KR102615753B1 - Method of preparing isoquercetin having excellent antibacterial and antioxidant activity. - Google Patents
Method of preparing isoquercetin having excellent antibacterial and antioxidant activity. Download PDFInfo
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- KR102615753B1 KR102615753B1 KR1020220163868A KR20220163868A KR102615753B1 KR 102615753 B1 KR102615753 B1 KR 102615753B1 KR 1020220163868 A KR1020220163868 A KR 1020220163868A KR 20220163868 A KR20220163868 A KR 20220163868A KR 102615753 B1 KR102615753 B1 KR 102615753B1
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- South Korea
- Prior art keywords
- dsf
- isoquercetin
- solution
- enzyme
- strain
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- OVSQVDMCBVZWGM-QSOFNFLRSA-N quercetin 3-O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-QSOFNFLRSA-N 0.000 title claims abstract description 89
- OVSQVDMCBVZWGM-IDRAQACASA-N Hirsutrin Natural products O([C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1)C1=C(c2cc(O)c(O)cc2)Oc2c(c(O)cc(O)c2)C1=O OVSQVDMCBVZWGM-IDRAQACASA-N 0.000 title claims abstract description 88
- FVQOMEDMFUMIMO-UHFFFAOYSA-N Hyperosid Natural products OC1C(O)C(O)C(CO)OC1OC1C(=O)C2=C(O)C=C(O)C=C2OC1C1=CC=C(O)C(O)=C1 FVQOMEDMFUMIMO-UHFFFAOYSA-N 0.000 title claims abstract description 88
- OVSQVDMCBVZWGM-QCKGUQPXSA-N isoquercetin Natural products OC[C@@H]1O[C@@H](OC2=C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)[C@H](O)[C@@H](O)[C@@H]1O OVSQVDMCBVZWGM-QCKGUQPXSA-N 0.000 title claims abstract description 85
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- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims description 24
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/0104—Alpha-L-rhamnosidase (3.2.1.40)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Abstract
본 발명은 이소케르세틴의 제조방법 및 이로부터 제조된 이소케르세틴에 관한 것이다. 보다 구제척으로, 본 발명은 식품 및 화장품 분야에 널리 사용되는 이소케르세틴의 제조방법에 있어서, 기존의 고가 효소의 사용 및 효소의 정제 과정을 생략하여 제조 비용이 최소화되면서도 항균성과 항산화 활성이 우수한 이소케르세틴을 제조할 수 있는 이소케르세틴의 제조방법 및 이로부터 제조된 이소케르세틴에 관한 것이다.The present invention relates to a method for producing isoquercetin and isoquercetin prepared therefrom. More specifically, the present invention relates to a method for producing isoquercetin, which is widely used in the food and cosmetics fields, by omitting the use of existing expensive enzymes and the enzyme purification process, thereby minimizing production costs and providing isoquercetin with excellent antibacterial and antioxidant activities. It relates to a method for producing isoquercetin, which can produce quercetin, and isoquercetin produced therefrom.
Description
본 발명은 항균성 및 항산화 활성이 우수한 이소케르세틴의 제조방법에 관한 것이다. 보다 구제척으로, 본 발명은 식품 및 화장품 분야에 널리 사용되는 이소케르세틴의 제조방법에 있어서, 기존의 값비싼 효소의 사용 및 효소의 정제 과정을 생략하여 제조 비용이 최소화되면서도 항균성과 항산화 활성이 우수한 이소케르세틴을 제조할 수 있는 이소케르세틴의 제조방법에 관한 것이다.The present invention relates to a method for producing isoquercetin, which has excellent antibacterial and antioxidant activities. More specifically, the present invention is a method for producing isoquercetin, which is widely used in the food and cosmetics fields, by omitting the use of existing expensive enzymes and the enzyme purification process, thereby minimizing manufacturing costs and exhibiting excellent antibacterial and antioxidant activities. It relates to a method for producing isoquercetin.
이소케르세틴(Isoquercetin, Isoquercitrin, Quercetin 3-O-beta-D-glucopyranoside)은 식물성 식품에서 발견되는 폴리페놀인 플라보노이드(flavonoid)의 일종으로 다양한 산업분야에서 산화방지제, 색소 안정화제, 향기변화 방지제, 항균제 등의 보존제로 사용되며, 약리적으로는 항염증, 항바이러스, 항암, 이뇨 작용, 혈관 보호, 신경 보호 등의 효능이 있는 것으로 알려져 있다.Isoquercetin (Isoquercetin, Isoquercitrin, Quercetin 3-O-beta-D-glucopyranoside) is a type of flavonoid, a polyphenol found in plant foods. It is used as an antioxidant, color stabilizer, aroma change inhibitor, and antibacterial agent in various industrial fields. It is used as a preservative, and is known to have pharmacological effects such as anti-inflammatory, antiviral, anticancer, diuretic, blood vessel protection, and neuroprotection.
천연 식물에서는 케르세틴(Quercetin)보다 그 배당체인 이소케르세틴(Isoquercetin)이나 루틴(rutin) 형태로 더 많이 분포되어 있으며, 생체 이용률은 랫드, 개, 돼지 및 인체에서 각각 이소케르세틴, 케르세틴, 루틴 순서로 높으므로 식품, 화장품, 건강기능성식품 등의 분야에서 이소케르세틴이 더 많이 이용되고 있다.In natural plants, its glycosides, isoquercetin and rutin, are more widely distributed than quercetin, and its bioavailability is highest in rats, dogs, pigs, and humans, respectively, in that order: isoquercetin, quercetin, and rutin. Therefore, isoquercetin is being used more in fields such as food, cosmetics, and health functional foods.
한편, 당전이효소를 이용하여 이소케르세틴에 포도당을 1~7개 더 결합시킨 효소처리루틴(Enzymatically Modified Isoquercitrin, EMIQ)은 수용화도가 높아서 음료용 항산화제로 가장 많이 이용되고 있으나, 효소처리루틴(EMIQ)은 항균 활성이 없는 단점이 있다.Meanwhile, Enzymatically Modified Isoquercitrin (EMIQ), which combines 1 to 7 glucose units with isoquercetin using glycosyltransferase, is most widely used as an antioxidant for beverages due to its high water solubility. ) has the disadvantage of having no antibacterial activity.
