KR102548168B1 - Method for producing hyaluronic acid oligomer containing ultra-low molecular weight hyaluronic acid - Google Patents
Method for producing hyaluronic acid oligomer containing ultra-low molecular weight hyaluronic acid Download PDFInfo
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 106
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 4
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- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 3
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- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 description 2
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- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
본 발명은 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법에 관한 것이다. 보다 상세하게는, 히알루론산을 에탄올에 분산 시킨 후 산 처리를 수행하는 것이므로, 히알루론산에 강산이 직접 처리되지 않아 히알루론산의 절대평균분자량을 효과적으로 낮출 수 있고, 히알루론산의 변색을 포함하는 품질 저하가 되지 않으며, 피부 투과율이 높아 생체 이용률을 향상시키고, 비침습형 진피층 필러 및 화장품 원료로 활용성이 높은 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법에 관한 것이다.The present invention relates to a method for preparing a hyaluronic acid oligomer comprising ultra-low molecular weight hyaluronic acid. More specifically, since acid treatment is performed after dispersing hyaluronic acid in ethanol, strong acid is not directly treated with hyaluronic acid, so that the absolute average molecular weight of hyaluronic acid can be effectively lowered, and quality degradation including discoloration of hyaluronic acid It does not, and the bioavailability is improved due to high skin permeability, and it relates to a method for producing hyaluronic acid oligomers containing ultra-low-molecular-weight hyaluronic acid that is highly usable as a non-invasive dermal layer filler and cosmetic raw material.
Description
본 발명은 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법에 관한 것이다. 보다 상세하게는, 히알루론산을 에탄올에 분산 시킨 후 산 처리를 수행하는 것이므로, 히알루론산에 강산이 직접 처리되지 않아 히알루론산의 절대평균분자량을 효과적으로 낮출 수 있고, 히알루론산의 변색을 포함하는 품질 저하가 되지 않으며, 피부 투과율이 높아 생체 이용률을 향상시키고, 비침습형 진피층 필러 및 화장품 원료로 활용성이 높은 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법에 관한 것이다.The present invention relates to a method for preparing a hyaluronic acid oligomer comprising ultra-low molecular weight hyaluronic acid. More specifically, since acid treatment is performed after dispersing hyaluronic acid in ethanol, strong acid is not directly treated with hyaluronic acid, so that the absolute average molecular weight of hyaluronic acid can be effectively lowered, and quality degradation including discoloration of hyaluronic acid It does not, and the bioavailability is improved due to high skin permeability, and it relates to a method for producing hyaluronic acid oligomers containing ultra-low-molecular-weight hyaluronic acid that is highly usable as a non-invasive dermal layer filler and cosmetic raw material.
도 1을 참조하면, 히알루론산(Hyaluronic acid)은 분자량이 500,000 내지 13,000,000Da에 이르는 무색의 고점도 다당류로서, 반복단위인 글루쿠론산과 N-아세틸 글루코오스아민이 (1-3)과 (1-4)로 번갈아 결합되어 있다. Referring to Figure 1, hyaluronic acid (Hyaluronic acid) is a colorless high-viscosity polysaccharide ranging from 500,000 to 13,000,000 Da in molecular weight, repeating units glucuronic acid and N-acetyl glucosamine are (1-3) and (1-4) ) are alternately combined.
히알루론산은 제조법, 정제방법에 따라 0.1×106~10×106 Da로 매우 다양하다. 수용액상에서 점성, 탄성 및 보습성을 갖으며 이러한 성질은 분자량과 농도에 밀접한 관계가 있다. 히알루론산의 분자량 저분자화 방법은 산 염기 촉매에 의한 방법, 고온가열, 초음파를 이용한 방법, 효소를 이용한 방법 등이 있으나 가장 작은 분자량의 범위가 5000이상으로 피부투과율이 15% 정도에 지나지 않는다. 또한 강산을 이용한 가수분해 과정에서 갈변이나 부산물이 생성되는 단점이 있다.Hyaluronic acid varies greatly from 0.1 × 10 6 to 10 × 10 6 Da depending on manufacturing and purification methods. In aqueous solution, it has viscosity, elasticity and moisture retention, and these properties are closely related to molecular weight and concentration. Methods for reducing the molecular weight of hyaluronic acid include a method using an acid-base catalyst, a method using high temperature heating, ultrasonic waves, and a method using enzymes. In addition, there is a disadvantage in that browning or by-products are generated in the hydrolysis process using strong acid.
따라서, 인체 피부에서의 히알루론산의 양은 노화와 함께 감소되는 것으로 보고되어 있으므로 초저분자량(평균분자량 1000Da이하)의 히알루론산을 제조함으로써 피부의 탄력 저하를 방지하고 수분 함유량을 유지시켜 줄 수 있으며 세포의 활발한 이동 및 증식을 나타낼 수 있다.Therefore, it is reported that the amount of hyaluronic acid in human skin decreases with aging, so by producing hyaluronic acid with an ultra-low molecular weight (average molecular weight of 1000 Da or less), it is possible to prevent deterioration of skin elasticity and maintain moisture content, May exhibit active migration and proliferation.
히알루론산은 보습효과를 가지며, 그 보습력은 히알루론산의 농도가 높을수록 증가하게 된다. 또한, 히알루론산은 물리적 마찰에 대한 윤활효과가 우수하며 진피층 수복에 도움을 줄 수도 있어 화장품 원료, 안면미용용 필러원료, 의료용 세포외기질 원료 등 다양한 미용의료산업 분야에 활용이 가능하다.Hyaluronic acid has a moisturizing effect, and the moisturizing power increases as the concentration of hyaluronic acid increases. In addition, hyaluronic acid has an excellent lubrication effect against physical friction and can help restore the dermal layer, so it can be used in various beauty and medical industries such as cosmetic raw materials, facial cosmetic filler raw materials, and medical extracellular matrix raw materials.
또한, 히알루론산은 안면미용 산업 중 진피층 필러(Dermal filler) 산업의 히알루론산 필러(HA filler)의 메인 원료로 사용되고 있으며 히알루론산의 진피층 투과 및 생체 이용률 상승을 목적하기 위해선 평균분자량의 저감화가 필수적이다.In addition, hyaluronic acid is used as the main raw material for HA filler in the dermal filler industry in the facial beauty industry, and it is essential to reduce the average molecular weight for the purpose of penetrating the dermal layer of hyaluronic acid and increasing bioavailability. .
