겐 샘플을 웨스턴 블럿 분석을 통하여 확인한 결과를 나타낸 것이다.Figure 5 shows the result confirmed by the Western blot analysis of the concentrated collagen sample.
발명의 상세한 설명 및 바람직한 Detailed description of the invention and preferred 구현예Embodiment
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명에서는 종래 성형외과적인 지방흡입시술 후에 버려지는 지방흡입 유출물에 함유되어 있는 양질의 콜라겐을 고순도로 추출하기 위하여, 이산화탄소를 초임계 유체로 이용하여 콜라겐을 추출하였다.In the present invention, in order to extract high quality collagen contained in the liposuction effluent discarded after the conventional plastic surgery liposuction, collagen was extracted using carbon dioxide as a supercritical fluid.
따라서, 본 발명은 일 관점에서, 콜라겐을 함유하는 지방흡입 유출물을 초임계 유체 존재 하에 처리하여 콜라겐을 추출하는 것을 특징으로 하는 지방흡입 유출물로부터 콜라겐의 추출방법에 관한 것이다.Thus, in one aspect, the present invention relates to a method for extracting collagen from a liposuction effluent, wherein the liposuction effluent containing collagen is treated in the presence of a supercritical fluid to extract collagen.
본 발명에서 '초임계유체'란 일반적인 조건에서는 기체 상태이나 임계 온도와 임계 압력 이상에서는 유체인 것을 말한다. In the present invention, the 'supercritical fluid' refers to a fluid in a gaseous state or a critical temperature and a critical pressure or more under general conditions.
본 발명에서 사용하기에 적합한 초임계유체로는 특별히 한정되지 않으나, 상기 초임계 유체는 이산화탄소 가스, 암모니아 가스, 질소가스, 일산화질소(NO) 가스, 이산화질소(NO2) 가스, 아산화질소(N2O) 가스, 이산화황 가스, 수소가스, 수증기, 메탄, 에틸렌, 프로판, 프로필렌 및 이들이 혼합가스로 구성되는 군, 에탄올 및 메탄올을 포함하는 알코올류, 벤젠 및 톨루엔을 포함하는 방향족 화합물에서 선택되는 초임계 유체를 사용할 수 있으며, 바람직하게는 임계온도가 31℃이고 임계압력이 72.8기압인 이산화탄소를 사용할 수 있다.The supercritical fluid suitable for use in the present invention is not particularly limited, but the supercritical fluid is carbon dioxide gas, ammonia gas, nitrogen gas, nitrogen monoxide (NO) gas, nitrogen dioxide (NO 2 ) gas, nitrous oxide (N 2). O) Supercritical selected from gas, sulfur dioxide gas, hydrogen gas, water vapor, methane, ethylene, propane, propylene and the group consisting of these gases, alcohols including ethanol and methanol, aromatic compounds including benzene and toluene A fluid may be used, and preferably carbon dioxide having a critical temperature of 31 ° C. and a critical pressure of 72.8 atm.
본 발명의 추출방법은 (a) 콜라겐을 함유하는 지방흡입 유출물을 초임계 유체 추출기에 충진하는 단계; (b)열교환기를 통하여 상기 추출기에 초임계 유체를 투입하여 콜라겐 함유 추출물을 추출하는 단계; 및 (c) 감압분리기에서 초임계 유체와 콜라겐 함유 추출물 혼합물을 감압시켜 분리하는 단계를 포함하는 지방흡입 유출물로부터 콜라겐을 추출하는 방법을 포함할 수 있다. The extraction method of the present invention comprises the steps of: (a) filling a supercritical fluid extractor with a liposuction effluent containing collagen; (b) extracting the collagen-containing extract by introducing a supercritical fluid into the extractor through a heat exchanger; And (c) extracting collagen from the liposuction effluent comprising separating the supercritical fluid and the collagen-containing extract mixture under reduced pressure in a pressure sensitive separator.
본 발명에 있어서, 상기 이산화탄소를 초임계 추추물로 사용할 경우, 상기 추출 단계의 온도는 10~50℃이고, 압력은 100~500bar 조건을 사용하는 것이 바람직하다. In the present invention, when using the carbon dioxide as a supercritical weight extract, the temperature of the extraction step is 10 ~ 50 ℃, the pressure is preferably used 100 ~ 500bar conditions.