도 1은 루틴의 전환물 및 루틴의 전환 방법을 도시한다. 도 1의 화학식에서 람노오스는 빨간색, 포도당은 파란색으로 표시되었다.1 shows a conversion product of a routine and a method of conversion of a routine. In the chemical formula of Figure 1, rhamnose is shown in red and glucose is shown in blue.
일반적으로, 이소케르세틴은 회화나무 꽃 유래의 루틴을 이용하여, 1) 산가수분해, 2)나린지네이즈 효소 처리(naringinase), 3)헤스페리디네이즈 효소 처리(Hesperidinase), 4)람노시데이즈 효소 처리(α-L-rhamnosidase) 방법에 의해 각각 제조할 수 있다.In general, isoquercetin is made using rutin derived from Prickly pear tree flowers, 1) acid hydrolysis, 2) naringinase enzyme treatment (naringinase), 3) hesperidinase enzyme treatment (Hesperidinase), 4) rhamnosidase. Each can be manufactured by enzyme treatment (α-L-rhamnosidase) method.
종래의 이소케르세틴의 제조방법 관련 기술에는 나린지네이즈(naringinase) 효소를 이용하는 “알파-글리코실이소퀘르시트린, 그의 제조 중간체, 및 부생성물의 조제 방법”(PCT/JP2004/013580, 산에이겐, 일본)과 “이소케르세틴 및 알파-글리코실이소케르세틴의 제조 방법”(특허출원 10-2018-0000228)이 소개되고 있으며, 상기 두 개의 특허는 Naringinase “”효소(Amano Enzyme Inc.)를 이용하여 루틴을 이소케르세틴으로 전환하는 기술을 개시한다.Conventional technology related to the production method of isoquercetin includes “Method for preparing alpha-glycosylisoquercitrin, production intermediates thereof, and by-products” using naringinase enzyme (PCT/JP2004/013580, San-Eigen, Japan) and “Method for producing isoquercetin and alpha-glycosylisoquercetin” (patent application 10-2018-0000228) are introduced, and the above two patents use Naringinase “” enzyme (Amano Enzyme Inc.) Discloses a technology for converting isoquercetin.
또한, 종래기술 중 Aspergillus niger KCTC 6906 유래의 람노시데이즈(rhamnosidase) 효소로 루틴을 이소케르세틴으로 전환하는 방법으로서 “아스퍼질러스 나이거 유래의 람노시데이즈를 이용한 루틴에서 쿼세틴 -3- 글루코사이드를 제조하는 방법”(특허출원 10-2008-0061997)이 소개되고 있으나, 상기 특허는 이소케르세틴의 구체적인 전환 방법/조건, 전환 수준에 관한 내용은 개시되어 있지 않다.In addition, as a method of converting rutin into isoquercetin using rhamnosidase enzyme derived from Aspergillus niger KCTC 6906 among the prior art, “quercetin-3-glucoside is produced from rutin using rhamnosidase derived from Aspergillus niger KCTC 6906.” However, the patent does not disclose specific conversion methods/conditions or conversion levels of isoquercetin.
그리고, 상기 특허에서 사용된 공시 균주(Aspergillus niger KCTC 6906)는 상업적 사용이 불가능하고, 시판 효소(Naringinase “”는 고가여서 이소케르세틴 제조 단가가 매우 높아지는 단점이 있다. 또한, 곰팡이가 생산하는 효소를 정제해서 사용할 경우 정제 비용이 추가되므로 생산된 이소케르세틴 가격이 매우 고가인 단점이 있다. 따라서, 효율적이고 경제적인 이소케르세틴의 제조방법에 대한 연구가 지속적으로 이루어지고 있다.In addition, the disclosed strain ( Aspergillus niger KCTC 6906) used in the above patent cannot be used commercially, and the commercially available enzyme (Naringinase “”) has the disadvantage of being expensive, which makes the production cost of isoquercetin very high. In addition, the enzyme produced by the fungus is When purified and used, purification costs are added, so the price of the produced isoquercetin is very expensive. Therefore, research is continuously being conducted on efficient and economical methods of producing isoquercetin.
이에, 종래 기술에서 고가 효소의 사용 및 효소의 정제 과정을 생략하여 제조 비용이 최소화되면서도 항균성과 항산화 활성이 우수한 이소케르세틴을 제조할 수 있는 이소케르세틴의 제조방법이 요구된다.Accordingly, there is a need for a method for producing isoquercetin that can produce isoquercetin with excellent antibacterial and antioxidant activity while minimizing production costs by omitting the use of expensive enzymes and enzyme purification processes in the prior art.
본 발명은 식품 및 화장품 분야에 널리 사용되는 이소케르세틴의 제조방법에 있어서, 기존의 고가 효소의 사용 및 효소의 정제 과정을 생략하여 제조 비용이 최소화되면서도 항균성과 항산화 활성이 우수한 이소케르세틴을 제조할 수 있는 이소케르세틴의 제조방법을 제공하는 것을 해결하고자 하는 과제로 한다.The present invention relates to a method for producing isoquercetin, which is widely used in the food and cosmetics fields, by omitting the use of existing expensive enzymes and the enzyme purification process, so that isoquercetin with excellent antibacterial and antioxidant activity can be produced while minimizing production costs. The problem to be solved is to provide a method for producing isoquercetin.
상기 과제를 해결하기 위해, 본 발명은,In order to solve the above problems, the present invention,
(A) 식품에서 유래하며, 나린지네이즈(naringinase) 또는 람노시데이즈(rhamnosidase)를 생산 가능한 균주를 배양하는 단계; (B) 상기 균주로부터 배양 효소액을 획득하는 단계; 및 (C) 상기 효소액으로 루틴을 효소 반응시켜 루틴을 이소케르세틴으로 전환하는 단계;를 포함하는 것을 특징으로 하는 이소케르세틴의 제조방법을 제공한다.(A) cultivating a strain derived from food and capable of producing naringinase or rhamnosidase; (B) obtaining a culture enzyme solution from the strain; and (C) converting rutin into isoquercetin by performing an enzyme reaction of rutin with the enzyme solution.
여기서, 상기 (A) 단계의 상기 균주는 Talaromyces funiculosus DSF-4 (KCTC15183BP), Aspergillus oryzae DSF-12 (KCTC15184BP) 및 Talaromyces verruculosus DSF-31 (KCTC15185BP) 중 어느 하나의 균주를 포함하는 것을 특징으로 하는 이소케르세틴의 제조방법을 제공한다.Here, the strain in step (A) is an isotype characterized in that it includes any one of Talaromyces funiculosus DSF-4 (KCTC15183BP) , Aspergillus oryzae DSF-12 (KCTC15184BP), and Talaromyces verruculosus DSF-31 (KCTC15185BP). A method for producing quercetin is provided.
