KR20070122059A - Phospholipid composition and manufacturing method thereof - Google Patents
Phospholipid composition and manufacturing method thereof Download PDFInfo
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- KR20070122059A KR20070122059A KR1020060057047A KR20060057047A KR20070122059A KR 20070122059 A KR20070122059 A KR 20070122059A KR 1020060057047 A KR1020060057047 A KR 1020060057047A KR 20060057047 A KR20060057047 A KR 20060057047A KR 20070122059 A KR20070122059 A KR 20070122059A
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- phospholipid
- fatty acid
- reactor
- acid
- polyunsaturated fatty
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- 150000003904 phospholipids Chemical class 0.000 title claims abstract description 165
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 claims abstract description 102
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- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 claims abstract description 51
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 claims abstract description 51
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-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
- A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Fats And Perfumes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
도 1은 인지질 조성물 내 DHA 및 EPA의 함량을 분석한 결과를 도시한 그래프이다. 1 is a graph showing the results of analyzing the content of DHA and EPA in the phospholipid composition.
도 2는 인지질 조성물 내 DHA 및 EPA의 함량을 분석한 결과를 도시한 그래프이다. Figure 2 is a graph showing the results of analyzing the content of DHA and EPA in the phospholipid composition.
도 3은 인지질 조성물 내 DHA, EPA 및 ARA의 함량을 분석한 결과를 도시한 그래프이다. Figure 3 is a graph showing the results of analyzing the content of DHA, EPA and ARA in the phospholipid composition.
도 4는 인지질 조성물 내 포스파티딜콜린 및 포스파티딜에탄올아민의 함량을 분석한 결과를 도시한 그래프이다. Figure 4 is a graph showing the results of analyzing the content of phosphatidylcholine and phosphatidylethanolamine in the phospholipid composition.
본 발명은 인지질 조성물 및 그 제조방법에 관한 것으로, 더욱 상세하게는 포스포리파아제 A1, 포스포리파아제 A2 또는 리파아제(Lipase)를 이용하여 에스테 르 교환반응(transesterfication) 또는 아실 교환반응(Transacylation)을 통해 인체 흡수율이 우수한 고도불포화지방산과 결합된 재조합 인지질을 제조하는 방법과 그 인지질 조성물에 관한 것이다. The present invention relates to a phospholipid composition and a method of preparing the same, and more particularly, to a phospholipid A1, a phospholipase A2, or a lipase, through an ester exchange reaction or an acyl exchange reaction. The present invention relates to a method for producing a recombinant phospholipid coupled with polyunsaturated fatty acid having excellent human absorption and a phospholipid composition thereof.
고도불포화지방산은 크게 오메가-6계와 오메가-3계로 나뉜다. 오메가-6계 지방산으로는 리놀레산(linoleic acid)(C18:3, LA), 리놀레산(C18:2, LA), γ-리놀레산(C18:3, GLA), 디호모(dihomo)-γ-리놀레산(C20:3, DHLA) 또는 아라키돈산(arachidonic acid)(C20:4, ARA)이 있다. 오메가-3계 지방산으로는 ω-3 계열의 지방산으로서는 α-리놀레산(C18:3, ALA), 에이코사펜타엔산(eicosapentaenoic acid)(C20:5, EPA) 및 도코사헥사엔산(docosahexaenoic acid)(C22:6, DHA) 등이 있다. 이들은 인체 대사과정 중 생리적으로 중요한 기능을 하는 2-프로스타글란딘(prostaglandins), 4-류코트리엔(leukotrienes), 2-트롬복산(thromboxanes), 5-류코트리엔(leukotriens), 3-프로스타글란딘(prostaglandins), 3-트롬복산(thromboxanes) 등을 합성한다. 이러한 인체 대사과정 중 발생한 물질들은 체내에서 순환기계, 호흡계, 소화계, 신장, 피부 및 면역계의 질병, 뇌와 안구의 미발달, 그리고 암의 발생에 이르기까지 다양하게 관여한다.Polyunsaturated fatty acids are largely divided into omega-6 and omega-3. Omega-6 fatty acids include linoleic acid (C18: 3, LA), linoleic acid (C18: 2, LA), γ-linoleic acid (C18: 3, GLA), dihomo-γ-linoleic acid ( C20: 3, DHLA) or arachidonic acid (C20: 4, ARA). Omega-3 fatty acids include ω-3 fatty acids such as α-linoleic acid (C18: 3, ALA), eicosapentaenoic acid (C20: 5, EPA), and docosahexaenoic acid. (C22: 6, DHA) and the like. They are 2-prostaglandins, 4-leukotrienes, 2-thromboxanes, 5-leukotriens, 3-prostaglandins, 3-throm, which play a physiologically important function during human metabolism. Synthesize thromboxanes and the like. Substances that occur during this metabolic process are involved in the body, ranging from diseases of the circulatory system, the respiratory system, the digestive system, the kidneys, the skin and the immune system, the development of the brain and the eye, and the development of cancer.
오메가-3 계열의 고도불포화지방산(Poly Unsaturated Fatty Acid; PUFA)의 일종인 에이코사펜타엔산(cis-5,8,11,14,17-Eicosapenta-enoic acid ; EPA, C20:5), 도코사헥사엔산(cis-4,7,10,13,16,19-Docosahexaenoic acid ; DHA, C22:6)과 오메가-6계열의 아라키돈산(cis-5,8,11,14- Arachidonic acid ; ARA, C20:4)은 일반적으로 널리 알려져 있다. Eicosapentaenoic acid (cis-5,8,11,14,17-Eicosapenta-enoic acid; EPA, C20: 5), doco, a type of polyunsaturated fatty acid (PUFA) of the omega-3 family Tetrahexanoic acid (cis-4,7,10,13,16,19-Docosahexaenoic acid; DHA, C22: 6) and omega-6-based arachidonic acid (cis-5,8,11,14-Arachidonic acid; ARA, C20: 4) are generally well known.
일반적으로 오메가-3 장쇄 고도불포화지방산인 EPA 및 DHA는 각각 혈소판 응집을 억제하고 혈전을 예방하며, 혈중 HDL 콜레스테롤을 낮추지 않고, VLDL, LDL-콜레스테롤을 저하시킨다. 또한, 혈중의 중성지질을 저하시키며, 혈액의 점도를 낮추고, 혈액순환을 좋게 하는 등의 약리 효능이 있는 것으로 알려지고 있다. In general, omega-3 long-chain polyunsaturated fatty acids, EPA and DHA, respectively, inhibit platelet aggregation, prevent blood clots, do not lower blood HDL cholesterol, and lower VLDL and LDL-cholesterol. In addition, it is known to have pharmacological effects such as lowering neutral lipids in the blood, lowering the viscosity of blood, and improving blood circulation.