피부 침투성이 우수하고, 세포증식효과를 극대화하며, 피부탄력을 개선 및 유지 시킬 수 있는 평균분자량 1,000Da 이하로 분해된 초 저분자 히알루론산을 포함하는 항노화 소재의 개발이 시급한 실정이다.There is an urgent need to develop an anti-aging material containing ultra-low-molecular hyaluronic acid decomposed to an average molecular weight of 1,000 Da or less, which has excellent skin permeability, maximizes cell proliferation, and improves and maintains skin elasticity.
선행기술문헌 : KR 공개특허공보 제10-2013-0078829호(2013.7.10 공개)Prior art literature: KR Patent Publication No. 10-2013-0078829 (published on July 10, 2013)
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로, 피부 침투성이 우수하고, 세포증식효과를 극대화하며, 피부탄력을 개선 및 유지 시킬 수 있는 평균분자량 1,000Da 이하로 분해된 초 저분자 히알루론산을 포함하는 항노화 소재를 화장품 소재로 적극적으로 활용할 수 있도록 하는 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법을 제공하는 데 그 목적이 있다.The present invention has been devised to solve the above problems, and provides ultra-low-molecular hyaluronic acid decomposed to an average molecular weight of 1,000 Da or less, which has excellent skin permeability, maximizes cell proliferation, and improves and maintains skin elasticity. The purpose is to provide a method for preparing a hyaluronic acid oligomer containing ultra-low molecular weight hyaluronic acid so that the containing anti-aging material can be actively used as a cosmetic material.
상기한 목적을 달성하기 위하여 본 발명에 따르는 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법은 반응조에 히알루론산 분말을 에틸알콜에 분산 시킨 후, 정제수을 투입하여 용해시키고, 팽윤화를 수행하여 겔화시키는 팽윤화단계; 팽윤화 단계에서 팽윤화가 완료된 히알루론산 용액에 인산(H3PO4)을 첨가하고, 혼합하여 균질화하며, 알콜분해반응을 이용한 탈아세틸화 반응을 수행하는 탈아세틸화 반응단계; 탈아세틸화 반응단계 후, 인산(H3PO4)을 추가 첨가하고, 승온시켜 잔류 알콜을 휘발시키며, 가수분해 반응을 수행하는 가수분해 반응단계; 가수분해 반응단계 후, 탄산칼슘(CaCO3)을 첨가하여 중화반응을 통해 가수분해를 종료시키고, 반응조 냉각 쟈킷에 냉수를 흘려 냉각시킴으로써 인산칼슘, 인산이수소칼슘(Ca(H2PO4)2·nH2O)을 포함하는 중화염류의 재결정화를 유도하는 중화 반응단계; 마이크로 필터를 이용하여 1차 여과하여 재결정화된 중화염류를 제거하고, 역삼투 필터를 이용하여 2차 여과하여 용액 내 이온화된 중화염류를 제거하는 중화염류 제거단계; 중화염류 제거단계를 수행한 가수분해 여과물을 질소 가스 기반으로 건식 가스 분무를 이용하여 건조하여 분말화하는 건식 가스분무 건조단계; 건식 가스분무 건조단계를 수행하여 응집된 분말 덩어리를 분쇄기로 미분쇄하여 미세 분말상으로 재가공하는 분쇄단계; 및 분쇄단계를 수행한 분쇄물의 입도 분포 표준화를 위해 특정 사이즈의 메쉬망을 이용하여 채걸음하는 입자표준화단계를 포함할 수 있다.In order to achieve the above object, the method for preparing a hyaluronic acid oligomer containing ultra-low-molecular-weight hyaluronic acid according to the present invention is to disperse hyaluronic acid powder in ethyl alcohol in a reaction tank, dissolve by adding purified water, and perform swelling to gel. swelling step; A deacetylation reaction step of adding phosphoric acid (H 3 PO 4 ) to the hyaluronic acid solution of which swelling has been completed in the swelling step, homogenizing by mixing, and performing a deacetylation reaction using an alcohol decomposition reaction; After the deacetylation reaction step, a hydrolysis reaction step in which phosphoric acid (H 3 PO 4 ) is additionally added, the temperature is raised to volatilize residual alcohol, and a hydrolysis reaction is performed; After the hydrolysis reaction step, calcium carbonate (CaCO 3 ) is added to terminate the hydrolysis through a neutralization reaction, and cold water is poured into the cooling jacket of the reaction tank to cool it, thereby producing calcium phosphate and dihydrogen phosphate (Ca(H 2 PO 4 ) 2 Neutralization reaction step of inducing recrystallization of neutral salts including nH 2 O); A neutral salt removal step of removing recrystallized neutral salts by primary filtration using a micro filter and secondary filtration using a reverse osmosis filter to remove ionized neutral salts in the solution; Dry gas spray drying step of drying and pulverizing the hydrolyzed filtrate after performing the neutral salt removal step using dry gas spray based on nitrogen gas; A pulverization step of performing a dry gas spray drying step to finely pulverize the agglomerated powder mass with a pulverizer and reprocessing it into fine powder; and a particle standardization step of sifting using a mesh network of a specific size in order to standardize the particle size distribution of the pulverized material after the pulverization step.
또한, 탈아세틸화 반응단계는 히알루론산 용액에 첨가하는 인산(H3PO4)은 히알루론산 원료 분말 질량 대비 0.1배수 이상 2배수 이하로 첨가하는 것을 포함할 수 있다.In addition, the deacetylation reaction step may include adding phosphoric acid (H 3 PO 4 ) added to the hyaluronic acid solution in an amount of 0.1 times or more and 2 times or less based on the mass of the hyaluronic acid raw material powder.
또한, 가수분해 반응단계는 탈아세틸화 반응이 종료된 히알루론산 용액에 첨가하는 인산(H3PO4)은 히알루론산 원료 분말 질량 대비 1배수 이상 2배수 이하로 첨가하는 것을 포함할 수 있다.In addition, the hydrolysis reaction step may include adding phosphoric acid (H 3 PO 4 ) added to the hyaluronic acid solution after the deacetylation reaction is 1-fold or more and 2-fold or less based on the mass of the hyaluronic acid raw material powder.