본 발명의 일 양태에 있어서, (c)단계에서 분리된 초임계 유체는 저장조를 순환한 후 외부에서 보충되는 초임계 유체와 함께 감압하여 재순환시키고, 분리된 추출물을 수거하는 단계를 추가적으로 포함할 수 있다.In one aspect of the present invention, the supercritical fluid separated in step (c) may further include the step of circulating the reservoir and recirculating under reduced pressure with the supercritical fluid supplemented from the outside, and collecting the separated extract. have.
본 발명에서 '보조용매'란 초임계유체 추출시의 첨가되는 초임계유체 이외의 용매를 말한다. In the present invention, the 'co-solvent' refers to a solvent other than the supercritical fluid added during supercritical fluid extraction.
본 발명에 있어서, 보조용매로서 에탄올, 메탄올, 아세톤, 헥산, 에칠아세테이트 및 메틸렌클로라이드로 구성된 군에서 선택되는 보조용매를 추가로 첨가하는 것을 특징으로 할 수 있다. In the present invention, the cosolvent may be further added with a cosolvent selected from the group consisting of ethanol, methanol, acetone, hexane, acetylacetate and methylene chloride.
본 발명에 있어서, 상기 초임계 유체 추출 온도는 10~50℃이고, 압력은 100~500bar 기압 조건인 것을 특징으로 할 수 있다. In the present invention, the supercritical fluid extraction temperature is 10 ~ 50 ℃, the pressure may be characterized in that the pressure condition 100 ~ 500bar.
본 발명에서 보조용매는 추출원료에 대하여 1∼500%(w/w)의 양으로 사용할 수 있다. 바람직하게는 상기 보조용매는 추출원료에 대하여 50∼200%(w/w)의 양으로 사용한다.In the present invention, the cosolvent may be used in an amount of 1 to 500% (w / w) based on the extracted raw material. Preferably, the co-solvent is used in an amount of 50 to 200% (w / w) based on the extract raw material.
또한, 상기 보조용매를 추출조에 투여하는 방식은 특별히 한정되지 않으며, 초임계유체와 보조용매를 추출조의 하부로부터 상부 방향으로 투여하여 추출물이 추출조의 상부로 유출되도록 하는 상향식 추출방법 또는 그 반대로 초임계유체와 보조용매를 추출조의 상부로부터 하부 방향으로 투여하여 추출물이 추출조의 하부로 유출되도록 하는 하향식 추출방법을 모두 사용할 수 있다.In addition, the manner in which the co-solvent is administered to the extraction tank is not particularly limited, and the supercritical fluid and the co-solvent in the upward direction from the bottom of the extraction tank to extract the extract to the top of the extraction tank or vice versa supercritical Both top-down extraction methods may be used in which the fluid and the cosolvent are administered from the top of the extraction tank to the downward direction so that the extract flows out to the bottom of the extraction tank.
본 발명에 따른 초임계유체 추출조건은 추출압력이 72.8∼500기압, 추출온도 31∼100℃인 것이 바람직하다. 보다 바람직하게는 추출압력이 100∼400기압, 추출온도 35∼65℃이다.In the supercritical fluid extraction condition according to the present invention, the extraction pressure is preferably 72.8 to 500 atm and the extraction temperature is 31 to 100 ° C. More preferably, extraction pressure is 100-400 atmospheres, and extraction temperature is 35-65 degreeC.
본 발명은 다른 관점에서, (a) 초임계 추출장치에 지방흡입 유출물을 투입하고, 추출반응기에 추출용매인 CO2를 공급하는 단계; (b) 상기 추출반응기에 공급된 CO2의 압력을 100~300bar, 온도를 10~50℃로 유지하여, 냉각기가 설치된 배출구를 통해 지질을 추출하여 제거하는 단계; 및 (c) 압력을 상압으로 조정하고 반응기를 분리하여 콜라겐을 수득하는 단계를 포함하는 초임계 유체 추출을 이용한 지방흡입 유출물로부터 콜라겐을 추출하는 방법에 관한 것이다. In another aspect, the present invention comprises the steps of: (a) injecting liposuction effluent into the supercritical extraction device, and supplying the extraction solvent CO 2 to the extraction reactor; (b) removing lipids by extraction by the pressure of the CO 2 fed to the extraction reactor with 100 ~ 300bar, 10 ~ 50 ℃ the temperature, via the discharge port is cooler installed; And (c) adjusting the pressure to atmospheric pressure and separating the reactor to obtain collagen. The method relates to extracting collagen from a liposuction effluent using supercritical fluid extraction.
