TWI461437B - Rapid extration method for fucoidan - Google Patents
Rapid extration method for fucoidan Download PDFInfo
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本發明係關於一種萃取褐藻糖膠之方法,特定言之,是一種使用少量褐藻快速解簡易萃取褐藻糖膠之方法。The invention relates to a method for extracting fucoidan gum, in particular, a method for rapidly extracting fucoidan gum by using a small amount of brown algae.
褐藻糖膠(Fucoidan)為近年來新興的保健飲食品,是一種水溶性食物纖維,主要存在於褐藻中,多取自海蘊和裙帶菜的孢子葉、海帶等,其中主要的多醣體為硫酸岩藻糖(Fucose)。Fucoidan is a new health food and beverage in recent years. It is a water-soluble dietary fiber mainly found in brown algae. It is derived from spore leaves and kelp of seaweed and wakame. The main polysaccharide is sulfuric acid. Fucose.
過去的萃取方式係以酸萃取以及鹽類沉澱萃取為主,一般為經過加熱煮沸、鹽類沉澱、冷凍乾燥等步驟,至少需時兩天的流程,且需1公斤以上較大量的褐藻原料。有文獻利用調整不同的酸鹼度及萃取溫度而萃取出多醣體,在進行醣類成分之檢測(J. Sci. Food Agric.,P. 122,1952 March;Glycoconj J.,16:19,1999 January)。但習知萃取方式耗費相當長的時程,且需耗費大量的褐藻原料。In the past, the extraction method was mainly acid extraction and salt precipitation extraction, generally followed by heating, boiling, salt precipitation, freeze-drying, etc., which took at least two days of processing, and required a larger amount of brown algae raw material of more than 1 kg. In the literature, polysaccharides are extracted by adjusting different pH and extraction temperature, and sugar components are detected (J. Sci. Food Agric., P. 122, 1952 March; Glycoconj J., 16:19, 1999 January) . However, conventional extraction methods take a relatively long time course and require a large amount of brown algae raw materials.
因褐藻的養殖方式不同以及季節的變化,每批收穫之褐藻中褐藻糖膠的含量不一,進行萃取褐藻糖膠前,如能以簡易之方法預前檢測該批褐藻中褐藻糖膠的含量,選出具相當高含量之褐藻糖膠脂原料進行褐藻糖膠萃取的工業製程,可使其更有效率。然而迄今,仍無任何可於短時間內使用少量褐藻萃取褐藻糖膠之方法。Due to the different culture methods of brown algae and seasonal changes, the content of fucoidan in each batch of brown algae harvested is different. Before extracting fucoidan, the content of fucoidan in the brown algae can be detected preliminarily by simple method. The industrial process of extracting fucoidan with a relatively high content of fucoidan gum raw material can be made more efficient. However, to date, there has been no method for extracting fucoidan using a small amount of brown algae in a short time.
本發明旨在解決習知褐藻糖膠成份含量檢測需耗費大量褐藻原料以及檢驗時間,而提供一種僅需少量之藻類原料,快速且簡易地萃取出褐藻糖膠之新穎方法,可製備得足供進一步分析檢測其中成份含量。The invention aims to solve the problem that the detection of the content of the fucoidan component requires a large amount of brown algae raw materials and the inspection time, and provides a novel method for extracting fucoidan quickly and easily by using only a small amount of algae raw materials, and the invention can be prepared sufficiently. Further analysis to determine the content of the ingredients.