본 발명에 의하면 히알루론산을 에탄올에 분산 시킨 후 산 처리를 수행하는 것이므로, 히알루론산에 강산이 직접 처리되지 않아 히알루론산의 절대평균분자량을 효과적으로 낮출 수 있고, 히알루론산의 변색을 포함하는 품질 저하가 되지 않으며, 피부 투과율이 높아 생체 이용률을 향상시키고, 비침습형 진피층 필러 및 화장품 원료로 활용성이 높은 데 그 효과가 있다. According to the present invention, since acid treatment is performed after dispersing hyaluronic acid in ethanol, strong acid is not directly treated with hyaluronic acid, so that the absolute average molecular weight of hyaluronic acid can be effectively lowered, and quality degradation including discoloration of hyaluronic acid is prevented. It is effective in improving bioavailability due to high skin permeability and high usability as a non-invasive dermal layer filler and cosmetic raw material.
도 1은 히알루론산 분자 구조를 나타낸 도면,
도 2는 본 발명의 바람직한 실시예에 따른 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법의 순서도,
도 3은 본 발명의 바람직한 실시예에 따른 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법의 제조 공정도, 및
도 4는 본 발명의 바람직한 실시예에 따른 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법의 건식 가스를 기반으로 하는 분무 건조를 이용한 히알루론산 분말을 제조하는 공정을 나타낸 제조공정도이다.1 is a view showing the molecular structure of hyaluronic acid;
2 is a flowchart of a method for preparing a hyaluronic acid oligomer including ultra-low molecular weight hyaluronic acid according to a preferred embodiment of the present invention;
3 is a manufacturing process diagram of a hyaluronic acid oligomer manufacturing method including ultra-low molecular weight hyaluronic acid according to a preferred embodiment of the present invention, and
4 is a manufacturing process diagram showing a process for manufacturing hyaluronic acid powder using spray drying based on dry gas in a method for preparing hyaluronic acid oligomers containing ultra-low molecular weight hyaluronic acid according to a preferred embodiment of the present invention.
이하, 본 발명의 바람직한 실시예를 첨부된 도면들을 참조하여 상세히 설명한다. 우선 각 도면의 구성 요소들에 참조 부호를 부가함에 있어서, 동일한 구성 요소들에 대해서는 비록 다른 도면상에 표시되더라도 가능한 한 동일한 부호를 가지도록 하고 있음에 유의해야 한다. 또한, 본 발명을 설명함에 있어, 관련된 공지 구성 또는 기능에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명은 생략한다. 또한, 이하에서 본 발명의 바람직한 실시예를 설명할 것이나, 본 발명의 기술적 사상은 이에 한정하거나 제한되지 않고 당업자에 의해 변형되어 다양하게 실시될 수 있음은 물론이다.Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. First, in adding reference numerals to components of each drawing, it should be noted that the same components have the same numerals as much as possible even if they are displayed on different drawings. In addition, in describing the present invention, if it is determined that a detailed description of a related known configuration or function may obscure the gist of the present invention, the detailed description will be omitted. In addition, although preferred embodiments of the present invention will be described below, the technical idea of the present invention is not limited or limited thereto and can be modified and implemented in various ways by those skilled in the art.
도 1은 히알루론산 분자 구조를 나타낸 도면이고, 도 2는 본 발명의 바람직한 실시예에 따른 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법(S100)의 순서도이며, 도 3은 본 발명의 바람직한 실시예에 따른 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법(S100)의 제조 공정도이고, 도 4는 본 발명의 바람직한 실시예에 따른 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법(S100)의 건식 가스를 기반으로 하는 분무 건조를 이용한 히알루론산 분말을 제조하는 공정을 나타낸 제조공정도이다.1 is a diagram showing the molecular structure of hyaluronic acid, FIG. 2 is a flowchart of a method for preparing a hyaluronic acid oligomer (S100) including ultra-low molecular weight hyaluronic acid according to a preferred embodiment of the present invention, and FIG. 3 is a preferred embodiment of the present invention. It is a manufacturing process diagram of the hyaluronic acid oligomer manufacturing method (S100) containing ultra-low-molecular-weight hyaluronic acid according to the example, and FIG. It is a manufacturing process diagram showing a process for manufacturing hyaluronic acid powder using dry gas-based spray drying.
본 발명은 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법(S100)에 관한 것이다. 보다 상세하게는, 히알루론산을 에탄올에 분산 시킨 후 산 처리를 수행하는 것이므로, 히알루론산에 강산이 직접 처리되지 않아 히알루론산의 절대평균분자량을 효과적으로 낮출 수 있고, 히알루론산의 변색을 포함하는 품질 저하가 되지 않으며, 피부 투과율이 높아 생체 이용률을 향상시키고, 비침습형 진피층 필러 및 화장품 원료로 활용성이 높은 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법(S100)에 관한 것이다.The present invention relates to a method for preparing a hyaluronic acid oligomer containing ultra-low molecular weight hyaluronic acid (S100). More specifically, since acid treatment is performed after dispersing hyaluronic acid in ethanol, strong acid is not directly treated with hyaluronic acid, so that the absolute average molecular weight of hyaluronic acid can be effectively lowered, and quality degradation including discoloration of hyaluronic acid It does not become, and the bioavailability is improved due to high skin permeability, and it relates to a method for producing hyaluronic acid oligomers (S100) including ultra-low-molecular-weight hyaluronic acid that is highly usable as a non-invasive dermal layer filler and cosmetic raw material.
도 2 내지 도 4를 참조하면, 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법(S100)은 팽윤화단계(S10), 탈아세틸화 반응단계(S20), 가수분해 반응단계(S30), 중화 반응단계(S40), 중화염류 제거단계(S50), 분쇄단계(S70), 및 입자표준화단계(S80)를 포함하여 이루어진다.Referring to FIGS. 2 to 4, the method for preparing a hyaluronic acid oligomer containing ultra-low molecular weight hyaluronic acid (S100) includes a swelling step (S10), a deacetylation reaction step (S20), a hydrolysis reaction step (S30), neutralization It comprises a reaction step (S40), a neutralized salt removal step (S50), a grinding step (S70), and a particle standardization step (S80).
이하, 팽윤화단계(S10)부터 설명하기로 한다.Hereinafter, the swelling step (S10) will be described.