인체의 뇌의 회백질부, 망막, 신경, 심장, 정자, 모유 중에 국소적으로 많이 함유되어 있는 DHA는 기억학습기능 향상 효과를 지닌다. 특히, 뇌를 구성하는 지방산을 분석해보면 DHA가 다량 함유되어 있다. 이는 인체 내의 오메가-3 지방산 중 뇌 관문을 통과할 수 있는 지방산은 DHA가 가장 많다는 것을 의미한다. 그러나, DHA가 뇌 관문을 통과하기 위해서는 트리글리세라이드(Triglyceride) 형태의 DHA 보다 인지질과 결합된 형태가 뇌 관문을 통과하기가 더 용이하다. 또한, DHA가 포함된 인지질의 경우, 세포의 막 유동성이 우수하고, 신경세포의 활성화 및 신경전달물질의 활성화가 향상되며, 뇌기능 향상, 항암 작용, 콜레스테롤 및 중성지질 저하작용, 항 알레르기 작용, 망막의 반사능력 향상 기능(시력 저하 억제 작용), 혈압 저하 작용, 항염증 작용 등의 약리 효능에 대해서도 많은 보고가 이루어지고 있어 산업적으로 식품 소재, 특히 건강 증진을 위한 기능성 식품소재, 화장품 소재, 의약품 소재 등으로 널리 사용되어지고 있다. DHA, which is contained locally in the gray matter, retina, nerve, heart, sperm and breast milk of the human brain, has the effect of improving memory learning function. In particular, the analysis of the fatty acids that make up the brain contains a large amount of DHA. This means that among the omega-3 fatty acids in the human body, fatty acids that can cross the brain barrier are the most DHA. However, in order for DHA to cross the brain barrier, it is easier for phospholipid-bound forms to cross the brain barrier than for triglyceride form of DHA. In addition, in the case of phospholipids containing DHA, the membrane fluidity of the cells is excellent, the activation of neurons and the activation of neurotransmitters is improved, the brain function, anticancer action, cholesterol and triglyceride lowering action, anti allergic action, Many reports have been made on the pharmacological effects of the retina's ability to improve reflexes (such as suppressing vision lowering), lowering blood pressure, and anti-inflammatory effects. It is widely used as a material.
전 세계적으로 EPA, DHA 및 ARA는 건강 보조 식품으로 애용되고 있다. 이 중에서 고순도 EPA(95%)는 고지혈증 치료제로 미국의 FDA에 의해 공인된 바 있다. 일반적으로 공급되는 EPA나 DHA는 이들의 에틸에스테르이거나 트리아실글리세롤(Tri acyl glycerol, TG)의 형태가 일반적이다. 그러나, 이들은 물에 대한 용해도가 떨 어져 인체 내의 소장에서 인지질과 결합된 형태로 만들어져 체내로 흡수된다. 즉, 인지질 형태의 고도불포화지방산은 체내 흡수가 매우 뛰어나게 된다. 일반적으로 DHA를 25% 함유한 인지질의 DHA 효능은 DHA를 95% 함유한 에틸에스테르와 유사한 것으로 보고되고 있다. 그러나, 그 제조방법에 있어서 대량의 유기용매를 이용하여 추출하는 방법이 주로 사용되므로 대규모로 수행하기에는 어려움이 있고 그 비용도 많이 드는 단점이 있다. EPA, DHA and ARA are being used as dietary supplements worldwide. Of these, high purity EPA (95%) has been approved by the US FDA for the treatment of hyperlipidemia. Generally supplied EPA or DHA is their ethyl ester or triacyl glycerol (TG) in general form. However, they lose their solubility in water, which is combined with phospholipids in the small intestine in the human body and absorbed into the body. In other words, polyunsaturated fatty acids in the form of phospholipids are highly absorbed by the body. In general, the DHA efficacy of phospholipids containing 25% DHA is reported to be similar to ethyl esters containing 95% DHA. However, since the extraction method using a large amount of organic solvent is mainly used in the manufacturing method, it is difficult to carry out on a large scale and has a disadvantage that the cost is high.
따라서, 체내 흡수가 용이한 EPA, DHA 또는 ARA를 다량 포함하고 있는 인지질을 보다 경제적인 방법으로 제조할 수 있는 기술을 개발해야 할 필요성이 끊임없이 대두되고 있다. 이를 위해 국내외에서는 인지질 내의 PUFA(EPA, DHA 및 ARA)의 함량을 높이기 위해 다양한 시도가 이루어져 왔다. Therefore, there is a constant need to develop a technology for producing phospholipids containing a large amount of EPA, DHA or ARA, which is easily absorbed in the body, in a more economical manner. To this end, various attempts have been made at home and abroad to increase the content of PUFAs (EPA, DHA and ARA) in phospholipids.
일본 순토리(Suntory)사 및 비젠(Bizen)화성(PCT/JP 96/01453)의 경우 오메가-3 및 오메가-6 계열 지방산의 함량이 높은 배양균체를 가금난에 첨가하여 ARA 및 EPA, DHA 함량이 증가한 난황 지질을 생산하는 기술을 소개하고 있다. 대한민국 공개특허공보(10-1998-013757)에서는 참깻묵, 들깻묵 등을 사료첨가제로 하여 오메가-3 지방산의 비율을 조정하려는 시도가 있으며, 대한민국 공개특허공보(10-1993-0005548)에서는 가쯔오부시나 오징어 제조시 얻어지는 폐기물을 사료에 0.5~5.0 wt% 첨가시켜 급이 및 사육함으로써 난황 100g 당 DHA를 0.5g 이상 함유하는 계란의 제조방법을 소개하고 있다. 그러나, EPA 및 DHA 함유량은 아직도 낮은 수준이며, 이들 오메가-3 지방산이 인지질에 결합된 형태의 인지질 조성물의 제조방법은 소개되지 않고 있다. 또한, 난황지질에 오메가-3 지방산의 함량이 높은 지방산을 배합 하여 오메가-3 지방산 함량을 증대하고자 하는 시도도 있었으나(대한민국 공개특허공보 10-2001-0035040 참조), 진정한 의미의 오메가-3 장쇄불포화지방산을 함유하는 인지질 조성물이라 하기 어렵다. In Japan, Suntory and Bizen Chemical (PCT / JP 96/01453), ARA, EPA and DHA contents were added to the poultry eggs by adding cultured cells with high content of omega-3 and omega-6 fatty acids. The technology to produce this increased yolk lipid is introduced. In the Republic of Korea Patent Publication (10-1998-013757), there is an attempt to adjust the ratio of omega-3 fatty acids by using sesame jelly, wild jelly, etc. as a feed additive, and in the Republic of Korea Patent Publication (10-1993-0005548) Katsuobushi B. The method for producing eggs containing 0.5g or more of DHA per 100g of egg yolk is introduced by feeding and breeding the waste obtained from the manufacture of squid by adding 0.5 to 5.0 wt% to the feed. However, the content of EPA and DHA is still low, and no method for preparing phospholipid compositions in the form in which these omega-3 fatty acids are bound to phospholipids has not been introduced. In addition, there have been attempts to increase the content of omega-3 fatty acids by combining fatty acids having a high content of omega-3 fatty acids with egg yolk lipids (see Korean Patent Application Laid-Open No. 10-2001-0035040). It is difficult to call it a phospholipid composition containing a fatty acid.