또한, 상기 (B) 단계는 균주를 효소 생산 배지에서 배양하여 균주 배양액을 획득하는 과정, 상기 균주 배양액을 여과하여 균주를 제거하는 과정 및 상기 균주가 제거된 배양액을 상기 배양 효소액으로서 획득하는 과정을 포함하는 것을 특징으로 하는 이소케르세틴의 제조방법을 제공한다.In addition, the step (B) includes a process of culturing the strain in an enzyme production medium to obtain a strain culture solution, a process of filtering the strain culture solution to remove the strain, and a process of obtaining the culture solution from which the strain has been removed as the culture enzyme solution. A method for producing isoquercetin comprising:
여기서, 상기 (B) 단계의 상기 배양 효소액은 정제되지 않는 것을 특징으로 하는 이소케르세틴의 제조방법을 제공한다.Here, a method for producing isoquercetin is provided, wherein the culture enzyme solution in step (B) is not purified.
한편, 상기 (C) 단계는 루틴 용액과 상기 효소액을 혼합하는 과정, 상기 혼합된 용액을 40℃내지 60℃에서 교반하여 효소 반응시키는 과정, 상기 효소 반응된 용액을 80℃내지 100℃에서 중탕시켜 효소 반응을 정지시키는 과정 및 상기 중탕된 용액에서 이소케르세틴을 수득하는 과정을 포함하는 것을 특징으로 하는 이소케르세틴의 제조방법을 제공한다.Meanwhile, step (C) is a process of mixing the rutin solution and the enzyme solution, stirring the mixed solution at 40°C to 60°C to cause an enzyme reaction, and bathing the enzyme-reacted solution at 80°C to 100°C. A method for producing isoquercetin is provided, comprising the steps of stopping the enzyme reaction and obtaining isoquercetin from the water-boiled solution.
본 발명은 이소케르세틴의 제조방법에 의하면, 식품 및 화장품 분야에 널리 사용되는 이소케르세틴의 제조방법에 있어서, 기존의 고가 효소의 사용 및 효소의 정제 과정을 생략하여 제조 비용이 최소화되면서도 항균성과 항산화 활성이 우수한 이소케르세틴을 제조할 수 있는 우수한 효과가 있다.According to the present invention, in the production method of isoquercetin, which is widely used in the food and cosmetic fields, the use of existing expensive enzymes and the enzyme purification process are omitted, thereby minimizing production costs and providing antibacterial and antioxidant activity. There is an excellent effect in producing this excellent isoquercetin.
도 1은 루틴의 전환물 및 루틴의 전환 방법을 도시한다.
도 2는 DSF-4 (Talaromyces funiculosus DSF-4)의 균주 계통도를 도시한다.
도 3은 DSF-12 (Aspergillus oryzae DSF-12)의 균주 계통도를 도시한다.
도 4는 DSF-31 (Talaromyces verruculosus DSF-31)의 균주 계통도를 도시한다.
도 5는 생물전환된 이소케르세틴의 HPLC 크로마토그램 분석결과를 도시한다.
도 6은 본 발명에 따른 이소케르세틴으로 제조된 이소케르세틴의 감자 무름병 방지 효과를 도시한다.1 shows a conversion product of a routine and a method of conversion of a routine.
Figure 2 shows the strain phylogenetic tree of Talaromyces funiculosus DSF-4 (DSF-4).
Figure 3 shows the strain phylogenetic tree of DSF-12 ( Aspergillus oryzae DSF-12).
Figure 4 shows the strain phylogenetic tree of Talaromyces verruculosus DSF-31 (DSF-31).
Figure 5 shows the results of HPLC chromatogram analysis of bioconverted isoquercetin.
Figure 6 shows the effect of isoquercetin prepared from isoquercetin according to the present invention in preventing potato soft rot.
이하, 첨부된 도면들을 참조하여 본 발명의 바람직한 실시예들을 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명된 실시예들에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 실시예들은 개시된 내용이 철저하고 완전해질 수 있도록, 그리고 당업자에게 발명의 사상이 충분히 전달될 수 있도록 하기 위해 제공되는 것이다. 명세서 전체에 걸쳐서 동일한 참조 번호들은 동일한 구성요소들을 나타낸다.Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosure will be thorough and complete, and so that the spirit of the invention can be sufficiently conveyed to those skilled in the art. Like reference numerals refer to like elements throughout the specification.
본 발명은 산화방지제, 색소 안정화제, 향기변화 방지제, 자외선 흡수제, 체지방 감소제 등으로서 식품, 화장품 분야에 널리 사용할 수 있는 이소케르세틴을 제조함에 있어서, 항균성 및 항산화 활성이 우수한 이소케르세틴을 비교적 저렴하면서도 수득율을 증가시켜 경제적으로 우수한 제조방법을 제공하는 것을 기술적 특징으로 한다.The present invention is to manufacture isoquercetin, which can be widely used in the food and cosmetics fields as an antioxidant, color stabilizer, fragrance change inhibitor, ultraviolet absorber, body fat reducer, etc., and isoquercetin, which has excellent antibacterial and antioxidant activities, is relatively inexpensive and Its technical feature is to provide an economically excellent manufacturing method by increasing the yield.
본 발명에 따른 이소케르세틴의 제조방법은 식품에서 유래하며, 나린지네이즈(naringinase) 또는 람노시데이즈(rhamnosidase)를 생산 가능한 균주를 배양하는 단계, (B) 상기 균주로부터 배양 효소액을 획득하는 단계 및 (C) 상기 효소액으로 루틴을 효소 반응시켜 루틴을 이소케르세틴으로 전환하는 단계를 포함한다.The method for producing isoquercetin according to the present invention is derived from food and includes the steps of cultivating a strain capable of producing naringinase or rhamnosidase, (B) obtaining a culture enzyme solution from the strain, and (C) comprising the step of converting rutin into isoquercetin by performing an enzyme reaction of rutin with the enzyme solution.