팽윤화단계(S10)는 반응조에 평균분자량 8000Da 이상 1,300,000Da 이하의 히알루론산 분말을 에틸알콜에 분산 시킨 후, 정제수을 투입하여 용해시키고, 10분 이상 1시간 이하의 시간 동안 팽윤화를 수행하여 겔화시킨다.In the swelling step (S10), hyaluronic acid powder having an average molecular weight of 8000 Da or more and 1,300,000 Da or less is dispersed in ethyl alcohol in a reaction tank, purified water is added to dissolve, and swelling is performed for 10 minutes or more and 1 hour or less to gel. .
또한, 팽윤화단계(S10)에서 원료로 사용되는 히알루론산의 평균 분자량은 8,000Da 미만일 경우, 원료 히알루론산의 가격이 급격히 상승하는 구간의 평균 분자량 범위 구간의 원료로써 공정 경제성이 나오지 않고, 1,300,000Da 초과일 경우, 용액의 점도가 높아져 5% 농도 이상의 용액은 교반, 이송 등의 가공공정의 운전이 용이 하지 않기에 8000Da 이상 1,300,000Da 이하의 히알루론산 분말을 사용함이 바람직하다.In addition, when the average molecular weight of hyaluronic acid used as a raw material in the swelling step (S10) is less than 8,000 Da, process economics do not come out as a raw material in the average molecular weight range section of the section where the price of hyaluronic acid as a raw material rises rapidly, and 1,300,000 Da If it exceeds, the viscosity of the solution increases, and it is not easy to operate the processing process such as stirring and transfer for solutions with a concentration of 5% or more.
또한, 팽윤화단계(S10)에서 사용되는 에틸알콜의 첨가 비율은 히알루론산 원료 분말 질량 대비 2배수 이상 5배수 이하로 첨가함이 바람직한데, 이는 2배수 미만일 경우, 히알루론산 분말의 분산이 용이하지 않아 히알루론산의 뭉침현상 제거가 완벽히 수행되지 않으며 이로 인한 겔화 속도가 지연되고 이후 탈아세틸화 반응에서의 알콜 소비량을 충족 못하는 문제가 있고, 5배수 초과일 경우, 히알루론산 분말의 분산은 용이하나 수화반응에 의한 겔화작용은 억제되는 문제점이 발생하며, 이후 단위 공정인 가수분해 반응 도입부에서의 알콜 제거 시간이 다량 소비되어 공정 운전시간이 늘어나기에 에틸알콜을 상기 범위로 첨가함이 바람직하다.In addition, the addition ratio of ethyl alcohol used in the swelling step (S10) is preferably 2 times or more and 5 times or less compared to the mass of the hyaluronic acid raw material powder. Therefore, the agglomeration of hyaluronic acid is not completely removed, and the gelation rate is delayed due to this, and there is a problem that the alcohol consumption in the subsequent deacetylation reaction cannot be met. It is preferable to add ethyl alcohol within the above range because the gelling effect caused by the reaction is inhibited, and since a large amount of alcohol removal time is consumed at the introduction of the hydrolysis reaction, which is a unit process, the process operation time is increased.
또한, 팽윤화단계(S10)에서 팽윤화를 수행하는 시간은 10분 이상 1시간 이하로 진행함이 바람직한데, 이는, 10분 미만일 경우, 팽윤화 시간이 짧아 히알루론산의 수화반응에 의한 겔화가 충분히 일어나지 않고, 1시간 초과일 경우, 유의적 차이가 없기에 상기 시간동안 팽윤화를 진행하는 것이 바람직하다.In addition, the swelling time in the swelling step (S10) is preferably 10 minutes or more and 1 hour or less, which is less than 10 minutes, the swelling time is short and gelation by the hydration reaction of hyaluronic acid If it does not occur sufficiently and exceeds 1 hour, there is no significant difference, so it is preferable to proceed with swelling during the above time.
탈아세틸화 반응단계(S20)는 팽윤화 단계에서 팽윤화가 완료된 히알루론산 용액에 인산(H3PO4)을 첨가하고, 혼합하여 균질화하며, 수소이온농도를 pH 1.3 이상 pH 1.5 이하로 조정하고, 60℃ 이상 90℃ 이하에서 알콜분해반응을 이용한 탈아세틸화 반응을 10분 이상 1시간 이하의 시간동안 수행한다.In the deacetylation reaction step (S20), phosphoric acid (H 3 PO 4 ) is added to the swollen hyaluronic acid solution in the swelling step, mixed and homogenized, and the hydrogen ion concentration is adjusted to pH 1.3 or more and pH 1.5 or less, The deacetylation reaction using the alcohol decomposition reaction is performed at 60° C. or more and 90° C. or less for a time of 10 minutes or more and 1 hour or less.
또한, 탈아세틸화 반응단계(S20)에서 탈아세틸화 반응은 히알루론산 구성분자인 N-아세틸글루코사민의 작용기인 아세틸기를 에틸기로 교환반응하여 탈아세틸화 반응을 수행하는 반응 구간으로써, 이후 가수분해에서 생산되는 초저분자 히알루론산의 생체내 생분해 속도를 지연 시키는 효과를 기대할 수 있다. In addition, in the deacetylation reaction step (S20), the deacetylation reaction is a reaction section in which an acetyl group, a functional group of N-acetylglucosamine, a component of hyaluronic acid, is exchanged for an ethyl group to perform a deacetylation reaction. The effect of delaying the biodegradation rate of the produced ultra-low-molecular-weight hyaluronic acid in vivo can be expected.