본 발명에 있어서, 공용매(보조용매)로 에탄올, 헥산, 메탄올, 아세톤, 헥산, 에칠아세테이트 및 메틸렌클로라이드로 구성된 군에서 선택되는 용매를 추가로 공급하는 것을 특징으로 할 수 있다.In the present invention, a solvent selected from the group consisting of ethanol, hexane, methanol, acetone, hexane, ethyl acetate and methylene chloride may be further supplied as a cosolvent (co-solvent).
본 발명에서 상기 지질은 오일상으로 추출되며, 상기 추출되는 지질은 지방흡입 유출물에 포함된 지방조직의 지방소적(lipid droplet) 유래의 지질로 트리아실글리세롤, 스테롤에스터 등을 포함한다. In the present invention, the lipid is extracted in the oil phase, and the extracted lipid is a lipid derived from a lipid droplet of adipose tissue contained in the liposuction effluent and includes triacylglycerol, sterol ester, and the like.
발명의 일 실시예에 따른 콜라겐 추출장치를 보다 구체적으로 살펴보면, 도 1에 도시된 바와 같이, 추출기①에 지방흡입 유출물을 충진하고, 열교환기⑨를 통하여 추출에 적합하게 가열된 초임계 이산화탄소를 추출기①의 하단부에 공급한다. 이렇게 공급된 초임계 이산화탄소는 충진된 지방흡입 유출물과 접촉하여 콜라겐을 추출하며 상승한 뒤 추출기 밖으로 방출되는데, 추출된 초임계 이산화탄소와 고순도 콜라겐의 혼합물은 감압밸브②를 경유하며 감압되면서 감압기③로 이송된다. 감압기③에서는 추출된 콜라겐과 이산화탄소가 분리되며, 분리된 이산화탄소는 열교환기④를 통하며 액화된 후 저장조⑤로 순환되어 재사용되며, 감압기③에서 분리된 콜라겐은 최종 제품⑩로 수거된다. 이산화탄소 저장조⑥에는 순환되어 공급되는 이산화탄소 외에 전 공정에서 발생하는 약간의 손실을 보충하도록 외부에서 이산화탄소가 보충될 수 있다. 저장조⑤에 저장된 이산화탄소는 펌프⑦를 통하여 가압되어 초임계 상태로 열교환기⑨를 통하여 다시 추출기에 공급된다. 보조용매는 필요에 따라 보조용매주입구⑧을 통하여 공급된다.Referring to the collagen extracting apparatus according to an embodiment of the present invention in more detail, as shown in Figure 1, the liposuction effluent is filled in the extractor ①, and supercritical carbon dioxide heated appropriately for extraction through the heat exchanger ⑨ Supply to the bottom of the extractor ①. The supercritical carbon dioxide supplied in this way is contacted with the packed liposuction effluent to extract collagen, then rises and is released out of the extractor. The mixture of extracted supercritical carbon dioxide and high-purity collagen is decompressed through the pressure reducing valve ② and reduced to Transferred. Extracted collagen and carbon dioxide are separated in the decompressor ③, the separated carbon dioxide is liquefied through the heat exchanger ④ circulated to the storage tank ⑤ reused, the collagen separated in the decompressor ③ is collected as a final product. The carbon dioxide storage tank ⑥ can be supplemented with carbon dioxide from the outside to compensate for some losses incurred in the entire process in addition to the carbon dioxide supplied in circulation. The carbon dioxide stored in the reservoir ⑤ is pressurized by the pump ⑦ and supplied to the extractor again through the heat exchanger ⑨ in a supercritical state. The cosolvent is supplied through the cosolvent inlet ⑧ as needed.
이러한 과정은 흡입된 지방으로부터 목표로 정한 콜라겐 추출 수율에 이를 때까지 연속적으로 진행될 수 있다. 또한 연속운전을 위하여, 추출기①은 2개 이상을 설치하여 다수의 공급밸브와 다수의 배출밸브를 조절하여 교대로 사용하는데, 사용하지 않는 추출기에서는 추출이 끝난 원료를 제거하고 새로운 지방흡입 유출물을 충진하여 다음번의 추출에 대비한다.This process can proceed continuously until the targeted collagen extraction yield is reached from the inhaled fat. In addition, for continuous operation, two or more extractors ① are installed to control a plurality of supply valves and a plurality of discharge valves alternately. In an extractor not used, the extracted raw material is removed and a new liposuction effluent is removed. Fill and prepare for next extraction.