최근의 연구 결과들에 따르면, 이들 인지질에 오메가-3 및 오메가-6 계열의 PUFA가 결합된 형태의 EPA, DHA(오메가-3) 및 ARA(오메가-6) 결합 인지질의 경우, 세포막의 유동성 향상, 염증유발물질(Eicosanoid 등) 생산 억제 등에 의해 생리효과를 발휘하며, 특히 뇌 부활기능인 수면시간 증가작용, 학습능력 향상작용, 기억력 향상작용에 대해서 구체적인 DHA와 EPA 결합 인지질의 효과들이 확인되어지고 있다. 이와 같이 인지질에 결합된 형태의 EPA 및 DHA의 경우, 천연 유지 내 EPA 및 DHA 보다 생리활성 효과가 높을 뿐만 아니라, EPA, DHA 결합 인지질로서의 독자적인 효과도 있어 그 유용성이 우수하다고 평가된다. 그러나, 천연으로 존재하는 인지질에는 EPA, DHA 및 ARA와 결합한 인지질의 함유량이 낮아 통상적인 섭취량을 기준으로 할 때 생리활성 효과를 기대하기 힘든 것이 현실이다. 또한 오메가-6 계열인 아라키돈산(ARA)도 인체에서는 대부분이 인지질 형태로 존재하고 있어 트리아실글리세롤(TG) 형태의 오일에 비해 섭취시 생리활성 효과가 우수할 것으로 기대되어진다. Recent studies have shown that EPA, DHA (Omega-3) and ARA (Omega-6) -bound phospholipids in combination with these phospholipids, omega-3 and omega-6 PUFAs, improve cell membrane fluidity. , And physiological effects by inhibiting the production of inflammatory substances (Eicosanoid, etc.), and in particular, the effects of DHA and EPA-bound phospholipids have been confirmed for brain reactivation function, increasing sleep time, improving learning ability, and improving memory. . As such, EPA and DHA bound to phospholipids are not only higher in physiological activity than EPA and DHA in natural fats and oils, but also have unique effects as EPA and DHA-bound phospholipids. However, the fact that the phospholipids present in nature is low in the content of phospholipids combined with EPA, DHA and ARA, it is difficult to expect a physiological activity effect based on the normal intake. In addition, most of the omega-6-based arachidonic acid (ARA) is present in the human body in the form of phospholipids, it is expected to have a superior physiological activity effect when ingested compared to triacylglycerol (TG) type oil.
본 발명이 이루고자 하는 기술적 과제는 포스포리파아제 A1, 포스포리파아제 A2 또는 리파아제(Lipase)를 이용하여 에스테르 교환반응(transesterfication) 또 는 아실 교환반응(Transacylation)을 통해 인체 흡수율이 우수한 고도불포화지방산과 결합된 재조합 인지질을 제조하는 방법을 제공함에 있다. The technical problem to be achieved by the present invention is to combine with a polyunsaturated fatty acid having excellent human absorption rate through transesterfication or acyl exchange reaction using phospholipase A1, phospholipase A2 or Lipase (Lipase) To provide a method for producing a recombinant phospholipid.
본 발명이 이루고자 하는 다른 기술적 과제는 인체 흡수율이 우수한 고도불포화지방산과 결합된 인지질 조성물을 제공함에 있다. Another object of the present invention is to provide a phospholipid composition combined with polyunsaturated fatty acid having excellent human absorption.
본 발명은, 반응기에 인지질과, 용매 및 고도불포화지방산을 투여하는 단계와, 상기 반응기에 포스포리파아제 A1, 포스포리파아제 A2 및 1-3 리파아제 중에서 선택된 적어도 하나의 효소를 투여하는 단계 및 상기 인지질에 결합된 지방산을 상기 효소에 의한 에스테르 교환반응(transesterification) 또는 아실 교환반응(transacylation)을 통해 고도불포화지방산으로 치환하거나 변화시켜 고도불포화지방산과 결합된 재조합 인지질을 얻는 단계를 포함하는 인지질 조성물 제조방법을 제공한다. The present invention comprises the steps of administering a phospholipid, a solvent and a polyunsaturated fatty acid to a reactor, administering at least one enzyme selected from phospholipase A1, phospholipase A2 and 1-3 lipase to the reactor and the phospholipid. Method of preparing a phospholipid composition comprising the step of replacing or changing the fatty acid bound to polyunsaturated fatty acid through transesterification or acyl exchange (transacylation) by the enzyme to obtain a recombinant phospholipid combined with polyunsaturated fatty acid. To provide.
상기 반응기에 투여하는 인지질로 포스파티딜콜린, 포스파티딜에탄올아민, 포스파티딜산, 포스파티딜세린, 포스파티딜이노시톨, 리소포스파티딜콜린, 리소포스파티딜에탄올아민, 리소포파티딜산, 리소포스파티딜세린 및 리소포스파티딜이노시톨을 적어도 한 종 포함하는 천연 또는 합성 인지질(phospholipid)을 사용할 수 있다. Natural or phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidyl inositol, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidyl inositol as phospholipids administered to the reactor. Synthetic phospholipids can be used.
상기 용매로 n-헥산, 에탄올, 아세톤, 클로로포름 및 에틸아세테이트를 적어도 하나 포함하는 유기용매를 사용할 수 있다. 상기 용매에 완충액인 아세트산 버 퍼(acetate buffer) 또는 인산 버퍼(phosphate buffer)가 더 포함될 수 있다. As the solvent, an organic solvent including at least one of n-hexane, ethanol, acetone, chloroform, and ethyl acetate may be used. Acetate buffer or phosphate buffer, which is a buffer, may be further included in the solvent.
상기 반응기에 투여하는 고도불포화지방산으로 에이코사펜타엔산(EPA), 도코사헥사엔산(DHA) 및 아라키돈산(ARA)을 적어도 하나 포함하는 아실(acyl)기를 갖는 고도불포화지방산을 사용할 수 있다. As the polyunsaturated fatty acid administered to the reactor, polyunsaturated fatty acids having an acyl group containing at least one of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA) may be used. .
상기 효소는 인지질의 sn-1번과 sn-2번의 위치에 있는 지방산을 에스테르 교환반응(transesterification) 또는 아실 교환반응(transacylation)을 통해 고도불포화지방산으로 치환하거나 변화시킬 수 있다. The enzyme may replace or change the fatty acid at the positions of sn-1 and sn-2 of the phospholipid to polyunsaturated fatty acid through transesterification or acyl exchange.