一方面,本發明提供一種自藻類快速萃取褐藻糖膠的方法,其包括下列步驟:In one aspect, the invention provides a method for rapidly extracting fucoidan from algae, comprising the steps of:
(1)取少量藻類原料溶解於水或水溶液中得藻類水溶液;(1) taking a small amount of algae raw material dissolved in water or an aqueous solution to obtain an aqueous algae solution;
(2)以核糖核酸水解酵素處理該藻類水溶液;(2) treating the aqueous solution of the algae with ribonuclease;
(3)再加入酚(phenol)/胍(guanidine)/氯仿(chloroform)試劑處理一段時間萃取;(3) further adding phenol/guanidine/chloroform reagent for a period of time;
(4)再加入氯仿快速劇烈混合均勻後,以高速離心取上清液;(4) After adding chloroform and mixing rapidly and vigorously, the supernatant is taken by high speed centrifugation;
(5)加入等量之第一醇類均勻混合,再以高速離心取底部之萃取物,去除上清液;(5) adding an equal amount of the first alcohol to be uniformly mixed, and then taking the bottom extract at a high speed to remove the supernatant;
(6)加入第二醇類水溶液萃取物,離心去上清液得褐藻糖膠。(6) Adding a second alcohol aqueous solution extract, and centrifuging the supernatant to obtain fucoidan.
根據本發明方法,所需之藻類原料量僅約0.1mg~10mg,因使用之量極少,且所需之處理時程僅約數小時以內。根據本發明師實施例為約1小時。而所萃取得的褐藻糖膠量足供用以分析比較其中之成份含量,藉此選取較優品質且較高含量之褐藻進行大規模之加工。According to the process of the present invention, the amount of algae material required is only about 0.1 mg to 10 mg, since the amount used is extremely small, and the required treatment time is only about several hours. According to the embodiment of the present invention, it is about 1 hour. The amount of fucoidan extracted is sufficient for analysis and comparison of the content of the components, thereby selecting a relatively high quality and high content of brown algae for large-scale processing.
於本文中使用之藻類原料,係為用以萃取褐藻糖膠之藻類原料,一般以褐藻為主,特別是海蘊。The algae raw material used in the present invention is an algae raw material for extracting fucoidan, and is generally mainly brown algae, especially seaweed.
本發明中,係使用以水或水溶液溶解藻類原料,其得以任何方式製成小顆粒,例如粉末狀,再以水或任何適當之水溶液溶解之;例如蒸餾水或二次蒸餾水。並得加溫,例如以37℃加熱,使其均勻溶解於水或水溶液中。In the present invention, the algae raw material is dissolved in water or an aqueous solution, which can be made into small particles, such as a powder, in any manner, and then dissolved in water or any suitable aqueous solution; for example, distilled water or double distilled water. It is heated, for example, by heating at 37 ° C to uniformly dissolve it in water or an aqueous solution.
根據本發明,為達水解核糖核酸之目的,可以核糖核酸水解酵素在適當溫度下靜置一段時間;本發明實施例中,可使用核糖核酸水解酵素(RNase A)在37℃水浴槽中靜置10分鐘。隨之,以一種酚/胍/氯仿試劑處理一段時間萃取出所需之多醣;本發明實施例中,係使用試劑混合到均勻為止。繼而,加入適量氯仿,劇烈混合均勻,以高速離心取上清液;再加入等量之第一醇類均勻混合,以高速離心取底部之萃取物,去除上清液;從而加入第二醇類水溶液,經適當時間萃取後離心去上清液得褐藻糖膠。依本發明實施例,以高速離心得以12,000rpm的速度離心;其中第一醇類及第二醇類水溶液可為任何萃取多醣之醇類試劑,其中較佳者,第一醇類為2-丙醇,而第二醇類水溶液為75%乙醇,以適當轉速離心,去除上清液,萃取得褐藻糖膠。此時可將萃取得之褐藻糖膠回溶於水中待分析。According to the present invention, for the purpose of hydrolyzing ribonucleic acid, the ribonuclease can be allowed to stand at a suitable temperature for a certain period of time; in the embodiment of the present invention, ribonucleotidase (RNase A) can be used to stand in a 37 ° C water bath. 10 minutes. Subsequently, the desired polysaccharide is extracted by treatment with a phenol/indole/chloroform reagent for a period of time; in the embodiment of the present invention, it is used. The reagents are mixed until homogeneous. Then, add appropriate amount of chloroform, mix thoroughly, and centrifuge at high speed to remove the supernatant; then add an equal amount of the first alcohol to mix uniformly, centrifuge at high speed to remove the supernatant, and remove the supernatant; thus adding the second alcohol. The aqueous solution is extracted after appropriate time, and then centrifuged to remove the supernatant to obtain fucoidan. According to an embodiment of the present invention, centrifugation at a speed of 12,000 rpm is performed by high speed centrifugation; wherein the first alcohol and the second alcohol aqueous solution may be any alcohol reagent for extracting polysaccharide, and preferably, the first alcohol is 2-prop. The alcohol is used, and the second alcohol solution is 75% ethanol, centrifuged at an appropriate rotation speed, the supernatant is removed, and fucoidan is extracted. At this point, the extracted fucoidan gel can be dissolved back into the water for analysis.