이와 관련하여, 본 발명에 따른 초임계유체를 이용하는 콜라겐 추출방법에 대해 설명한다.In this regard, the collagen extraction method using a supercritical fluid according to the present invention will be described.
본 발명에 따른 초임계 유체를 이용하는 고함량, 고순도 콜라겐 추출방법은 기본적으로 지방흡입 유출물을 추출기에 충진하고, 지방흡입 유출물이 충진된 추출기에 초임계 유체를 투입하여 콜라겐을 추출하고, 이렇게 추출된 초임계 유체와 콜라겐의 혼합물을 감압시켜 분리하며, 분리된 유체를 펌프에 의해 가압하여 재순환시키는 공정으로 구성된다.The high content and high purity collagen extraction method using the supercritical fluid according to the present invention basically fills the liposuction effluent into the extractor, and inputs the supercritical fluid to the extractor filled with the liposuction effluent to extract collagen. A mixture of the extracted supercritical fluid and collagen is separated under reduced pressure, and the separated fluid is pressurized by a pump to recycle.
이때, 콜라겐의 추출효율을 향상시키기 위하여 지방흡입 유출물을 미세입자로 파쇄하는 전처리 공정이 더 추가되는 것이 바람직하다. 또한, 상기 초임계 유체는 여러 가지가 사용될 수 있지만, 이산화탄소를 사용하는 것이 가장 바람직하며, 압력은 100~500bar, 온도는 10~50℃로 유지하는 것이 바람직하며, 더욱 바람직하게는 온도는 20~50℃, 압력은 200~400 bar로 유지하는 것이 바람직하다.At this time, in order to improve the extraction efficiency of collagen, it is preferable that a pretreatment step of crushing the liposuction effluent into fine particles is further added. In addition, the supercritical fluid may be used in various ways, it is most preferred to use carbon dioxide, the pressure is 100 ~ 500bar, the temperature is preferably maintained at 10 ~ 50 ℃, more preferably the temperature is 20 ~ It is preferable to maintain the pressure at 50 ° C and 200 to 400 bar.
본 발명에 의해 추출된 콜라겐은 종래의 방법으로 추출된 콜라겐에 비하여 고분자 상태로 추출이 가능한 장점이 있다.Collagen extracted by the present invention has the advantage that can be extracted in a polymer state as compared to the collagen extracted by a conventional method.
본 발명의 다른 양태에서는 초임계추출에 의하여 분리된 콜라겐을 전기영동한 결과 고분자의 콜라겐 밴드를 확인하였으며(도 2 및 도 4), 웨스턴 블럿 분석을 통하여 타입 I 콜라겐임을 확인하였다(도 5). In another embodiment of the present invention, the collagen band of the polymer was confirmed by electrophoresis of the collagen isolated by supercritical extraction (FIGS. 2 and 4), and it was confirmed that it was type I collagen by Western blot analysis (FIG. 5).
또한, 본 발명의 방법에 따라 제조된 초임계유체 추출물은 공지의 방법에 따라 화장수, 에센스 및 팩과 같은 형태의 화장품으로 제조될 수 있다. 화장수는 일반적으로 정제수, 에탄올 및 보습제를 기본으로 한다. 여기에 목적에 따라 산, 알칼리 또는 수렴제를 첨가한다. 에센스는 피부 보습 빛 노화억제 효과를 갖는 화장료 성분을 고농축으로 함유한 것을 말한다. 에센스의 주요 구성성분은 피부유연제, 보습제, 용매, 유화제 및 점증제이다. 팩은 피부에 적절한 긴장감을 주고 외부로부터 공기를 차단하여 영양성분의 흡수를 용이하게 하는 목적으로 사용된다. 상기 팩의 주요 성분으로는 피막형성제, 보습제,유화제 및 용매이다.In addition, the supercritical fluid extract prepared according to the method of the present invention may be prepared in the form of cosmetics such as lotion, essence and pack according to a known method. The lotion is generally based on purified water, ethanol and humectants. To this is added an acid, alkali or astringent depending on the purpose. Essence refers to a high concentration of a cosmetic ingredient having a skin moisturizing light aging inhibitory effect. The main components of the essence are skin softeners, moisturizers, solvents, emulsifiers and thickeners. The pack is used for the purpose of providing proper tension to the skin and blocking the air from the outside to facilitate the absorption of nutrients. The main components of the pack are film forming agents, humectants, emulsifiers and solvents.