본 발명에 따른 이소케르세틴의 제조방법의 (A) 단계는 효소로서 나린지네이즈(naringinase) 또는 람노시데이즈(rhamnosidase)를 생산하는 균주를 배양할 수 있다. 상기 효소는 효소 반응하는 동안 루틴에서 람노오스를 제거하여 이소케르세틴으로 전환시킬 수 있다.Step (A) of the method for producing isoquercetin according to the present invention can be performed by cultivating a strain that produces naringinase or rhamnosidase as an enzyme. The enzyme can remove rhamnose from rutin and convert it into isoquercetin during the enzymatic reaction.
여기서, 상기 균주는 다양한 미생물이 포함된 각종 식품, 예를 들면 농산물, 과일, 채소, 누룩 등을 포함할 수 있다. 바람직하게, 상기 균주는 루틴의 전환율이 높고 전환 과정에서 독소를 생산하지 않는 균주, 예를 들면 Talaromyces funiculosus, Aspergillus oryzae 및 Talaromyces verruculosus 중 어느 하나의 균주를 포함할 수 있다.Here, the strain may include various foods containing various microorganisms, such as agricultural products, fruits, vegetables, yeast, etc. Preferably, the strain may include a strain that has a high conversion rate of rutin and does not produce toxins during the conversion process, for example, any one of Talaromyces funiculosus, Aspergillus oryzae , and Talaromyces verruculosus .
Talaromyces funiculosus는 파인애플을 감염시키는 식물 병원체이며, 또한 돼지 사료 로바비오 엑셀에 사용되는 비전분 다당류 가수분해 효소인 자일라나아제 및 베타글루카나아제 효소의 공급원으로 사용되는 균주이다. Talaromyces funiculosus is a plant pathogen that infects pineapple and is also a strain used as a source of xylanase and beta-glucanase enzymes, which are non-starch polysaccharide hydrolyzing enzymes used in swine feed Robabio Excel.
Aspergillus oryzae는 누룩곰팡이로도 불리우며, 자낭균류 누룩곰팡이속에 속하는 생물로 많은 종류가 있다. Aspergillus oryzae는 여러 가지 물질로부터 영양분을 섭취하여 성장할 수 있으며, 이 중에는 공업에 이용되는 것이 많다. 유성 생식을 할 때는 다세포인 균사체로부터 길쭉한 장낭기와 장정기가 이웃하여 생기며 이들이 서로 얽혀 접합하는 특징을 갖는다. Aspergillus oryzae , also called Aspergillus oryzae, is an organism belonging to the Aspergillus aspergillus genus. There are many types of Aspergillus oryzae. Aspergillus oryzae can grow by taking in nutrients from various substances, many of which are used in industry. During sexual reproduction, elongated sacs and stomata grow adjacent to each other from the multicellular mycelium, and these have the characteristic of intertwining and joining together.
Talaromyces verruculosus는 미국 토양에서 분리된 Penicillium 속의 아나모프 종의 곰팡이입니다. 그것은 베루쿨로겐, 베루코시딘, 베루쿨로톡신, 데칼펜산, 데하이드로알테누신, cyciooctasulfur, 아트로베네티논, 알테누신 및 페니트렘 A를 생성한다. Talaromyces verruculosus is a fungus of the anamorph species of the genus Penicillium isolated from soil in the United States. It produces verruculogen, verrucocidin, verruculotoxin, decalpenic acid, dehydroaltenusin, cyciooctasulfur, atrobenethinon, altenusin and fenitreme A.
본 발명에 따른 이소케르세틴의 제조방법의 (B) 단계는 균주를 효소 생산 배지에서 배양하여 균주 배양액을 획득하는 과정, 상기 균주 배양액을 여과하여 균주를 제거하는 과정 및 상기 균주가 제거된 배양액을 상기 배양 효소액으로서 획득하는 과정을 포함할 수 있다.Step (B) of the method for producing isoquercetin according to the present invention includes culturing the strain in an enzyme production medium to obtain a strain culture medium, filtering the strain culture medium to remove the strain, and filtering the strain culture medium to remove the strain. It may include a process of obtaining a culture enzyme solution.
여기서, 상기 배양 효소액은 정제되지 않은 상태의 효소액이다. 따라서, 종래의 기술과 같이 곰팡이가 생산하는 효소를 정제해서 사용할 경우 필수적으로 수반되던 정제공정이 생략되므로, 제조과정이 간소화되며, 이로부터 제조되는 이소케르세틴의 비용이 감소될 수 있으므로 효율적이고 경제적이다.Here, the culture enzyme solution is an enzyme solution in an unrefined state. Therefore, the purification process that is essential when using an enzyme produced by a fungus by purifying it as in the prior art is omitted, thereby simplifying the manufacturing process and reducing the cost of isoquercetin produced from it, making it efficient and economical. .
본 발명에 따른 이소케르세틴의 제조방법의 (C) 단계는 루틴 용액과 상기 효소액을 혼합하는 과정, 상기 혼합된 용액을 40℃내지 60℃에서 교반하여 효소 반응시키는 과정, 상기 효소 반응된 용액을 80℃내지 100℃에서 중탕시켜 효소 반응을 정지시키는 과정 및 상기 중탕된 용액에서 최종 생물전환된 이소케르세틴을 수득하는 과정을 포함할 수 있다.Step (C) of the method for producing isoquercetin according to the present invention is a process of mixing the rutin solution and the enzyme solution, stirring the mixed solution at 40°C to 60°C to perform an enzyme reaction, and heating the enzyme-reacted solution for 80 minutes. It may include a process of stopping the enzyme reaction by bathing at ℃ to 100℃ and obtaining the final bioconverted isoquercetin from the bathed solution.
본 발명은 하기 실험을 통해 식품 및 농산물 유래 곰팡이를 99종 분리하였고, 정제되지 않은 배양 효소액으로 루틴을 이소케르세틴으로 전환하는 효율을 분석한 후, 전환율이 우수한 균주를 동정하였다. 이 중에서 전환율이 높으면서 곰팡이 독소 생산 정보가 없는 균주를 선발하여 이소케르세틴의 제조방법을 완성하였다.The present invention isolated 99 types of fungi derived from food and agricultural products through the following experiment, analyzed the efficiency of converting rutin into isoquercetin using an unpurified culture enzyme solution, and identified a strain with an excellent conversion rate. Among these, a strain with a high conversion rate and no information on mycotoxin production was selected to complete the production method of isoquercetin.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail through examples.