고도불포화지방산과 결합된 상기 재조합 인지질은 포스파티딜콜린의 함량이 상기 고도불포화지방산과 결합된 재조합 인지질에 대하여 30∼95%가 되도록 제어하는 것이 바람직하다. The recombinant phospholipid bound to the polyunsaturated fatty acid is preferably controlled so that the content of phosphatidylcholine is 30 to 95% relative to the recombinant phospholipid bound to the polyunsaturated fatty acid.
상기 반응기의 분위기를 질소 가스 분위기로 만들고, 상기 반응기의 온도는 효소의 활성화를 고려하여 20∼95℃ 범위로 설정하여 12∼90 시간 반응시켜 상기 재조합 인지질을 얻을 수 있다. An atmosphere of the reactor may be made into a nitrogen gas atmosphere, and the temperature of the reactor may be set in a range of 20 to 95 ° C. for 12 to 90 hours in consideration of activation of enzymes to obtain the recombinant phospholipid.
고도불포화지방산과 결합된 상기 재조합 인지질은 고도불포화지방산인 EPA의 함량이 재조합 인지질의 전체 지방산에 대하여 15∼75%, 고도불포화지방산인 DHA의 함량이 재조합 인지질의 전체 지방산에 대하여 15∼75%, 또는 고도불포화지방산인 ARA의 함량이 재조합 인지질의 전체 지방산에 대하여 5∼45%가 되도록 제어하는 것이 바람직하다. The recombinant phospholipid combined with polyunsaturated fatty acid has 15 to 75% of the total polyunsaturated fatty acid of EPA, 15 to 75% of the total fatty acid of recombinant phospholipid, and 15 to 75% of the total fatty acid of recombinant phospholipid. Or it is preferable to control so that content of ARA which is polyunsaturated fatty acid may be 5 to 45% with respect to the whole fatty acid of recombinant phospholipid.
또한, 본 발명은, 본 발명에 따른 인지질 조성물 제조방법으로 제조된 식품, 화장품 또는 의약품 소재로서의 인지질 조성물을 제공한다.The present invention also provides a phospholipid composition as a food, cosmetic or pharmaceutical material prepared by the method for producing a phospholipid composition according to the present invention.
이하, 첨부된 도면을 참조하여 본 발명에 따른 바람직한 실시예를 상세하게 설명하기로 한다. 그러나, 이하의 실시예는 이 기술분야에서 통상적인 지식을 가진 자에게 본 발명이 충분히 이해되도록 제공되는 것으로서 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 다음에 기술되는 실시예에 한정되는 것은 아니다. 도면상에서 동일 부호는 동일한 요소를 지칭한다. Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following embodiments are provided to those skilled in the art to fully understand the present invention, and may be modified in various forms, and the scope of the present invention is limited to the embodiments described below. It doesn't happen. Like numbers refer to like elements in the figures.
본 발명은 인지질의 지방산을 효소를 이용하여 에스테르화 교환반응(transesterification)과 아실 교환반응(transacylation)을 통해 고도불포화지방산으로 전환시켜 DHA(docosahexaenoic acid), EPA(Eicosapentaenoic acid) 또는 ARA(Arachidonic acid)의 함량이 풍부한 인지질을 제조하는 방법을 제시한다. The present invention converts phospholipid fatty acids into polyunsaturated fatty acids through transesterification and acyl exchange using enzymes, and then DHA (docosahexaenoic acid), EPA (Eicosapentaenoic acid) or ARA (Arachidonic acid). A method of preparing phospholipids rich in content is presented.
본 발명의 바람직한 실시예에 따른 인지질 조성물 제조방법은, 대두 또는 난황 인지질에 포스포리파아제(Phospholipase) A1, 포스포리파아제 A2 또는 리파아제(Lipase)를 사용하여 인지질의 sn-1과 sn-2 부위의 포화지방산 또는 불포화지방산을 에스테르 교환반응(transesterification) 또는 아실 교환반응(transacylation)을 통하여 EPA, DHA 또는 ARA와 같은 고도불포화지방산으로 전환시켜 DHA, EPA 또는 ARA와 결합된 인지질을 얻는다. Phospholipid composition according to a preferred embodiment of the present invention, phospholipid (Phospholipase) A1, phospholipase A2 or lipase (Lipase) using the phospholipids sn-1 and sn-2 site of the phospholipids Saturated fatty acids or unsaturated fatty acids are converted to polyunsaturated fatty acids such as EPA, DHA or ARA by transesterification or acyl exchange to obtain phospholipids bound to DHA, EPA or ARA.
인지질은 인체의 뇌, 시신경 및 중추신경계 조직의 주요 구성물질로 알려져 있으며, 인지질은 포스파티딜콜린(PC), 포스파티딜에탄올아민(PE) 등 수종의 인지질 동족체들의 혼합체 형태로 존재한다. 기존 생산 또는 판매되는 인지질에 효소를 이용한 지방산의 전환 기술을 바탕으로 인지질 내의 PUFA(DHA, EPA 및 ARA)의 함량 이 매우 높은 재조합 인지질을 제조할 수 있다. Phospholipids are known as major components of human brain, optic nerve and central nervous system tissues. Phospholipids exist in the form of a mixture of several phospholipid homologues such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Recombinant phospholipids with a very high content of PUFAs (DHA, EPA and ARA) in phospholipids can be prepared based on the conversion of fatty acids using enzymes to existing phospholipids.
이하, 본 발명에 의한 EPA, DHA 또는 ARA와 결합된 인지질 조성물의 제조방법에 대하여 구체적으로 살펴본다.Hereinafter, the method for preparing the phospholipid composition combined with EPA, DHA or ARA according to the present invention will be described in detail.
반응기에 인지질을 투여한다. 상기 인지질은 포스파티딜콜린, 포스파티딜에탄올아민, 포스파티딜산, 포스파티딜세린, 포스파티딜이노시톨, 리소포스파티딜콜린, 리소포스파티딜에탄올아민, 리소포파티딜산, 리소포스파티딜세린 및 리소포스파티딜이노시톨을 적어도 한 종 이상 포함하는 천연 또는 합성 인지질(phospholipid)로서, 예컨대 대두 또는 난황의 인지질일 수 있다. Administer phospholipids to the reactor. The phospholipid is a natural or synthetic phospholipid comprising at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidyl inositol, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidyl acid, lysophosphatidylserine and lysophosphatidyl inositol ( phospholipid), for example soybean or egg yolk phospholipid.