또한, 탈아세틸화 반응단계(S20)는 히알루론산 용액에 첨가하는 인산(H3PO4)은 히알루론산 원료 분말 질량 대비 0.1배수 이상 2배수 이하로 첨가하는 것이 바람직한데, 이는, 인산(H3PO4) 첨가 질량비가 히알루론산 원료 분말 질량 대비 0.1배수 미만일 경우, 탈아세틸화 반응에 요구되는 인산(H3PO4) 반응 몰수 양이 부족하여 탈아세틸화 반응의 수행이 원활하지 못하고, 히알루론산 원료 분말 질량 대비 2배수 초과일 경우, 탈아세틸화 반응에 요구되는 인산(H3PO4) 반응 몰수 양을 초과하는 범위로써 탈아세틸화 반응에 있어서 에틸아민과 초산 생성 반응인 정반응 뿐 아니라 생성된 에틸아민과 초산이 재반응하여 아세틸기 생성 반응인 역반응도 동반 수행되므로 반응 효율이 불량한 문제점이 있기에 인산(H3PO4)을 상기 범위 내에서 첨가하도록 한다.In addition, in the deacetylation reaction step (S20), phosphoric acid (H 3 PO 4 ) added to the hyaluronic acid solution is preferably added in an amount of 0.1 times or more and 2 times or less based on the mass of the hyaluronic acid raw material powder, which is, phosphoric acid (H 3 When the mass ratio of PO 4 ) is less than 0.1 times the mass of the hyaluronic acid raw material powder, the amount of moles of phosphoric acid (H 3 PO 4 ) required for the deacetylation reaction is insufficient, making the deacetylation reaction difficult to perform. If it is more than twice the mass of the raw material powder, it exceeds the amount of moles of phosphoric acid (H 3 PO 4 ) required for the deacetylation reaction, and in the deacetylation reaction, it is not only the forward reaction that generates ethylamine and acetic acid, but also the generated Phosphoric acid (H 3 PO 4 ) is added within the above range because ethylamine and acetic acid re-react and the reverse reaction, which is an acetyl group generation reaction, is also performed, resulting in poor reaction efficiency.
또한, 탈아세틸화 반응단계(S20)에서 반응온도를 60℃ 이상 90℃ 이하에서 수행하는 것은, 60℃ 미만인 경우, 반응에 요구되는 온도구간에 미치지 못하여 반응이 수행되지않고, 90℃ 초과인 경우, 용매인 물과 에탄올의 공비 구간을 초과하는 온도 구간으로써 에탄올의 급격한 증발이 초래되어 반응수율이 저감하는 문제점이 발생하기에 상기 온도범위 내에서 반응시킴이 바람직하다.In addition, in the deacetylation reaction step (S20), the reaction temperature is carried out at 60 ° C or more and 90 ° C or less, when the temperature is less than 60 ° C, the reaction is not performed because it does not reach the temperature range required for the reaction, and when the temperature exceeds 90 ° C , It is preferable to react within the above temperature range because the temperature range exceeding the azeotrope range of water and ethanol, which are solvents, causes rapid evaporation of ethanol and reduces the reaction yield.
또한, 탈아세틸화 반응단계(S20)에서 알콜분해반응을 이용한 탈아세틸화 반응 시간을 10분 이상 1시간 이하의 시간동안 수행하도록 하는 데, 반응시간이 10분 미만일 경우, 반응이 완료되지않고, 60분 초과일 경우, 유의적 차이가 없기 때문에 상기 반응 시간 동안 탈아세틸화 반응을 수행하도록 한다.In addition, in the deacetylation reaction step (S20), the deacetylation reaction time using the alcohol decomposition reaction is carried out for a time of 10 minutes or more and 1 hour or less. When the reaction time is less than 10 minutes, the reaction is not completed, If it exceeds 60 minutes, since there is no significant difference, the deacetylation reaction is performed during the reaction time.
가수분해 반응단계(S30)는 가수분해 반응단계(S30)는 탈아세틸화 반응단계(S20) 후, 인산(H3PO4)을 추가 첨가하고, 100℃까지 승온시켜 잔류 알콜을 휘발시켜, 2시간 동안 가수분해 반응을 수행한다. In the hydrolysis reaction step (S30), after the deacetylation reaction step (S20), phosphoric acid (H 3 PO 4 ) is additionally added, and the temperature is raised to 100 ° C. to volatilize residual alcohol, 2 The hydrolysis reaction is carried out for a period of time.
특히, 가수분해 반응단계(S30)는 탈아세틸화 반응이 종료된 히알루론산 용액에 첨가하는 인산(H3PO4)은 히알루론산 원료 분말 질량 대비 1배수 이상 2배수 이하로 첨가하도록 하는 데, 첨가하는 인산(H3PO4)의 질량이 히알루론산 원료 분말 질량 대비 1배수 미만일 경우, 히알루론산 고분자중합결합인 에테르 결합의 가수분해에 요구되는 인산(H3PO4)의 반응 몰비가 작아 가수분해 반응 속도가 매우 느리며 본 발명에서 목적하는 평균분자량 1,000Da 이하까지 가수분해하기가 용이하지 않은 문제점이 있고, 히알루론산 원료 분말 질량 대비 2배수 초과일 경우, 히알루론산 고분자중합결합인 에테르 결합의 가수분해 속도는 상승하나 최종 가수분해 조성물내 단량체 함량 조성이 높아지는 문제점이 있기에 인산(H3PO4)을 상기 범위 내에서 첨가함이 바람직하다.In particular, in the hydrolysis reaction step (S30), phosphoric acid (H 3 PO 4 ) added to the hyaluronic acid solution after the deacetylation reaction is added in an amount of 1 to 2 times the mass of the hyaluronic acid raw material powder. When the mass of phosphoric acid (H 3 PO 4 ) is less than 1 times the mass of the hyaluronic acid raw material powder, the reaction molar ratio of phosphoric acid (H 3 PO 4 ) required for hydrolysis of the ether bond, which is the polymeric bond of hyaluronic acid, is small and hydrolysis There is a problem that the reaction rate is very slow and it is not easy to hydrolyze to the average molecular weight of 1,000 Da or less, which is the purpose of the present invention, and when it exceeds 2 times the mass of the hyaluronic acid raw material powder, hydrolysis of the ether bond, which is a polymeric bond of hyaluronic acid, Although the speed is increased, it is preferable to add phosphoric acid (H 3 PO 4 ) within the above range because there is a problem in that the monomer content of the final hydrolysis composition is increased.