本發明之另一特徵在於僅需使用相當低量之藻類原料。本發明之一實施例中,係取約5mg海蘊粉末,加入約100μl二次蒸餾水,繼之加入約1μl之RNase A。再加入約1ml之試劑混合均勻以萃取之。再加入約200μl氯仿,劇烈混合均勻,以高速離心後取上清液,加入等量的2-丙醇均勻混合。再以高速離心取底部之萃取物。加入約1ml冰的75%乙醇,離心去除上清液得萃取之褐藻糖膠萃取物,約3mg。Another feature of the invention is that only a relatively low amount of algae material needs to be used. In one embodiment of the invention, about 5 mg of marine powder is taken, about 100 μl of secondary distilled water is added, followed by about 1 μl of RNase A. Add about 1ml The reagents were mixed well to extract. Further, about 200 μl of chloroform was added, and the mixture was vigorously mixed uniformly. After centrifugation at a high speed, the supernatant was taken, and an equal amount of 2-propanol was added and uniformly mixed. The bottom extract was then centrifuged at high speed. About 1 ml of ice 75% ethanol was added, and the supernatant was removed by centrifugation to obtain an extracted fucoidan extract of about 3 mg.
另一方面,本發明提供一種自藻類快速檢測褐藻糖膠成份含量的方法,其包括下列步驟:In another aspect, the present invention provides a method for rapidly detecting the content of fucoidan from algae, comprising the steps of:
(1)取少量藻類原料溶解於水或水溶液中得藻類水溶液;(1) taking a small amount of algae raw material dissolved in water or an aqueous solution to obtain an aqueous algae solution;
(2)以核糖核酸水解酵素處理該藻類水溶液;(2) treating the aqueous solution of the algae with ribonuclease;
(3)再加入酚/胍/氯仿試劑處理一段時間萃取;(3) further adding phenol / hydrazine / chloroform reagent for a period of time extraction;
(4)再加入氯仿快速劇烈混合均勻後,以高速離心取上清液;(4) After adding chloroform and mixing rapidly and vigorously, the supernatant is taken by high speed centrifugation;
(5)加入等量之第一醇類均勻混合,再以高速離心取底部之萃取物,去除上清液;(5) adding an equal amount of the first alcohol to be uniformly mixed, and then taking the bottom extract at a high speed to remove the supernatant;
(6)加入第二醇類水溶液萃取物,離心去上清液得褐藻糖膠;(6) adding a second alcohol aqueous solution extract, and centrifuging the supernatant to obtain fucoidan;
(7)將所得褐藻糖膠萃取物回溶於水或水溶液中檢測其中醣類成分及含量。(7) The obtained fucoidan extract is dissolved in water or an aqueous solution to detect the sugar component and content thereof.
根據本發明,可以任何以之方法檢測萃取物中之醣類成分及含量。於本發明之實施例中,係使用酵素免疫微盤分析儀檢測所得萃取物中之多醣含量;使用分光光度計檢測所得萃取物中之硫酸根含量;及使用醣類分析儀(Sugar analysis system Dionex DX-500)分析其中單糖種類及含量。According to the present invention, the saccharide component and content in the extract can be detected by any method. In the embodiment of the present invention, the polysaccharide content in the obtained extract is detected by using an enzyme immuno-microplate analyzer; the sulfate content in the obtained extract is detected by using a spectrophotometer; and a sugar analyzer (Sugar analysis system Dionex) is used. DX-500) analyzes the type and content of monosaccharides.