상기 반응기에 용매를 투여한다. 상기 용매는 n-헥산, 에탄올, 아세톤, 클로로포름 및 에틸아세테이트를 적어도 하나 포함하는 유기용매일 수 있다. 인지질과 상기 용매의 무게비는 1:1∼1:5 정도인 것이 바람직하다. 한편, 상기 용매에 완충액(Buffer)이 더 포함될 수 있다. 예컨대, 상기 용매로서 n-헥산(hexane)과 완충액이 혼합된 이상계 시스템을 사용할 수 있다. n-헥산과 완충액의 비는 무게비로 5:1 내지 50:1인 것이 바람직하다. 완충액으로는 아세트산 버퍼(acetate buffer) 또는 인산 버퍼(phosphate buffer) 등이 사용될 수 있으며, pH 3.5 내지 8.5인 것이 바람직하다. 상기 용매는 인지질을 용해하는데 사용할 수 있으며, 그 결과 용해가 원활하며 반응 후 반응물 회수에도 용이하다. The solvent is administered to the reactor. The solvent may be an organic solvent including at least one of n-hexane, ethanol, acetone, chloroform and ethyl acetate. The weight ratio of the phospholipid and the solvent is preferably 1: 1 to 1: 5. Meanwhile, a buffer may be further included in the solvent. For example, an ideal system in which n-hexane and a buffer are mixed may be used as the solvent. The ratio of n-hexane and the buffer is preferably 5: 1 to 50: 1 by weight. Acetate buffer or phosphate buffer may be used as the buffer, and the pH is preferably 3.5 to 8.5. The solvent can be used to dissolve the phospholipids, and as a result, the dissolution is smooth and easy to recover the reactants after the reaction.
일반적으로 인지질은 포스파티딜콜린(Phosphatidyl choline; PC)의 함량에 의해 제품의 등급이 결정된다. 그 중에서 포스파티딜콜린(Phosphatidyl choline)의 함량이 20∼40%, 포스파티딜에탄올아민(Phosphatidyl ethanolamine; PE)의 함량이 20∼30% 정도인 저순도 인지질과 포스파티딜콜린의 함량이 40∼95%인 고순도 인지질은 유기용매의 특성에 따라 잘 녹지 않는 경우도 있다. 특히, 에탄올, 에틸아세테이트, 아세톤의 경우 인지질을 일정 농도 이상 사용할 경우, 교반이 원활하지 않아 인지질의 응집 현상이 일어나며 반응성이 매우 떨어진다. Phospholipids are generally graded by the content of phosphatidyl choline (PC). Among them, low-purity phospholipids containing 20-40% of phosphatidyl choline and 20-30% of phosphatidylethanolamine (PE) and high-purity phospholipids having 40-95% of phosphatidylcholine are organic. It may not be easily dissolved depending on the characteristics of the solvent. In particular, in the case of ethanol, ethyl acetate, acetone, when a phospholipid is used at a certain concentration or more, agitation is not smooth and phospholipid aggregation occurs and the reactivity is very low.
그러나 본 발명의 바람직한 실시예에서는 용매로서 n-헥산, 에탄올, 아세톤, 클로로포름 또는 에틸아세테이트 등을 사용하며, 동시에 적절한 용해성을 얻기 위해 온도 변화 및 용매의 조성 변화를 고려한다. 본 발명은 중저순도의 인지질(인지질 함량 50% 이하) 뿐만 아니라 고순도의 인지질의 지방산 조성도 변화시켜 고순도 인지질과 결합한 PUFA의 함량을 높이고자 한다. 또한, 반응 후 반응 잔여물로 생성되는 인지질 반응물의 회수가 용이하도록 한다. In a preferred embodiment of the present invention, however, n-hexane, ethanol, acetone, chloroform or ethyl acetate is used as the solvent, and at the same time, temperature change and composition change of the solvent are considered to obtain proper solubility. The present invention is to increase the content of PUFA combined with high-purity phospholipids by changing the fatty acid composition of the high-purity phospholipids as well as medium-low purity phospholipids (50% or less of phospholipids). It also facilitates the recovery of the phospholipid reactants produced as reaction residues after the reaction.
인지질이 투여된 반응기에 아실(acyl)기를 갖는 고도불포화지방산을 투여한다. 상기 아실(acyl)기를 갖는 고도불포화지방산은 오메가-3 계열의 고도불포화지방산 또는 오메가-6 계열의 고도불포화지방산일 수 있다. 상기 오메가-3 계열의 고도불포화지방산은 에이코사펜타엔산(EPA) 또는 도코사헥사엔산(DHA)이고, 상기 오메가-6 계열의 고도불포화지방산은 아라키돈산(ARA)일 수 있다. The polyunsaturated fatty acid having an acyl group is administered to the reactor to which the phospholipid is administered. The polyunsaturated fatty acid having an acyl group may be an omega-3 polyunsaturated fatty acid or an omega-6 polyunsaturated fatty acid. The polyunsaturated fatty acid of the omega-3 series may be eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the polyunsaturated fatty acid of the omega-6 series may be arachidonic acid (ARA).
상기 반응기에 포스포리파아제 A1, 포스포리파아제 A2 및 1-3 리파아제 중에서 선택된 적어도 하나 이상의 효소를 투여한다. 상기 효소는 인지질과 결합한 지방산의 sn-1번과 sn-2번의 위치에 있는 지방산을 에스테르 교환반응(transesterification) 또는 아실 교환반응(transacylation)을 통해 인체 흡수율이 뛰어난 고도불포화지방산으로 치환하거나 변화시킨다. The reactor is administered with at least one enzyme selected from phospholipase A1, phospholipase A2 and 1-3 lipases. The enzyme replaces or changes fatty acids at positions sn-1 and sn-2 of fatty acids bound with phospholipids to polyunsaturated fatty acids with excellent human absorption through transesterification or acyl exchange.
본 발명의 바람직한 실시예에 사용되는 효소는 표 1에 나타내었다.The enzymes used in the preferred examples of the present invention are shown in Table 1.
표 1의 공급자(Supplier)에서, A는 아마노 제약사(Amano Pharmaceutical Co.)(Nagoya, Japan), B는 바이오캐털리스트사(Biocatalysts)(Pontyprid, UK), C는 기스트-브로캐이드사(Gist-Brocades)(Delft, Netherlands), D는 메이토산요사(Meito Sangyo)(Aichi, Japan), E는 노보자임 A/S사(Novozymes A/S)(Bagsvaerd, Denmark), F는 세이카가쿠 코요사(Seikagaku Kogyo Co.)(Tokyo, Japan), G는 시그마 케이칼사(Sigma Chemical)(St. Louis, MO), H는 타나베 세이야쿠(Tanabe Seiyaku)(Osaka, Japan)의 약칭을 말한다. In the suppliers of Table 1, A is Amano Pharmaceutical Co. (Nagoya, Japan), B is Biocatalysts (Pontyprid, UK), and C is Gist-Brocade. Brocades (Delft, Netherlands), D for Meito Sangyo (Aichi, Japan), E for Novozymes A / S (Bagsvaerd, Denmark), F for Seikagaku Koyosa Seikagaku Kogyo Co. (Tokyo, Japan), G stands for Sigma Chemical (St. Louis, MO), and H stands for Tanabe Seiyaku (Osaka, Japan).