마지막으로 본 발명의 방법에 따라 제조된 초임계유체 추출물은 공지의 방법에 따라 겔 또는 고체형태의 화장품으로 제조될 수 있다. 겔 조성물은 상술한 용액 조성물에 농후제를 추가로 혼합함으로써 제조할 수 있다. 고체 형태의 조성물은 피부연화제, 농후제, 유화제 및 용매로 구성되어 있다.Finally, the supercritical fluid extract prepared according to the method of the present invention may be prepared in a gel or solid cosmetic form according to a known method. The gel composition can be prepared by further mixing the thickener with the solution composition described above. The composition in solid form consists of emollients, thickeners, emulsifiers and solvents.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
실시예Example
실시예 1: 지방흡입 유출물로부터 초임계 유체를 이용한 콜라겐 분리Example 1 Collagen Separation Using Supercritical Fluid from Liposuction Effluent
도 1과 같이 구성한 초임계 유체 추출장치의 추출기에 상온 상압에서 지방흡입샘플 100 ml을 투입하였다. 초임계유체인 이산화타소는 고압펌프를 이용하여 60 bar, 및 -40C에서 주입하고 배출구의 밸브를 닫아 교반기의 압력을 서서히 상승시켰다. 교반기 내부에 온도센서를 설치하고 내부온도가 31℃의 초임계 조건이 되도록 유지시켰다. 내부압력이 250bar가 되면 배출구의 밸브를 열고 동시에 부스터를 작동하여 고압의 이산화탄소를 주입하며 내부압력이 250bar로 유지시켰다. 100 ml of the liposuction sample was added to the extractor of the supercritical fluid extracting apparatus configured as shown in FIG. Titanium dioxide, a supercritical fluid, was injected at 60 bar and -40C using a high pressure pump and the valve of the outlet was closed to gradually increase the pressure of the stirrer. A temperature sensor was installed inside the stirrer and maintained at a supercritical condition of 31 ° C. When the internal pressure reached 250bar, the outlet valve was opened and the booster was operated at the same time to inject high pressure carbon dioxide and maintain the internal pressure at 250bar.
이 경우, 냉각기(chiller)가 설치된 배출구에 분리된 오일상의 지질이 추출되기 시작한다. 오일의 분출이 낮아지면 배출구의 밸브를 조정하여 압력을 300 바까지 상승하여 15분간 유지한다. 배출구에서 오일이 추가로 제거되면 오일이 완전히 제거될 때까지 30분간 대기한다.In this case, the separated oily lipids begin to be extracted at the outlet where the chiller is installed. When the oil flows down, adjust the valve on the outlet to raise the pressure to 300 bar and hold for 15 minutes. If additional oil is removed from the outlet, wait 30 minutes for the oil to be completely removed.
압력을 서서히 상압으로 조정하고 반응기를 분리하여 잔존하는 조직을 추출하여 콜라겐의 유무를 확인하였다.The pressure was slowly adjusted to atmospheric pressure, the reactor was separated, and the remaining tissue was extracted to check for the presence of collagen.
공용매(cosolvent)를 사용하는 경우 교반기 전단 보조탱크에 지방 100 ml당 10 ml의 99.9% EtOH를 채우고 위와 동일한 공정으로 오일을 추출한 후 콜라겐을 분리하였다.In the case of using a cosolvent, the agitator shear auxiliary tank was filled with 10 ml of 99.9% EtOH per 100 ml of fat, oil was extracted in the same process as above, and collagen was separated.
실시예 2: 전기영동을 통한 초임계 추출된 콜라겐의 확인Example 2: Identification of Supercritical Extracted Collagen by Electrophoresis
초임계 추출에 의해 분리된 콜라겐을 SDS-PAGE를 이용하여 전기영동한 결과, 도 2에 나타난 바와 같이 콜라겐 밴드를 확인할 수 있었다.The collagen separated by supercritical extraction was electrophoresed using SDS-PAGE. As shown in FIG. 2, the collagen band was confirmed.
초임계 추출 원액은 원심분리하여, 상등액과 침전물로 각각 분리하고, 한외여과장치를 이용하여 탈염 및 농축하여 20배로 농축하였다(도 3). Supercritical extraction stock solution was centrifuged, separated into supernatant and precipitate, respectively, and concentrated 20 times by desalting and concentrating using an ultrafiltration apparatus (FIG. 3).