<실험예 1> 식품 및 농산물 유래 효소 생산 곰팡이 분리<Experimental Example 1> Isolation of enzyme-producing fungi derived from food and agricultural products
루틴에서 람노오스를 제거하고 이소케르세틴으로 전환하는 효소로는 나린지네이즈(naringinase)와 람노시데이즈(rhamnosidase)가 있으며, 이 효소를 생산하는 곰팡이를 분리하기 위해서 나린진과 람노스를 각각 탄소원으로 포함하는 선택배지를 사용하였다. 다양한 미생물이 포함된 식품 및 농산물인 메주(순창, 해남, 예산, 나주 등), 누룩, 부패 진행중인 과일(귤, 한라봉, 딸기 등), 겨울 보관중인 채소(고구마, 마, 배추)에서 99종의 곰팡이를 분리하였다.The enzymes that remove rhamnose from rutin and convert it to isoquercetin include naringinase and rhamnosidase, and in order to isolate the fungus that produces this enzyme, naringin and rhamnose were included as carbon sources, respectively. A selective medium was used. 99 types of food and agricultural products containing various microorganisms, such as meju (Sunchang, Haenam, Yesan, Naju, etc.), yeast, fruits in the process of decay (tangerines, Hallabong, strawberries, etc.), and vegetables stored in winter (sweet potatoes, yam, cabbage) The mold was isolated.
미생물 분리원 10g을 멸균 생리식염수 90g에 현탁 후 분쇄한 여액을 나린진 선택배지(Naringin 10g/1L, Corn steep liquor 10g/L, K2HPO4 1g/L, KH2PO4 2g/L, MgSO4 0.1g /L, Agar 15g/L) 또는 람노스 선택배지(Rhamnose 10g/1L, Corn steep liquor 10g/L, K2HPO4 1g/L, KH2PO4 2g/L, MgSO4 0.1g/L, Agar 15g/L)에 100㎕씩 도말 후 30℃48시간 동안 배양하였다. 배양된 미생물의 형태적 특성이 상이한 균주들을 1차 선별한 다음, PDA(Potato Dextrose Agar) 배지에 계대 배양하여 순수 분리하였으며, 분리균의 특성을 아래 표 1에 나타냈다.10 g of the microbial isolate was suspended in 90 g of sterilized saline solution and the pulverized filtrate was cultured on Naringin selective medium (Naringin 10 g/1L, Corn steep liquor 10 g/L, K2HPO4 1g/L, KH2PO4 2g/L, MgSO4 0.1g/L, Agar 15g). /L) or smear 100㎕ each on rhamnose selective medium (Rhamnose 10g/1L, Corn steep liquor 10g/L, K2HPO4 1g/L, KH2PO4 2g/L, MgSO4 0.1g/L, Agar 15g/L) at 30℃. Cultured for 48 hours. Strains with different morphological characteristics of the cultured microorganisms were first selected, then subcultured on PDA (Potato Dextrose Agar) medium to pure isolate, and the characteristics of the isolates are shown in Table 1 below.
<실험예 2> 루틴 생물전환 곰팡이 선발 <Experimental Example 2> Selection of routine bioconversion fungi
상기에서 분리된 각각의 균주에 대해서 루틴에서 이소케르세틴으로 전환하는 효율을 평가하였다. 분리균들을 효소 생산 배지(Rhamnose 10g/L, Casamino acid 5g/L, FeSO4·7H2O 0.01g/L, MgSO4·7H2O 0.5g/L, K2HPO4 1g/L, NaNO3 0.5g/L)에서 30℃120 rpm, 7일간 배양 후, 배양액을 여과하여(Whatman No.1) 균체를 제거한 배양 여액을 생물전환 효소액으로 사용하였다.For each of the strains isolated above, the efficiency of conversion from rutin to isoquercetin was evaluated. The isolated bacteria were grown in enzyme production medium (Rhamnose 10g/L, Casamino acid 5g/L, FeSO4·7H2O 0.01g/L, MgSO4·7H2O 0.5g/L, K2HPO4 1g/L, NaNO3 0.5g/L) at 30℃120 rpm. , After culturing for 7 days, the culture solution was filtered (Whatman No. 1) to remove bacterial cells, and the culture filtrate was used as a bioconversion enzyme solution.
9 g 배양 효소액과 1 g 루틴 기질 용액을{10% Rutin, 10% MeOH, 90% 0.1M PBS buffer(pH7.0)} 혼합 후, 50℃에서 24시간, 150 rpm으로 효소 반응을 시킨 후, 90℃에서 10분간 중탕 가온하여 효소 반응을 정지시켰다.After mixing 9 g culture enzyme solution and 1 g rutin substrate solution {10% Rutin, 10% MeOH, 90% 0.1M PBS buffer (pH7.0)}, the enzyme reaction was performed at 50°C for 24 hours at 150 rpm, The enzyme reaction was stopped by heating in a double boiler at 90°C for 10 minutes.
생물전환 효율을 분석하기 위해서 기질인 루틴과 전환된 이소케르세틴 함량을 아래 표 2의 분석 조건을 만족하는 HPLC로 확인하였고, 루틴과 이소케르세틴의 피크 면적의 합에 대한 이소케르세틴의 피크 면적비율을 산출하여 이소케르세틴 전환율(%)을 구하였다.To analyze the bioconversion efficiency, the contents of the substrate, rutin, and converted isoquercetin were confirmed by HPLC satisfying the analysis conditions in Table 2 below, and the peak area ratio of isoquercetin to the sum of the peak areas of rutin and isoquercetin was calculated. The isoquercetin conversion rate (%) was obtained.
분리균 99종 전체의 이소케르세틴 전환율을 평가한 결과는 아래 표 3과 같으며, 전환율이 높은 균주들을 동정한 후, 곰팡이 독소 생산 정보가 없는 균주인 DSF-4, DSF-12, DSF-31 세 균주를 최종적으로 선발하였다. 전환되지 않은 균주는 제외하였다. 특히 DSF-4 균주는 상업 효소(Naringinase 'Amano')를 0.6% 첨가하여 반응시킨 이소케르세틴 생성양과 유사하였다. 더욱이, 상기 균주들은 효소 생산 배지 최적화를 통해서 이소케르세틴 생산량을 증가시킬 수 있었다.The results of evaluating the isoquercetin conversion rate of all 99 isolates are shown in Table 3 below. After identifying strains with high conversion rates, DSF-4, DSF-12, and DSF-31, which are strains without mycotoxin production information, are shown in Table 3 below. Strains were finally selected. Strains that did not convert were excluded. In particular, the DSF-4 strain produced a similar amount of isoquercetin when reacted with 0.6% of commercial enzyme (Naringinase 'Amano'). Moreover, the strains were able to increase isoquercetin production through optimization of the enzyme production medium.