반응기 내의 분위기를 질소 가스 분위기로 만들고 반응시킨다. 즉, 질소 가스로 반응기를 퍼지(purge)하여 반응기 내의 산소를 제거하고 질소 가스 분위기에서 반응시킨다. 상기 반응에 의해, 인지질의 sn-1 및 sn-2 부위에 결합된 포화 또는 불포화 지방산의 조성이 에스테르 교환반응(transesterification) 또는 아실 교환반응(transacylation)을 통해 EPA, DHA 또는 ARA로 치환되거나 변화되게 되고, DHA(docosahexaenoic acid), EPA(Eicosapentaenoic acid) 또는 ARA(Arachidonic acid)의 함량이 많은 재조합 인지질을 얻을 수 있다. 상기 반응의 온도는 효소의 활성화 온도를 고려하여 20℃∼95℃의 범위인 것이 바람직하며, 반응 시간은 효소에 의한 에스테르 교환반응(transesterification) 또는 아실 교환반응(transacylation)을 고려하여 12hr∼90hr인 것이 바람직하다. 또한, 반응기의 RPM(revolution per minute)은 효소에 의한 에스테르 교환반응(transesterification) 또는 아실 교환반응(transacylation)을 고려하여 200∼1000 정도인 것이 바람직하다. 또한, 반응에 의해 얻어진 재조합 인지질은 포스파티딜콜린의 함량이 상기 재조합 인지질에 대하여 30∼95%가 되도록 제어하는 것이 바람직하다. 또한, 반응에 의해 얻어진 고도불포화지방산과 결합된 재조합 인지질은 고도불포화지방산인 EPA의 함량이 재조합 인지질의 전체 지방산에 대하여 15∼75%, 고도불포화지방산인 DHA의 함량이 재조합 인지질의 전체 지방산에 대하여 15∼75%, 또는 고도불포화지방산인 ARA의 함량이 재조합 인지질의 전체 지방산에 대하여 5∼45%가 되도록 제어하는 것이 바람직하다. The atmosphere in the reactor is made into a nitrogen gas atmosphere and reacted. That is, the reactor is purged with nitrogen gas to remove oxygen in the reactor and reacted in a nitrogen gas atmosphere. By this reaction, the composition of saturated or unsaturated fatty acids bound to the sn-1 and sn-2 sites of the phospholipid is replaced or changed to EPA, DHA or ARA by transesterification or acyl exchange. In addition, a recombinant phospholipid with a high content of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or arachidonic acid (ARA) can be obtained. The temperature of the reaction is preferably in the range of 20 ℃ to 95 ℃ considering the activation temperature of the enzyme, the reaction time is 12hr ~ 90hr in consideration of transesterification or acyl exchange (transacylation) by the enzyme It is preferable. In addition, RPM (revolution per minute) of the reactor is preferably about 200 to 1000 in consideration of transesterification or acyl exchange (transacylation) by the enzyme. In addition, the recombinant phospholipid obtained by the reaction is preferably controlled so that the content of phosphatidylcholine is 30 to 95% relative to the recombinant phospholipid. In addition, the recombinant phospholipid obtained by the reaction was 15 to 75% of the total fatty acid of the recombinant phospholipid and 15% to 75% of the total fatty acid of the recombinant phospholipid. It is preferable to control so that content of ARA which is 15 to 75% or polyunsaturated fatty acid becomes 5 to 45% with respect to the whole fatty acid of a recombinant phospholipid.
본 발명이 목적으로 하는 최종산물의 EPA, DHA 및 ARA의 양을 측정하기 위한 분석 조건은 다음과 같다. Analytical conditions for measuring the amount of EPA, DHA and ARA of the final product of the present invention is as follows.
<분석조건><Analysis Condition>
인지질과 결합한 EPA, DHA 및 ARA의 함량을 측정하기 위하여 반응물을 식품공전상의 인지질 함량 측정법 중 아세톤 불용물 측정법으로 인지질을 추출 정제한 후, 이를 메틸-에스테르화(methyl-esterification) 가스-크로마토그래피(gas-chromatography)를 이용한다. In order to measure the content of EPA, DHA and ARA bound to phospholipids, the reactants were extracted and purified by acetone insolubles in the phospholipid content measurement method in food industry, and then methyl-esterification gas-chromatography ( gas-chromatography).
먼저 추출된 시료 0.2g에 0.5N NaOH가 포함된 메탄올 6㎖를 가하여 100℃에서 중탕한 다음, 10% BF3가 포함된 메탄올(스위스의 플루카사(Fluka Co.) 제품) 7㎖를 가한다. 5분 후, 6㎖의 n-헥산(hexane)을 가하여 2분간 방치한 다음, 이 액을 분액여두에 옮긴다. 분액여두에 옮겨진 액에 포화 NaCl용액 2㎖을 가하여 30초간 흔든 다음 방치한 후, 원심분리한다. 원심분리된 상층액에 무수황산나트륨을 첨가하여 수분을 제거한 후 1㎕ 가스-크로마토그래피(gas-chromatography)(미국의 휴렛패커드(Hewlett Packard)사의 5890Ⅱ 제품)에 주입하여 분석한다. To 0.2 g of the extracted sample, 6 ml of methanol containing 0.5 N NaOH was added, followed by agitation at 100 ° C., followed by 7 ml of methanol containing 10% BF 3 (Fluka Co., Switzerland). . After 5 minutes, 6 ml of n-hexane was added and allowed to stand for 2 minutes, after which the solution was transferred to a separatory filter. 2 ml of saturated NaCl solution is added to the transferred solution, shaken for 30 seconds, and then centrifuged. Water was removed by adding anhydrous sodium sulfate to the centrifuged supernatant, followed by injection into 1 μl gas-chromatography (Hewlett Packard, USA, 5890 II).
표 2는 가스 크로마토그래피에 대한 분석 조건을 나타낸 표이다. Table 2 is a table showing the analysis conditions for gas chromatography.
본 발명은 하기의 실험예들을 참고로 더욱 상세히 설명되며, 이 실험예들이 본 발명을 제한하는 것은 아니다.The invention is described in more detail with reference to the following experimental examples, which do not limit the invention.
<실험예 1>Experimental Example 1
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 10g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 DHA(70%) 20g을 투여하고, 효소인 1-3 리파아제(Lipase) 10g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 2%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 45℃의 온도에서 400 RPM으로 30시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 DHA의 함량이 재조합 인지질의 전체 지방산에 대하여 25%였고, 수율은 78%였다. 10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase (Lipase) was administered. In addition, 2% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was conducted at 400 RPM for 30 hours at a temperature of 45 ° C. in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to have a DHA content of 25% relative to the total fatty acids of the recombinant phospholipids, and a yield of 78%.