농축 전 전체 원액에 대한 단백질 농도를 브래드포드 방법으로 측정하였으며, 브래드포드 법에 의한 단백질 정량으로는 콜라겐이 측정되지 않는다. 전체 원액의 단백질 농도는 0.239(mg/ml)이었으며, 상등액의 단백질 농도는 0.179 mg/ml이었고, 침전물에서는 단백질이 검출되지 않았다(표 1).Protein concentration of the entire stock solution before concentration was measured by the Bradford method, collagen was not measured by protein quantification by the Bradford method. The protein concentration of the total stock solution was 0.239 (mg / ml), the protein concentration of the supernatant was 0.179 mg / ml, and no protein was detected in the precipitate (Table 1).
단백질 농도 (mg/ml)Protein concentration (mg / ml)
양성대조군 (positive collagen)Positive collagen 00
원액 전체Whole liquid 0.2390.239
상등액 (supernatant)Supernatant 0.1790.179
침전물 (precipitate)Precipitate 00
농축 후의 상등액과 침전물의 콜라겐 유무를 확인하기 위하여, SDS-PAGE를 수행하였으며, 그 결과, 도 4에 나타난 바와 같이, 농축 전 및 농축 후의 상등액과 침전물에서 콜라겐이 확인되었다.In order to confirm the presence or absence of collagen of the supernatant and the precipitate after concentration, SDS-PAGE was performed. As a result, collagen was confirmed in the supernatant and the precipitate before and after concentration.
실시예 3: 웨스턴 블럿 분석을 통한 초임계 추출된 콜라겐의 확인Example 3: Identification of Supercritical Extracted Collagen by Western Blot Analysis
실시예 2에서 농축된 콜라겐 추출물 상등액 및 침전물에 대하여 웨스턴 블럿 분석을 수행하여, 타입 I 콜라겐의 유무를 확인하였다. Western blot analysis was performed on the concentrated collagen extract supernatant and precipitate in Example 2 to confirm the presence or absence of type I collagen.
20배 농축한 상등액 및 침전물을 8% SDS-PAGE 젤에 전기영동한 후, 콜라겐 밴드를 니트로셀룰로오스 막으로 이동시킨 후, 타입 1 콜라겐 단클론항체(Abcam, Cambridge, UK)를 처리하여 웨스턴 블럿을 수행하였다. 20-fold concentrated supernatants and precipitates were electrophoresed on 8% SDS-PAGE gels, and then the collagen bands were transferred to nitrocellulose membranes followed by Western blot treatment with type 1 collagen monoclonal antibodies (Abcam, Cambridge, UK). It was.
그 결과, 도 5에 나타난 바와 같이, 20배 침전물에서 뚜렷한 타입 1 콜라겐 단백질 밴드를 확인할 수 있었다.As a result, as shown in Figure 5, it was possible to confirm the distinct type 1 collagen protein band in 20-fold precipitate.
본 발명에 따르면, 종래 버려지는 지방흡입 유출물의 콜라겐을 고분자 상태로 고순도로 추출할 수 있으며, 추출된 고분자의 고순도 콜라겐은 의료, 제약 및 화장품 등에 널리 사용될 수 있다.According to the present invention, it is possible to extract the collagen of the liposuction effluent, which is conventionally discarded, in high molecular state, and the high purity collagen of the extracted polymer may be widely used in medicine, pharmaceuticals, and cosmetics.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따르면, 종래 버려지는 지방흡입 유출물의 콜라겐을 고분자 상태로 고순도로 추출할 수 있으며, 추출된 고분자의 고순도 콜라겐은 의료, 제약 및 화장품 등에 널리 사용될 수 있다.According to the present invention, it is possible to extract the collagen of the liposuction effluent, which is conventionally discarded, in high molecular state, and the high purity collagen of the extracted polymer may be widely used in medicine, pharmaceuticals, and cosmetics.