또한, 도 5는 각각 표준물질, DSF-4 (Talaromyces funiculosus) 배양 효소액, DSF-12 (Aspergillus oryzae) 배양 효소액, DSF-31 (Talaromyces verruculosus) 배양 효소액으로 루틴을 효소 반응시켜 최종적으로 생물 전환된 이소케르세틴의 HPLC 크로마토그램 분석결과를 도시한다.In addition, Figure 5 shows the final bioconversion of rutin by enzymatic reaction with standard materials, DSF-4 ( Talaromyces funiculosus ) culture enzyme solution, DSF-12 ( Aspergillus oryzae ) culture enzyme solution, and DSF-31 ( Talaromyces verruculosus ) culture enzyme solution, respectively. The results of HPLC chromatogram analysis of quercetin are shown.
<실험예 3> 루틴 생물전환 균주 동정<Experimental Example 3> Routine bioconversion strain identification
상기 선발된 DSF-4, DSF-12, DSF-31 균주의 18S rRNA 유전자 염기서열을 분석한 결과, DSF-4는탈라로마이세스 푸니큘로서스(KCTC15183BP, Talaromyces funiculosus DSF-4), DSF-12는 아스퍼질러스 오리제(KCTC15184BP, Aspergillus oryzae DSF-12) DSF-31은 탈라로마이세스 베르큘로서스(KCTC15185BP, Talaromyces verruculosus DSF-31)로 각각 동정하였고, 한국생명공학연구원 생물자원센터(KCTC, Korean Collection for Type Cultures)에 균주를 특허기탁하였다.As a result of analyzing the 18S rRNA gene sequences of the selected strains of DSF-4, DSF-12, and DSF-31, DSF-4 was Talaromyces funiculosus (KCTC15183BP, Talaromyces funiculosus DSF-4), DSF- 12 was identified as Aspergillus oryzae (KCTC15184BP, Aspergillus oryzae DSF-12) and DSF-31 was identified as Talaromyces verruculosus (KCTC15185BP, Talaromyces verruculosus DSF-31), and the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center ( The strain was patented and deposited in KCTC, Korean Collection for Type Cultures.
도 2는 DSF-4 (Talaromyces funiculosus DSF-4)의 균주 계통도를 도시하고, 도 3은 DSF-12 (Aspergillus oryzae DSF-12)의 균주 계통도를 도시하고, 도 4는 DSF-31 (Talaromyces verruculosus DSF-31)의 균주 계통도를 도시한다.Figure 2 shows the strain tree of DSF-4 ( Talaromyces funiculosus DSF-4), Figure 3 shows the strain tree of DSF-12 ( Aspergillus oryzae DSF-12), and Figure 4 shows the strain tree of DSF-31 ( Talaromyces verruculosus DSF). -31) shows the strain phylogeny.
<실험예 4> 람노시데이즈 역가 분석 <Experimental Example 4> Rhamnosidase titer analysis
곰팡이 배양액의 람노시데이즈 효소 활성을 평가하였다. 기질 용액(0.5 ㎖)에 완충액(0.5 ㎖)을 넣고 잘 혼합한 다음, 배양 여액(효소액, 0.1 ㎖)을 넣고 50℃10분 반응 후에 반응 종료액(3 ㎖)을 첨가하여 효소 반응을 정지시켰다. 시판 효소액은 약 1,000~2,000배 희석 후 반응시켰으며, 역가 산정 시 희석배수를 반영하였다. 형성된 p-nitrophenol은 405 nm에서 흡광도를 측정하고, 검량선에 대입하여 정량하였다. 여기서, 기질용액, 완충액, 반응 종료액 및 검량용 표면물질에 사용된 시약 종류는 아래 표 4와 같다.The rhamnosidase enzyme activity of the fungal culture was evaluated. Add buffer solution (0.5 ml) to substrate solution (0.5 ml) and mix well, then add culture filtrate (enzyme solution, 0.1 ml), and after reaction at 50°C for 10 minutes, reaction termination solution (3 ml) was added to stop the enzyme reaction. . Commercially available enzyme solutions were reacted after being diluted approximately 1,000 to 2,000 times, and the dilution factor was reflected when calculating the titer. The formed p-nitrophenol was quantified by measuring the absorbance at 405 nm and substituting it into the calibration curve. Here, the types of reagents used in the substrate solution, buffer solution, reaction completion solution, and surface material for calibration are shown in Table 4 below.
효소 1 Unit은 기질로부터 분당 1 μmol의 p-nitrophenol을 생성하는 효소의 양을 의미하며, 효소 역가 계산법은 아래 [식 1]과 같다. [식 1]을 통해 계산된 배양 효소액 및 시판 효소의 람노시데이즈 역가는 아래 표 5에 나타난 바와 같다.1 Enzyme Unit refers to the amount of enzyme that produces 1 μmol of p-nitrophenol per minute from the substrate, and the enzyme titer calculation method is as follows [Equation 1]. The rhamnosidase titers of the cultured enzyme solution and commercially available enzymes calculated using [Equation 1] are shown in Table 5 below.
[식 1][Equation 1]
C: 검량선에서 얻은 시험용액의 p-nitrophenyl의 농도(μmol/㎖)C: Concentration of p-nitrophenyl in the test solution obtained from the calibration curve (μmol/ml)
D: 시험용액의 희석배수(㎖)D: Dilution factor of test solution (ml)
W: 검체의 채취량(g)W: sample collection amount (g)
10: 반응시간(min)10: Response time (min)
<실험예 5> 이소케르세틴의 항산화 및 항균 활성<Experimental Example 5> Antioxidant and antibacterial activity of isoquercetin
5-1. 이소케르세틴의 항산화 활성5-1. Antioxidant activity of isoquercetin
이소케르세틴 (Isoquercetin)의 항산화 활성은 자유라디칼 소거능 측정법인 DPPH (2,2-Diphenyl-1-picrylhydrazyl) 분석법을 사용하였다. 농도별로 희석된 샘플에 DPPH 시약을 가하여 반응은 빛이 차단된 35℃에서 30분 동안 반응시켰으며 517nm에서 흡광도를 측정하였다. 측정된 흡광도값을 이용하여 DPPH 라디칼 소거능(%)을 계산하였다.Isoquercetin's antioxidant activity was measured using DPPH (2,2-Diphenyl-1-picrylhydrazyl) analysis, a free radical scavenging ability measurement method. DPPH reagent was added to the sample diluted by concentration, reaction was carried out for 30 minutes at 35°C with light blocked, and absorbance was measured at 517 nm. DPPH radical scavenging ability (%) was calculated using the measured absorbance value.