<실험예 2>Experimental Example 2
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 10g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 DHA(70%) 20g을 투여하고, 효소인 1-3 리파아제(Lipase) 10g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 4%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 35℃의 온도에서 400 RPM으로 45시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 DHA의 함량이 재조합 인지질의 전체 지방산에 대하여 22%였고, 수율은 71%였다. 10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase (Lipase) was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 45 hours at 400 RPM at a temperature of 35 ° C. in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. Recombinant phospholipids thus obtained were found to have a DHA content of 22% relative to the total fatty acids of the recombinant phospholipids, and a yield of 71%.
<실험예 3>Experimental Example 3
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 30g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 DHA(70%) 20g을 투여하고, 효소인 포스포리파아제 A2 2g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 4%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 40℃의 온도에서 400 RPM으로 40시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 DHA의 함량이 재조합 인지질의 전체 지방산에 대하여 36%였고, 수율은 64%였다. 10 g of phospholipid was administered to the reactor, and 30 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 2 g of phospholipase A2, an enzyme, was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 40 hours at 400 RPM at a temperature of 40 ℃ in a closed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to have a DHA content of 36% and a yield of 64% for the total fatty acids of the recombinant phospholipids by gas-chromatographic analysis.
<실험예 4>Experimental Example 4
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 30g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 DHA(70%) 20g을 투여하고, 효소인 포스포리파아제 A2 10g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 4%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 45℃의 온도에서 400 RPM으로 30시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 DHA의 함량이 재조합 인지질의 전체 지방산에 대하여 52%였고, 수율은 70%였다. 10 g of phospholipid was administered to the reactor, and 30 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 10 g of phospholipase A2, an enzyme, was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was conducted at 400 RPM for 30 hours at a temperature of 45 ° C. in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipid thus obtained was found to have 52% of the total fatty acid of the recombinant phospholipid and the yield was 70% by gas-chromatographic analysis.
<실험예 5>Experimental Example 5
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 10g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 DHA(70%) 20g을 투여하고, 효소인 1-3 리파아제(Lipase) 5g과 포스포리파아제 A2 0.1g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 4%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 60℃의 온도에서 400 RPM으로 90시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 DHA의 함량이 재조합 인지질의 전체 지방산에 대하여 36%였고, 수율은 63%였다. 10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid DHA (70%) was administered to the reactor, and 5 g of enzyme 1-3 Lipase and 0.1 g of phospholipase A2 were administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was performed at 400 RPM at a temperature of 60 ° C. for 90 hours in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to have a DHA content of 36% with respect to the total fatty acids of the recombinant phospholipids, and a yield of 63%.
<실험예 6>Experimental Example 6
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 10g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 EPA(70%) 20g을 투여하고, 효소인 1-3 리파아제(Lipase) 10g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 2%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 60℃의 온도에서 400 RPM으로 72시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 EPA의 함량이 재조합 인지질의 전체 지방산에 대하여 28.3%였고, 수율은 76.4%였다. 10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase (Lipase) was administered. In addition, 2% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 72 hours at 400 RPM at a temperature of 60 ℃ in a closed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipid thus obtained was found to be 28.3% with respect to the total fatty acid of the recombinant phospholipid and the yield was 76.4% by gas-chromatographic analysis.
<실험예 7>Experimental Example 7
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 10g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 EPA(70%) 10g을 투여하고, 효소인 1-3 리파아제(Lipase) 10g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 4%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 60℃의 온도에서 400 RPM으로 24시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 EPA의 함량이 재조합 인지질의 전체 지방산에 대하여 24.2%였고, 수율은 71%였다. 10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 10 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase (Lipase) was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out at 400 RPM at a temperature of 60 ° C. for 24 hours in a closed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to be 24.2% with respect to the total fatty acids of the recombinant phospholipids and the yield was 71% by gas-chromatographic analysis.
<실험예 8>Experimental Example 8
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 30g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 EPA(70%) 20g을 투여하고, 효소인 포스포리파아제 A2 10g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 4%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 60℃의 온도에서 1000 RPM으로 60시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 EPA의 함량이 재조합 인지질의 전체 지방산에 대하여 44.9%였고, 수율은 64%였다. 10 g of phospholipid was administered to the reactor, and 30 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 10 g of phospholipase A2, an enzyme, was administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. The reactor was filled with nitrogen gas to remove oxygen in the reactor, and the reaction was carried out at 1000 RPM for 60 hours at a temperature of 60 ° C. in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to be 44.9% with respect to the total fatty acids of the recombinant phospholipids, and the yield was 64% by gas-chromatographic analysis.
<실험예 9>Experimental Example 9
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 10g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 EPA(70%) 20g을 투여하고, 효소인 1-3 리파아제 5g과 포스포리파아제 A2 10g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 4%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 40℃의 온도에서 1000 RPM으로 48시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 EPA의 함량이 재조합 인지질의 전체 지방산에 대하여 53.8%였고, 수율은 63%였다. 10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 5 g of enzymes 1-3 lipase and 10 g of phospholipase A2 were administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 48 hours at 1000 RPM at a temperature of 40 ℃ in a closed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were found to be 53.8%, and the yield was 63% of the total fatty acids of the recombinant phospholipids by gas-chromatographic analysis.
<실험예 10>Experimental Example 10
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 10g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 EPA(70%) 20g을 투여하고, 효소인 1-3 리파아제 5g과 포스포리파아제 A2 0.1g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 4%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 50℃의 온도에서 1000 RPM으로 72시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 EPA의 함량이 재조합 인지질의 전체 지방산에 대하여 42.2%였고, 수율은 63%였다. 10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid EPA (70%) was administered to the reactor, and 5 g of enzymes 1-3 lipase and 0.1 g of phospholipase A2 were administered. In addition, 4% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 72 hours at 1000 RPM at a temperature of 50 ℃ in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. The recombinant phospholipids thus obtained were gas-chromatographically analyzed. The EPA content was 42.2% with respect to the total fatty acids of the recombinant phospholipids, and the yield was 63%.