중화 반응단계(S40)는 가수분해 반응단계(S30) 후, 탄산칼슘(CaCO3)을 첨가하여 80℃를 유지하는 온도환경에서 중화반응을 통해 가수분해를 종료시키고, 이 때의 수소이온농도는 pH 6.5 이상 pH 7.5 이하로 조정하며, 이후, 반응조 냉각 쟈킷에 냉수를 흘려 10℃ 이상 60℃ 이하로 냉각시킴으로써 인산칼슘, 인산이수소칼슘(Ca(H2PO4)2·nH2O)을 포함하는 중화염류의 재결정화를 유도한다.In the neutralization reaction step (S40), after the hydrolysis reaction step (S30), calcium carbonate (CaCO 3 ) is added to terminate hydrolysis through a neutralization reaction in a temperature environment maintaining 80 ° C., and the hydrogen ion concentration at this time is After adjusting the pH to 6.5 or more and pH 7.5 or less, cold water was poured into the cooling jacket of the reaction tank to cool to 10 ° C or more and 60 ° C or less, thereby preparing calcium phosphate and calcium dihydrogen phosphate (Ca(H 2 PO 4 ) 2 nH 2 O). It induces recrystallization of heavy salts containing
또한, 중화 반응단계(S40)에서 가수분해 종료 시 요구되는 탄산칼슘(CaCO3)의 중화반응 몰수는 인산(H3PO4) 몰수의 2배수가 요구되며, 탄산칼슘(CaCO3)의 첨가 몰수 대비 인산(H3PO4)의 첨가 몰수의 2배수 미만일 경우, 인산(H3PO4) 반응몰수 보다 부족하여 중화염류 생성 이외 유리 인산이온의 존재로 인한 수소이온농도의 상승을 초래할 수 있어 추가적인 수소이온 농도의 조정이 요구된다.In addition, the number of moles of neutralization reaction of calcium carbonate (CaCO 3 ) required at the end of hydrolysis in the neutralization reaction step (S40) is twice the number of moles of phosphoric acid (H 3 PO 4 ), and the number of moles of calcium carbonate (CaCO 3 ) added When less than twice the number of moles of phosphoric acid (H 3 PO 4 ) added, phosphoric acid (H 3 PO 4 ) It is less than the number of moles of reaction, which may lead to an increase in hydrogen ion concentration due to the presence of free phosphate ions in addition to the formation of neutral salts, so additional adjustment of the hydrogen ion concentration is required.
또한, 중화 반응단계(S40)에서 탄산칼슘(CaCO3)의 첨가 몰수는 인산(H3PO4)의 첨가 몰수 대비 2배수로 첨가하는 것이 바람직한 데, 인산(H3PO4)첨가 몰수의 2배수 미만일 경우, 인산(H3PO4) 반응 몰수 보다 부족하여 중화염류 생성 이외 유리 인산이온의 존재로 인한 수소이온농도의 상승을 초래할 수 있어 추가적인 수소이온 농도의 조정이 요구되고, 인산(H3PO4) 첨가 몰수의 2배수 초과일 경우, 인산(H3PO4) 반응 몰수 보다 초과하여 중화염류 생성 이외 유리 칼슘이온의 존재로 인한 수산이온농도의 상승을 초래할 수 있어 추가적인 수소이온 농도의 조정이 요구되기에 탄산칼슘(CaCO3)의 첨가 몰수는 인산(H3PO4)의 첨가 몰수 대비 2배수로 첨가하는 것이 바람직하다.In addition, in the neutralization reaction step (S40), the number of moles of calcium carbonate (CaCO 3 ) added is preferably doubled compared to the number of moles of phosphoric acid (H 3 PO 4 ) added . If less than, it is less than the number of moles of phosphoric acid (H 3 PO 4 ) reaction, which may lead to an increase in hydrogen ion concentration due to the presence of free phosphate ions in addition to the formation of neutral salts, requiring additional adjustment of the hydrogen ion concentration. 4 ) If the number of moles added is more than twice the number of moles of phosphoric acid (H 3 PO 4 ) reaction, it may cause an increase in the concentration of hydroxide ions due to the presence of free calcium ions in addition to the formation of neutral salts, requiring additional adjustment of the concentration of hydrogen ions. As required, the number of moles of calcium carbonate (CaCO 3 ) added is preferably doubled compared to the number of moles added of phosphoric acid (H 3 PO 4 ).
또한, 중화 반응단계(S40)에서 반응조 냉각 쟈킷에 냉수를 흘려 10℃ 이상 60℃ 이하로 냉각시키도록 하는 데, 냉각 온도가 10℃ 미만일 경우, 중화염류의 결정화물 응집에서 결정물 계면간 포집되는 가수분해물의 함량이 증가하여 생산로스가 증가하는 문제점이 있고, 60℃ 초과일 경우, 중화염류의 결정화가 발생하지 않기에 상기 온도범위로 냉각시키는 것이 바람직하다.In addition, in the neutralization reaction step (S40), cold water is poured into the reaction tank cooling jacket to cool it to 10 ° C or more and 60 ° C or less. There is a problem that the production loss increases due to the increase in the content of the hydrolyzate, and when the temperature exceeds 60 ° C., it is preferable to cool to the above temperature range because the crystallization of neutral salts does not occur.
중화염류 제거단계(S50)는 마이크로 필터를 이용하여 1차 여과하여 재결정화된 중화염류를 제거하고, 역삼투 필터를 이용하여 2차 여과하여 용액 내 이온화된 중화염류를 제거한다.In the step of removing neutral salts (S50), recrystallized neutral salts are removed by primary filtration using a micro filter, and ionized neutral salts in the solution are removed by secondary filtration using a reverse osmosis filter.
도 4를 참조하면, 건식 가스분무 건조단계(S60)는 중화염류 제거단계(S50)를 수행한 가수분해 여과물을 질소 가스 기반으로 건식 가스 분무를 이용하여 건조하여 분말화한다.Referring to FIG. 4, in the dry gas spray drying step (S60), the hydrolyzed filtrate subjected to the neutralized salt removal step (S50) is dried using dry gas spray based on nitrogen gas to pulverize.
분쇄단계(S70)는 건식 가스분무 건조단계(S60)를 수행하여 응집된 분말 덩어리를 분쇄기로 미분쇄하여 미세 분말상으로 재가공한다.In the crushing step (S70), the dry gas spray drying step (S60) is performed to pulverize the agglomerated powder mass with a grinder to reprocess it into fine powder.
입자표준화단계(S80)는 분쇄단계(S70)를 수행한 분쇄물의 입도 분포 표준화를 위해 특정 사이즈(200~400mesh)의 메쉬망을 이용하여 채걸음하여 최종 수득한다.In the particle standardization step (S80), the pulverized material subjected to the pulverization step (S70) is finally obtained by sifting using a mesh network of a specific size (200 to 400 mesh) to standardize the particle size distribution.
