WO2018030748A1 - Procédé de séparation de collagène d'un effluent de liposuccion à l'aide d'un procédé supercritique - Google Patents

Procédé de séparation de collagène d'un effluent de liposuccion à l'aide d'un procédé supercritique Download PDF

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Publication number
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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WIPO (PCT)
Prior art keywords
collagen
extraction
gas
supercritical fluid
supercritical
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PCT/KR2017/008543
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English (en)
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/ko
Publication of WO2018030748A1 publication Critical patent/WO2018030748A1/fr

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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

Definitions

  • 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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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Cosmetics (AREA)

Abstract

La présente invention concerne un procédé d'extraction de collagène à partir d'un effluent de liposuccion, le collagène étant extrait par traitement d'un effluent de liposuccion contenant du collagène en présence d'un fluide supercritique. Selon la présente invention, le collagène rejeté de manière classique dans un effluent de liposuccion peut être extrait à une pureté élevée, et le collagène de haute pureté extrait peut être largement utilisé dans des produits médicaux, pharmaceutiques et cosmétiques.
PCT/KR2017/008543 2016-08-08 2017-08-08 Procédé de séparation de collagène d'un effluent de liposuccion à l'aide d'un procédé supercritique WO2018030748A1 (fr)

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Application Number Priority Date Filing Date Title
US16/322,894 US11655270B2 (en) 2016-08-08 2017-08-08 Method for separating collagen from liposuction effluent using supercritical process

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20160100641 2016-08-08
KR10-2016-0100641 2016-08-08
KR1020170100339A KR101954905B1 (ko) 2016-08-08 2017-08-08 지방흡입 유출물로부터 초임계공정을 이용하여 콜라겐을 분리하는 방법
KR10-2017-0100339 2017-08-08

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021057936A1 (fr) * 2019-09-26 2021-04-01 上海复璐帝流体技术有限公司 Appareil d'extraction au dioxyde de carbone supercritique et procédé associé

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030072677A1 (en) * 2001-10-17 2003-04-17 Ralph Kafesjian Supercritical fluid extraction process for tissue preparation
US20030162707A1 (en) * 2001-12-20 2003-08-28 Fraser John K. Systems and methods for treating patients with collagen-rich material extracted from adipose tissue
KR20110040977A (ko) * 2008-08-07 2011-04-20 우데 하이 프레셔 테크놀로지 게엠베하 세포내 유용물질의 선택적 추출 분리를 위해 분무와 감압의 공정조합에 의한 식물 또는 동물 출발물질의 세포용해
US20130079494A1 (en) * 2011-09-22 2013-03-28 Tyco Healthcare Group Lp Processing Tissue Utilizing Supercritical Fluid
US20160159884A1 (en) * 2011-06-28 2016-06-09 Veris Medical, Inc. System and Method for Collagen Isolation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030072677A1 (en) * 2001-10-17 2003-04-17 Ralph Kafesjian Supercritical fluid extraction process for tissue preparation
US20030162707A1 (en) * 2001-12-20 2003-08-28 Fraser John K. Systems and methods for treating patients with collagen-rich material extracted from adipose tissue
KR20110040977A (ko) * 2008-08-07 2011-04-20 우데 하이 프레셔 테크놀로지 게엠베하 세포내 유용물질의 선택적 추출 분리를 위해 분무와 감압의 공정조합에 의한 식물 또는 동물 출발물질의 세포용해
US20160159884A1 (en) * 2011-06-28 2016-06-09 Veris Medical, Inc. System and Method for Collagen Isolation
US20130079494A1 (en) * 2011-09-22 2013-03-28 Tyco Healthcare Group Lp Processing Tissue Utilizing Supercritical Fluid

Non-Patent Citations (1)

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Title
WANG, J. K. ET AL.: "Supercritical Carbon Dioxide Extracted Extracellular Matrix Material from Adipose Tissue", MATERIAL SCIENCE AND ENGINEERING C, vol. 75, 5 February 2017 (2017-02-05), pages 349 - 358, XP029977984 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021057936A1 (fr) * 2019-09-26 2021-04-01 上海复璐帝流体技术有限公司 Appareil d'extraction au dioxyde de carbone supercritique et procédé associé

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