DPPH 라디칼을 50% 소거하는 농도를 계산하여 항산화능력을 평가하였으며, 이를 IC50 (Inhibitory concentration of 50%)로 나타냈으며, 이 값이 낮을수록 물질의 항산화능이 우수함을 뜻한다.Antioxidant ability was evaluated by calculating the concentration that eliminates 50% of DPPH radicals, and this was expressed as IC50 (Inhibitory concentration of 50%). The lower this value, the better the antioxidant ability of the substance.
DPPH 라디칼 소거능은 아래 [식 2]에 따라 산출되었다.DPPH radical scavenging ability was calculated according to [Equation 2] below.
[식 2][Equation 2]
DPPH 소거능(%) = {1-(A-C)/B} X 100DPPH scavenging ability (%) = {1-(A-C)/B}
A : 시료+시약 흡광도A: Sample + reagent absorbance
C : 시료+에탄올 흡광도C: sample + ethanol absorbance
B : 에탄올+시약 흡광도B: Ethanol + reagent absorbance
이소케르세틴 시약(Sigma, 90%) 및 DSF-4(Talaromyces funiculosus DSF-4) 균주 효소액으로 전환한 이소케르세틴(60%)으로 DPPH 라디칼 소거능을 측정한 결과, 아래 표 6과 같이 각각 IC50값이 10.6 ppm, 12.7 ppm으로 매우 우수한 항산화 활성을 나타냈다. 이는 항산화 활성으로 가장 잘 알려진 차추출물과 유사한 활성이다.As a result of measuring the DPPH radical scavenging ability with isoquercetin reagent (Sigma, 90%) and isoquercetin (60%) converted to DSF-4 ( Talaromyces funiculosus DSF-4) strain enzyme solution, the IC50 value was 10.6, respectively, as shown in Table 6 below. ppm, showing very excellent antioxidant activity at 12.7 ppm. This activity is similar to that of tea extract, which is best known for its antioxidant activity.
5-2. 이소케르세틴의 항균 활성5-2. Antibacterial activity of isoquercetin
DSF-4(Talaromyces funiculosus DSF-4) 균주 배양 여액으로 전환한 이소케르세틴(60%)의 항균 활성을 최소생육저해 농도값(MIC, Minimum inhibition concentration, CLSI microdilution method)으로 평가하였으며, 그 결과값은 아래 표 7과 같다.The antibacterial activity of isoquercetin (60%) converted into DSF-4 ( Talaromyces funiculosus DSF-4) strain culture filtrate was evaluated using the minimum growth inhibition concentration (MIC) value (CLSI microdilution method), and the results were It is as shown in Table 7 below.
<실험예 6> 이소케르세틴을 포함한 코팅액의 감자 무름병 방지 효과<Experimental Example 6> Potato soft rot prevention effect of coating liquid containing isoquercetin
과일, 채소의 무름병 원인균인 Pectobacterium carotovorum에 의해 유발되는 무름병 방지 효능을 확인하였다. DSF-4(Talaromyces funiculosus DSF-4) 배양 효소액으로 전환한 이소케르세틴을 DMSO(Dimethyl sulfoxide)에 용해한 다음, 가용화한 1% 카복시메틸셀룰로스 용액(w/w, 400 rpm, 70℃최종 0.05%, 0.1%가 되도록 각각 첨가하고, 마지막으로 글리세린(0.2%)을 넣고 교반하여 코팅액을 제조하였다. 홈을 판 감자에 무름병균인 Pectobacterium carotovorum 균주를 도포한 후, 그 위에 제조한 코팅액으로 코팅하여 보관하면서 무름병 발생 정도를 확인하였다.The efficacy of preventing soft rot caused by Pectobacterium carotovorum, a pathogen causing soft rot of fruits and vegetables, was confirmed. Isoquercetin converted to DSF-4 ( Talaromyces funiculosus DSF-4) culture enzyme solution was dissolved in DMSO (Dimethyl sulfoxide) and then solubilized in 1% carboxymethyl cellulose solution (w/w, 400 rpm, 70°C, final 0.05%, 0.1%). %, and finally, glycerin (0.2%) was added and stirred to prepare a coating solution. After applying the soft rot fungus Pectobacterium carotovorum strain to the grooved potato, it was coated with the prepared coating solution and stored to prevent soft rot. The extent of occurrence was confirmed.
구체적으로 감자는 흐르는 수돗물에 세척하여 감자 표면에 붙은 흙과 먼지를 제거한 뒤 1% 차아염소산나트륨 용액(v/v)에 15분간 침지 하여 표면 부착균을 제거하고, 증류수로 1회 세척 진행하여 잔여 이물과 차아염소산나트륨을 제거한 뒤 자연 건조하여 감자 표면의 물기를 제거했다. 감자는 껍질을 제거하지 않은 채로 껍질 부분에 cork boarder를 이용해 직경 7mm, 깊이 1mm의 홈을 파고, 배양된 Pectobacterium carotovorum PCC3 배양액(PDB 배지, 30℃ 24hr)을 균수가 105~6 CFU/ml 이 되도록 홈에 접종한 후, 균이 잘 부착될 수 있도록 1시간 동안 자연 건조하였다. 건조된 감자 홈에 상기 이소케르세틴을 포함하는 코탱액을 적정량 도포한 후, 2~3 시간 동안 완전히 건조한 다음, 항온항습기(30 ℃85% RH)에 보관하며 저장시간 경과에 따른 무름 발생을 확인하였다.Specifically, potatoes were washed in running tap water to remove dirt and dust attached to the potato surface, then immersed in 1% sodium hypochlorite solution (v/v) for 15 minutes to remove surface-adhering bacteria, and washed once with distilled water to remove any remaining bacteria. After removing foreign substances and sodium hypochlorite, the potatoes were naturally dried to remove moisture from the surface of the potatoes. Without removing the skin, a groove with a diameter of 7 mm and a depth of 1 mm was dug in the skin of the potato using a cork boarder, and the cultured Pectobacterium carotovorum PCC3 culture medium (PDB medium, 30°C for 24 hours) was cultured until the bacterial count was 10 5 to 6 CFU/ml. After inoculating as much of the groove as possible, it was naturally dried for 1 hour to allow the bacteria to adhere well. After applying an appropriate amount of Cotin solution containing the isoquercetin to the dried potato grooves, drying completely for 2 to 3 hours, storing in a constant temperature and humidity chamber (30°C 85% RH), the occurrence of softness over storage time was confirmed. .