<실시예><Example>
팽윤화 단계 : 반응조에 히알루론산(평균분자량 : 1,300,000Da)분말 3kg을 95%에틸알콜 6kg에 분산 시킨 후 정제수 97kg을 투입하여 완전 용해시키고, 이후 1시간 동안 팽윤화를 수행하여 겔화를 시킨다.Swelling step: After dispersing 3kg of hyaluronic acid (average molecular weight: 1,300,000Da) powder in 6kg of 95% ethyl alcohol in a reaction tank, 97kg of purified water is added to completely dissolve it, and then swelling is performed for 1 hour to gelation.
탈아세틸화 반응단계(S20) : 상기 팽윤화가 완료된 히알루론산 용액에 H3PO4 0.6kg을 첨가한 후 10분간 혼합하여 균질화를 수행한다. 이때, 수소이온농도는 pH1.3~1.5로 조정한다. 이후, 72℃에서 알콜분해반응을 이용하여 탈아세틸화 반응을 1시간 동안 수행한다.Deacetylation reaction step (S20): After adding 0.6 kg of H 3 PO 4 to the swollen hyaluronic acid solution, homogenization is performed by mixing for 10 minutes. At this time, the hydrogen ion concentration is adjusted to pH 1.3 to 1.5. Thereafter, a deacetylation reaction is performed for 1 hour using alcohol decomposition at 72°C.
가수분해 반응단계(S30) : 상기 탈아세틸화 반응이 종료되면 H3PO4 2.4kg을 추가 첨가한 후 100℃까지 승온 시켜 잔류 알콜을 모두 휘발시켜 종료시키고 이후, 2시간동안 가수분해 반응을 수행한다.Hydrolysis reaction step (S30): When the deacetylation reaction is completed, 2.4 kg of H 3 PO 4 is additionally added, and the temperature is raised to 100 ° C. do.
중화 반응단계(S40) : 가수분해 반응 종료는 CaCO3 6kg을 첨가하여 중화반응을 수행함으로써 종료시키고, 이때 반응 온도는 80℃를 유지한다. 이때 수소이온농도는 pH6.5~7.5로 조정한다. 이후 반응조 냉각 쟈킷에 냉수를 흘려 반응조 온도를 30℃이하로 냉각시킴으로써 중화염류(인산칼슘, 인산이수소칼슘(Ca(H2PO4)2·nH2O)의 재결정화를 유도한다.Neutralization reaction step (S40): The hydrolysis reaction is terminated by adding 6 kg of CaCO 3 to carry out a neutralization reaction, at which time the reaction temperature is maintained at 80 °C. At this time, the hydrogen ion concentration is adjusted to pH 6.5 ~ 7.5. Thereafter, cold water is poured into the cooling jacket of the reaction vessel to cool the temperature of the reaction vessel to 30° C. or lower, thereby inducing recrystallization of neutralized salts (calcium phosphate, calcium dihydrogen phosphate (Ca(H 2 PO 4 ) 2 nH 2 O).
중화염류 제거단계(S50) : 재결정화된 중화 염류 제거는 마이크로 필터를 이용하여 1차 여과하고 이후 용액내 이온화된 염류는 역삼투 필터를 이용하여 2차 중화염류 제거를 수행한다.Neutralized salts removal step (S50): The recrystallized neutralized salts are removed by first filtration using a micro filter, and then the ionized salts in the solution are secondarily removed by using a reverse osmosis filter.
건식 가스분무 건조단계(S60) : 가수분해 여과물의 건조는 질소 가스 기반 건식 가스 분무 건조를 이용하여 분말화를 수행한다.Dry gas spray drying step (S60): Drying of the hydrolyzed filtrate is powdered using nitrogen gas-based dry gas spray drying.
분쇄 단계 : 상기 분무 건조 수행 이후 응집된 분말 덩어리는 분쇄기로 미분쇄하여 미세 분말상으로 재가공 한다.Grinding step: After performing the spray drying, the agglomerated powder mass is finely pulverized with a pulverizer and reprocessed into fine powder.
입자 표준화 단계 : 분쇄물의 입도 분포 표준화를 위해 표준망을 이용한 채걸음을 수행한다. 이때, 표준 메쉬망의 규격은 200~400메쉬를 기준으로 한다.Particle standardization step: To standardize the particle size distribution of the pulverized material, sifting is performed using a standard net. At this time, the specification of the standard mesh network is based on 200 to 400 mesh.
<분석예><Analysis Example>
본 발명에서 규정되는 분자량 분포는 겔 여과 컬럼을 이용하여 시료를 액체 크로마토그래피 분석하는 것으로부터 얻을 수 있다. 상기 저분자량 히알루론산은 반복 구조 단위(N-아세틸 D-글루코사민 및 D-글루코산)의 수에 따라서 다른 분자량을 가지는 복수 성분의 혼합물이다. 따라서, 겔 여과 컬럼을 이용하여 시료에 대하여 액체 크로마토그래피 분석을 수행하는 것으로, 상기 저분자량 히알루론산을 구성하는 성분을 분자 사이즈에 따라 분리할 수 있다. 따라서 본 발명에서의 히알루론산 가수분해물의 평균분자량 측정은 Gel permeation chromatography(GPC)를 이용하여 분석을 수행하였다. 분석결과는 하기 도표에 나타내었다.The molecular weight distribution defined in the present invention can be obtained from liquid chromatography analysis of a sample using a gel filtration column. The low molecular weight hyaluronic acid is a mixture of plural components having different molecular weights according to the number of repeating structural units (N-acetyl D-glucosamine and D-glucosan). Therefore, by performing liquid chromatography analysis on the sample using a gel filtration column, components constituting the low molecular weight hyaluronic acid can be separated according to molecular size. Therefore, the average molecular weight of the hyaluronic acid hydrolyzate in the present invention was analyzed using gel permeation chromatography (GPC). The analysis results are shown in the chart below.