부호의 설명Explanation of the sign
① 추출기 ② 감압 조절기① Extractor ② Pressure reducer
③ CO2 - 콜라겐 분리기 ④ 냉각기(chiller)③ CO 2 -collagen separator ④ chiller
⑤ 액화CO2 저장조 ⑥ CO2 보충 공급조⑤ Liquefied CO 2 reservoir ⑥ CO 2 replenishment supply tank
⑦ CO2 순환 펌프 ⑧ 보조용매 주입구⑦ CO 2 circulation pump ⑧ Co-solvent inlet
⑨ 열교환기 ⑩ 고순도 콜라겐 함유 추출물⑨ Heat exchanger 추출물 Extract containing high purity collagen
⑪ CO2 vent ⑫ 응축수 제거⑪ CO 2 vent 제거 Remove condensate

Claims (7)

  1. 콜라겐을 함유하는 지방흡입 유출물을 초임계 유체 존재하에 처리하여 콜라겐을 추출하는 것을 특징으로 하는 지방흡입 유출물로부터 콜라겐의 추출방법.A method of extracting collagen from a liposuction effluent, wherein the liposuction effluent containing collagen is treated in the presence of a supercritical fluid to extract collagen.
  2. 제1항에 있어서, 상기 초임계 유체는 이산화탄소 가스, 암모니아 가스, 질소가스,일산화질소(NO) 가스, 이산화질소(NO2) 가스, 아산화질소(N2O) 가스, 이산화황 가스, 수소가스, 수증기, 포화탄화수소, 불포화탄화수소, 방향족 화합물 및 이들이 혼합가스로 구성되는 군에서 선택되는 것을 특징으로 하는 방법.The method of claim 1, wherein the supercritical fluid is carbon dioxide gas, ammonia gas, nitrogen gas, nitrogen monoxide (NO) gas, nitrogen dioxide (NO 2 ) gas, nitrous oxide (N 2 O) gas, sulfur dioxide gas, hydrogen gas, water vapor , Saturated hydrocarbons, unsaturated hydrocarbons, aromatic compounds and the method characterized in that they are selected from the group consisting of mixed gas.
  3. 제2항에 있어서, 상기 초임계유체는 이산화탄소 가스인 것을 특징으로 하는 방법.The method of claim 2, wherein the supercritical fluid is carbon dioxide gas.
  4. 제3항에 있어서, 상기 초임계 추출의 온도 및 압력은 -10~200℃ 및 0~1000 bar인 것을 특징으로 하는 방법.The method of claim 3, wherein the temperature and pressure of the supercritical extraction are -10 to 200 ° C. and 0 to 1000 bar.
  5. 제1항에 있어서, 공용매로서 에탄올, 메탄올, 아세톤, 헥산, 에칠아세테이트 및 메틸렌클로라이드로 구성된 군에서 선택되는 용매를 추가로 첨가하는 것을 특징으로 하는 방법.The method according to claim 1, wherein a solvent selected from the group consisting of ethanol, methanol, acetone, hexane, ethyl acetate and methylene chloride is further added as a cosolvent.
  6. 다음 단계를 포함하는 초임계 유체 추출을 이용한 지방흡입 유출물로부터 콜라겐을 추출하는 방법:A method for extracting collagen from a liposuction effluent using supercritical fluid extraction comprising the following steps:
    (a) 초임계 추출장치에 지방흡입 유출물을 투입하고, 추출장치에 추출용매인 CO2를 공급하는 단계;(a) injecting a liposuction effluent into a supercritical extraction device and supplying CO 2 as an extraction solvent to the extraction device;
    (b) 상기 추출장치에 공급된 CO2의 압력 및 온도를 100~500bar 및 10~50℃로 유지하여, 지질을 추출하고, 냉각기가 설치된 배출구를 통해 추출된 지질을 제거하는 단계; 및(b) maintaining the pressure and temperature of the CO 2 supplied to the extraction apparatus at 100 to 500 bar and 10 to 50 ° C., extracting lipids, and removing the extracted lipids through an outlet provided with a cooler; And
    (c) 압력을 상압으로 조정하고 추출장치를 분리하여 콜라겐을 수득하는 단계.(c) adjusting the pressure to atmospheric pressure and separating the extractor to obtain collagen.
  7. 제6항에 있어서, 공용매로 에탄올, 헥산, 메탄올, 아세톤, 헥산, 에칠아세테이트 및 메틸렌클로라이드로 구성된 군에서 선택되는 용매를 추가로 공급하는 것을 특징으로 하는 방법.The method of claim 6, further comprising supplying a solvent selected from the group consisting of ethanol, hexane, methanol, acetone, hexane, ethyl acetate and methylene chloride as a cosolvent.
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2021057936A1 (en) * 2019-09-26 2021-04-01 上海复璐帝流体技术有限公司 Supercritical carbon dioxide extraction apparatus and method thereof

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