도 6은 본 발명에 따른 이소케르세틴으로 제조된 이소케르세틴의 감자 무름병 방지 효과를 도시한다. 구체적으로, 비교예 1인 음성대조구(Negative control)는 무처리, 비교예 2인 양성대조구(Positive control)는 무름병 균액만 처리한 경우, 실시예는 0.05% 이소케르세틴이 포함된 코팅액을 감자 시편에 처리한 경우 건조된 감자 시편의 단면 상태를 촬영하였다. 도 6에 도시된 바와 같이, 비교예 1 및 무름병 균액만 처리한 비교예 2(양성대조군) 대비 실시예(이소케르세틴이 포함된 코팅액 처리구)의 무름병 발생이 50% 이상 저하되는 것을 확인했다.Figure 6 shows the effect of isoquercetin prepared from isoquercetin according to the present invention in preventing potato soft rot. Specifically, Comparative Example 1, the negative control, was untreated, Comparative Example 2, the positive control, was treated with only the soft rot bacterial solution, and in the Example, a coating solution containing 0.05% isoquercetin was applied to the potato specimen. When treated, the cross-sectional state of the dried potato specimen was photographed. As shown in Figure 6, it was confirmed that the occurrence of soft rot in Example (treated with coating solution containing isoquercetin) was reduced by more than 50% compared to Comparative Example 1 and Comparative Example 2 (positive control) treated only with soft rot bacterial solution.
본 명세서는 본 발명의 바람직한 실시예를 참조하여 설명하였지만, 해당 기술분야의 당업자는 이하에서 서술하는 특허청구범위에 기재된 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경 실시할 수 있을 것이다. 그러므로 변형된 실시가 기본적으로 본 발명의 특허청구범위의 구성요소를 포함한다면 모두 본 발명의 기술적 범주에 포함된다고 보아야 한다.Although this specification has been described with reference to preferred embodiments of the present invention, those skilled in the art may make various modifications and changes to the present invention without departing from the spirit and scope of the present invention as set forth in the claims described below. It will be possible to implement it. Therefore, if the modified implementation basically includes the elements of the claims of the present invention, it should be considered to be included in the technical scope of the present invention.
Claims (5)
(B) 상기 균주로부터 배양 효소액을 획득하는 단계; 및
(C) 상기 효소액으로 루틴을 효소 반응시켜 루틴을 이소케르세틴으로 전환하는 단계;를 포함하고,
상기 (A) 단계의 상기 균주는 Talaromyces funiculosus DSF-4 (KCTC15183BP) 또는 Talaromyces verruculosus DSF-31 (KCTC15185BP)을 포함하는 것을 특징으로 하는, 이소케르세틴의 제조방법.(A) cultivating a strain derived from food and capable of producing naringinase or rhamnosidase;
(B) obtaining a culture enzyme solution from the strain; and
(C) converting rutin into isoquercetin by enzymatically reacting rutin with the enzyme solution;
A method for producing isoquercetin, wherein the strain in step (A) includes Talaromyces funiculosus DSF-4 (KCTC15183BP) or Talaromyces verruculosus DSF-31 (KCTC15185BP).
상기 (B) 단계는 균주를 효소 생산 배지에서 배양하여 균주 배양액을 획득하는 과정, 상기 균주 배양액을 여과하여 균주를 제거하는 과정 및 상기 균주가 제거된 배양액을 상기 배양 효소액으로서 획득하는 과정을 포함하는 것을 특징으로 하는 이소케르세틴의 제조방법.According to paragraph 1,
The step (B) includes the steps of cultivating the strain in an enzyme production medium to obtain a strain culture solution, filtering the strain culture solution to remove the strain, and obtaining the culture solution from which the strain has been removed as the culture enzyme solution. A method for producing isoquercetin, characterized in that.
상기 (B) 단계의 상기 배양 효소액은 정제되지 않는 것을 특징으로 하는 이소케르세틴의 제조방법.According to paragraph 3,
A method for producing isoquercetin, characterized in that the culture enzyme solution in step (B) is not purified.
상기 (C) 단계는 루틴 용액과 상기 효소액을 혼합하는 과정, 상기 혼합된 용액을 40℃내지 60℃에서 교반하여 효소 반응시키는 과정, 상기 효소 반응된 용액을 80℃내지 100℃에서 중탕시켜 효소 반응을 정지시키는 과정 및 상기 중탕된 용액에서 이소케르세틴을 수득하는 과정을 포함하는 것을 특징으로 하는 이소케르세틴의 제조방법.According to paragraph 1,
The step (C) is a process of mixing the rutin solution and the enzyme solution, stirring the mixed solution at 40 ℃ to 60 ℃ for enzyme reaction, and boiling the enzyme-reacted solution at 80 ℃ to 100 ℃ for enzyme reaction. A method for producing isoquercetin, comprising the steps of stopping and obtaining isoquercetin from the water-boiled solution.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030013371A (en) * | 2000-02-11 | 2003-02-14 | 메르크 파텐트 게엠베하 | Method for producing monoglycosidated flavonoids |
| CN1261585C (en) * | 2003-08-01 | 2006-06-28 | 金凤燮 | Method for preparing isoquercetin and quercetin by enzymatic method and hydrolyzing rutin |
| KR20100001907A (en) * | 2008-06-27 | 2010-01-06 | 재단법인서울대학교산학협력재단 | Method for preparation of quercetin -3-glucoside from rutin using rhamnosidase originated from aspergillus niger |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030013371A (en) * | 2000-02-11 | 2003-02-14 | 메르크 파텐트 게엠베하 | Method for producing monoglycosidated flavonoids |
| CN1261585C (en) * | 2003-08-01 | 2006-06-28 | 金凤燮 | Method for preparing isoquercetin and quercetin by enzymatic method and hydrolyzing rutin |
| KR20100001907A (en) * | 2008-06-27 | 2010-01-06 | 재단법인서울대학교산학협력재단 | Method for preparation of quercetin -3-glucoside from rutin using rhamnosidase originated from aspergillus niger |
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