가수분해 전 후 Gel permeation chromatography(GPC) 비교 그래프Gel permeation chromatography (GPC) comparison graph before and after hydrolysis
이상의 설명은 본 발명의 기술 사상을 예시적으로 설명한 것에 불과한 것으로서, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자라면 본 발명의 본질적인 특성에서 벗어나지 않는 범위 내에서 다양한 수정, 변경 및 치환이 가능할 것이다. 따라서, 본 발명에 개시된 실시예 및 첨부된 도면들은 본 발명의 기술 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시예 및 첨부된 도면에 의하여 본 발명의 기술 사상의 범위가 한정되는 것은 아니다. 본 발명의 보호 범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리범위에 포함되는 것으로 해석되어야 할 것이다.The above description is merely an example of the technical idea of the present invention, and those skilled in the art can make various modifications, changes, and substitutions without departing from the essential characteristics of the present invention. will be. Therefore, the embodiments disclosed in the present invention and the accompanying drawings are not intended to limit the technical idea of the present invention, but to explain, and the scope of the technical idea of the present invention is not limited by these embodiments and the accompanying drawings. . The protection scope of the present invention should be construed according to the claims below, and all technical ideas within the equivalent range should be construed as being included in the scope of the present invention.
S10 - 팽윤화단계
S20 - 탈아세틸화 반응단계
S30 - 가수분해 반응단계
S40 - 중화 반응단계
S50 - 중화염류 제거단계
S60 - 건식 가스분무 건조단계
S70 - 분쇄단계
S80 - 입자표준화단계
S100 - 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법S10 - Swelling step
S20 - Deacetylation reaction step
S30 - Hydrolysis reaction step
S40 - neutralization reaction step
S50 - Removal of heavy salts
S60 - dry gas spray drying step
S70 - crushing step
S80 - particle standardization step
S100 - Method for producing hyaluronic acid oligomers including ultra-low molecular weight hyaluronic acid
Claims (3)
팽윤화 단계에서 팽윤화가 완료된 히알루론산 용액에 인산(H3PO4)을 첨가하고, 혼합하여 균질화하며, 알콜분해반응을 이용한 탈아세틸화 반응을 수행하는 탈아세틸화 반응단계;
탈아세틸화 반응단계 후, 인산(H3PO4)을 추가 첨가하고, 승온시켜 잔류 알콜을 휘발시키며, 가수분해 반응을 수행하는 가수분해 반응단계;
가수분해 반응단계 후, 탄산칼슘(CaCO3)을 첨가하여 중화반응을 통해 가수분해를 종료시키고, 반응조 냉각 쟈킷에 냉수를 흘려 냉각시킴으로써 인산칼슘, 인산이수소칼슘(Ca(H2PO4)2·nH2O)을 포함하는 중화염류의 재결정화를 유도하는 중화 반응단계;
마이크로 필터를 이용하여 1차 여과하여 재결정화된 중화염류를 제거하고, 역삼투 필터를 이용하여 2차 여과하여 용액 내 이온화된 중화염류를 제거하는 중화염류 제거단계;
중화염류 제거단계를 수행한 가수분해 여과물을 질소 가스 기반으로 건식 가스 분무를 이용하여 건조하여 분말화하는 건식 가스분무 건조단계;
건식 가스분무 건조단계를 수행하여 응집된 분말 덩어리를 분쇄기로 미분쇄하여 미세 분말상으로 재가공하는 분쇄단계; 및
분쇄단계를 수행한 분쇄물의 입도 분포 표준화를 위해 특정 사이즈의 메쉬망을 이용하여 채걸음하는 입자표준화단계
를 포함하는 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법.A swelling step of dispersing hyaluronic acid powder in ethyl alcohol in a reaction vessel, dissolving it by adding purified water, and performing swelling to gelate;
A deacetylation reaction step of adding phosphoric acid (H 3 PO 4 ) to the hyaluronic acid solution of which swelling has been completed in the swelling step, homogenizing by mixing, and performing a deacetylation reaction using an alcohol decomposition reaction;
After the deacetylation reaction step, a hydrolysis reaction step in which phosphoric acid (H 3 PO 4 ) is additionally added, the temperature is raised to volatilize residual alcohol, and a hydrolysis reaction is performed;
After the hydrolysis reaction step, calcium carbonate (CaCO 3 ) is added to terminate the hydrolysis through a neutralization reaction, and cold water is poured into the cooling jacket of the reaction tank to cool it, thereby producing calcium phosphate and dihydrogen phosphate (Ca(H 2 PO 4 ) 2 Neutralization reaction step of inducing recrystallization of neutral salts including nH 2 O);
A neutral salt removal step of removing recrystallized neutral salts by primary filtration using a micro filter and secondary filtration using a reverse osmosis filter to remove ionized neutral salts in the solution;
Dry gas spray drying step of drying and pulverizing the hydrolyzed filtrate after performing the neutral salt removal step using dry gas spray based on nitrogen gas;
A pulverization step of performing a dry gas spray drying step to finely pulverize the agglomerated powder mass with a pulverizer and reprocessing it into fine powder; and
Particle standardization step of sifting using a mesh network of a specific size to standardize the particle size distribution of the pulverized material that has undergone the pulverization step
Method for producing a hyaluronic acid oligomer comprising ultra-low molecular weight hyaluronic acid comprising a.
탈아세틸화 반응단계는
히알루론산 용액에 첨가하는 인산(H3PO4)은 히알루론산 원료 분말 질량 대비 0.1배수 이상 2배수 이하로 첨가하는 것
을 포함하는 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법.According to claim 1,
The deacetylation reaction step is
Phosphoric acid (H 3 PO 4 ) added to the hyaluronic acid solution is added in an amount of 0.1 times or more and 2 times or less based on the mass of the hyaluronic acid raw material powder.
A method for producing a hyaluronic acid oligomer comprising ultra-low molecular weight hyaluronic acid comprising a.
가수분해 반응단계는
탈아세틸화 반응이 종료된 히알루론산 용액에 첨가하는 인산(H3PO4)은 히알루론산 원료 분말 질량 대비 1배수 이상 2배수 이하로 첨가하는 것
을 포함하는 초 저분자 히알루론산을 포함하는 히알루론산 올리고머 제조방법.According to claim 1,
The hydrolysis reaction step is
Phosphoric acid (H 3 PO 4 ) added to the hyaluronic acid solution after the deacetylation reaction is added in an amount of 1-fold or more and 2-fold or less based on the mass of the hyaluronic acid raw material powder
A method for producing a hyaluronic acid oligomer comprising ultra-low molecular weight hyaluronic acid comprising a.
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