<실험예 11>Experimental Example 11
반응기에 인지질 10g을 투여하고, 용매인 n-헥산(hexane) 10g을 상기 반응기에 투여하였다. 또한, 상기 반응기에 고도불포화지방산인 ARA(70%) 20g을 투여하고, 효소인 1-3 리파아제 10g을 투여하였다. 또한, 상기 반응기에 완충액으로 인산 버퍼(Phosphate Buffer)를 총 기질 반응액의 2%(weight/volume)를 투여하였다. 반응기 내를 질소 가스로 충진하여 반응기 내 산소를 제거한 후, 밀폐된 상태에서 60℃의 온도에서 1000 RPM으로 72시간 반응을 시켰다. 반응 후, 기공 크기(pore size) 100㎛의 필터를 이용하여 반응이 완료된 효소를 제거하고, 질소 하에서 진공농축하여 n-헥산을 제거하였으며, 이를 -20℃의 아세톤을 이용하여 2시간 침지시킨 후, 상온의 아세톤 500㎖을 이용하여 수세한 다음, 아세톤 불용물인 인지질을 회수하였다. 이렇게 얻은 재조합 인지질은 가스-크로마토그래피 분석결과 ARA의 함량이 재조합 인지질의 전체 지방산에 대하여 28.3%였고, 수율은 76.4%였다. 10 g of phospholipid was administered to the reactor, and 10 g of a solvent, n-hexane, was administered to the reactor. In addition, 20 g of polyunsaturated fatty acid ARA (70%) was administered to the reactor, and 10 g of enzyme 1-3 lipase was administered. In addition, 2% (weight / volume) of the total substrate reaction solution was administered to the reactor using a phosphate buffer (Phosphate Buffer) as a buffer. After filling the reactor with nitrogen gas to remove oxygen in the reactor, the reaction was carried out for 72 hours at 1000 RPM at a temperature of 60 ℃ in a sealed state. After the reaction, the enzyme having completed the reaction was removed by using a filter having a pore size of 100 μm, and concentrated in vacuo under nitrogen to remove n-hexane, which was immersed in acetone at −20 ° C. for 2 hours. After washing with 500 ml of acetone at room temperature, phospholipids were recovered as an acetone insoluble matter. Thus obtained recombinant phospholipid was gas. Chromatography analysis showed that the content of ARA was 28.3% and the yield was 76.4% of the total fatty acid of the recombinant phospholipid.
도 1은 인지질 조성물 내 DHA 및 EPA의 함량을 분석한 결과를 도시한 그래프로서, 상기 실험예 4에 따라 실험한 경우의 결과이다. 1 is a graph showing the results of analyzing the content of DHA and EPA in the phospholipid composition, which is the result when the experiment according to the Experimental Example 4.
도 2는 인지질 조성물 내 DHA 및 EPA의 함량을 분석한 결과를 도시한 그래프로서, 상기 실험예 9에 따라 실험한 경우의 결과이다. Figure 2 is a graph showing the results of analyzing the content of DHA and EPA in the phospholipid composition, which is the result when the experiment according to the experimental example 9.
도 3은 인지질 조성물 내 DHA, EPA 및 ARA의 함량을 분석한 결과를 도시한 그래프로서, 상기 실험예 10에 따라 실험한 경우의 결과이다. Figure 3 is a graph showing the results of analyzing the content of DHA, EPA and ARA in the phospholipid composition, which is the result when the experiment according to Experimental Example 10.
도 4는 인지질 조성물 내 포스파티딜콜린 및 포스파티딜에탄올아민의 함량을 분석한 결과를 도시한 그래프이다. Figure 4 is a graph showing the results of analyzing the content of phosphatidylcholine and phosphatidylethanolamine in the phospholipid composition.
도 4는 실험예들을 통해 얻어진 시료(인지질)를 0.5g씩 채취하여 메탄올에 녹이고 초음파로 5분간 추출한 후 100㎖로 정용하였으며, 이렇게 얻어진 용액을 10㎖ 취하여 메탄올로 100㎖가 되게 정용하고 여과하여 시험 용액으로 사용하여 고압 액상 크로마토그래피(High Pressure Liquid Chromatography; HPLC)로 분석한 결과이다. 고압 액상 크로마토그래피를 이용한 장비 분석 조건은 아래의 표 3에 나타내었다. 4 is 0.5g of samples (phospholipids) obtained through the experimental examples, dissolved in methanol and extracted for 5 minutes with ultrasonic wave, and then justified to 100ml. The solution thus obtained is taken up to 10ml with methanol and filtered to 100ml. As a test solution, the result was analyzed by High Pressure Liquid Chromatography (HPLC). Equipment analysis conditions using high pressure liquid chromatography are shown in Table 3 below.
본 발명에 의하면, 식용 및 기타 산업에 적용함에도 안전한 고도불포화지방산(PUFA)과 결합된 인지질을 얻을 수 있다. According to the present invention, phospholipids combined with polyunsaturated fatty acids (PUFA) that are safe even for application to food and other industries can be obtained.
본 발명은 현재 생산되거나 또는 판매되는 인지질의 지방산 조성을 1,3-리파아제, 포스포리파아제 A1 또는 포스포리파아제 A2와 같은 효소를 이용하여 인지질 의 sn-1과 sn-2 site 부위의 포화 또는 불포화지방산을 에스테르화 교환반응 또는 아실 교환반응을 통하여 오메가-3계열 장쇄고도불포화지방산인 EPA, DHA 또는 오메가-6계열 장쇄고도불포화지방산인 ARA(아라키돈산)이 인지질에 고농도로 함유된 형태의 인지질 조성물을 얻을 수 있다.The present invention uses saturated or unsaturated fatty acids at the sn-1 and sn-2 site sites of phospholipids using enzymes such as 1,3-lipase, phospholipase A1 or phospholipase A2. EPA, DHA or omega-6 long chain highly unsaturated fatty acids ARA (arachidonic acid), which is a high concentration of phospholipids, are contained in a phospholipid by esterification or acyl exchange reaction. You can get it.
또한, 본 발명에 따른 인지질 조성물은 식품, 화장품 또는 의약품 소재로서 사용될 수 있다. In addition, the phospholipid composition according to the present invention can be used as a food, cosmetic or pharmaceutical material.
이상, 본 발명의 바람직한 실시예를 들어 상세하게 설명하였으나, 본 발명은 상기 실시예에 한정되는 것은 아니며, 본 발명의 기술적 사상의 범위내에서 당 분야에서 통상의 지식을 가진 자에 의하여 여러 가지 변형이 가능하다.As mentioned above, although preferred embodiment of this invention was described in detail, this invention is not limited to the said embodiment, A various deformation | transformation by a person of ordinary skill in the art within the scope of the technical idea of this invention is carried out. This is possible.
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CN106609286A (en) * | 2015-10-22 | 2017-05-03 | 丰益(上海)生物技术研发中心有限公司 | Preparation method for phosphatide containing long-chain polyunsaturated fatty acid |
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CN106609286A (en) * | 2015-10-22 | 2017-05-03 | 丰益(上海)生物技术研发中心有限公司 | Preparation method for phosphatide containing long-chain polyunsaturated fatty acid |
CN106609286B (en) * | 2015-10-22 | 2022-01-25 | 丰益(上海)生物技术研发中心有限公司 | Preparation method of phospholipid containing long-chain polyunsaturated fatty acid |
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