首先將乾燥的海蘊用研缽研磨成細小的粉末,取5mg海蘊粉末,置入1.5ml之微量離心管(eppendrof tube)中。加入100μl二次蒸餾水,於37℃加熱使其均勻溶解。加入1μl之RNase A(Sigma Co,美國),在37℃水浴槽中靜置10分鐘。再加入1ml之試劑(Invitrogen,美國)混合到均勻為止,約3分鐘。加入200μl氯仿,劇烈混合均勻,約30秒。以高速離心機,以12,000rpm的速度離心後將上清液移置新的1.5ml微量離心管中,並加入等量的2-丙醇均勻混合。再使用高速離心機,以12,000rpm的速度離心,取底部之萃取物,去除上清液。加入1ml冰的75%乙醇,以7,500rpm的速度離心,去除上清液,將離心下來的萃取物風乾。得到約3mg萃取物。將萃取物回溶於100μl二次蒸餾水中,待分析。First, the dried seaweed was ground into a fine powder with a mortar, and 5 mg of seaweed powder was taken and placed in a 1.5 ml eppendrof tube. 100 μl of double distilled water was added and heated at 37 ° C to dissolve uniformly. 1 μl of RNase A (Sigma Co, USA) was added and allowed to stand in a 37 ° C water bath for 10 minutes. Add 1ml again The reagent (Invitrogen, USA) was mixed until homogeneous for about 3 minutes. Add 200 μl of chloroform and mix vigorously for about 30 seconds. After centrifugation at 12,000 rpm in a high speed centrifuge, the supernatant was transferred to a new 1.5 ml microcentrifuge tube and uniformly mixed with an equal amount of 2-propanol. Then, using a high speed centrifuge, centrifuge at 12,000 rpm, take the bottom extract, and remove the supernatant. 1 ml of ice 75% ethanol was added, and the mixture was centrifuged at 7,500 rpm to remove the supernatant, and the centrifuged extract was air-dried. Approximately 3 mg of extract was obtained. The extract was dissolved back in 100 μl of secondary distilled water for analysis.
參考先前文獻分析萃取物中之多醣體(F. Anal. Chem 1956;28:350)。取1μl樣品,混合50μl酚(Sigma Co,美國)及250μl H2 SO4 (Kanto Chemical CO.,INC.,日本),室溫水解反應24小時,使用酵素免疫微盤分析儀(BioTek EL808)判讀波長490nm。以Sucrose(Sigma Co,美國)作標準品,檢測所得萃取物中之多醣含量。The polysaccharides in the extract were analyzed with reference to previous literature (F. Anal. Chem 1956; 28:350). Take 1 μl of the sample, mix 50 μl of phenol (Sigma Co, USA) and 250 μl of H 2 SO 4 (Kanto Chemical CO., INC., Japan), hydrolyze at room temperature for 24 hours, and interpret using an enzyme immunoplate analyzer (BioTek EL808). The wavelength is 490 nm. The polysaccharide content in the obtained extract was measured using Sucrose (Sigma Co, USA) as a standard.
參考先前文獻分析萃取物中之硫酸根含量(J Biol Chem 1968;243: 1536)。取10μl樣品,加入840μl二次蒸餾水,靜置於37℃水浴槽中24小時。加入150μl之三氟乙酸(Trifluoroacetic Acid),靜置於37℃水浴槽中4小時。取300μl水解完全的樣品,加入700μl反應呈色劑,混合均勻後,室溫靜置10分鐘。取0.5g Gelatin(Sigma Co,美國)溶於100ml二次水中,並置於60%水浴槽中使其完全溶解,加入1.48ml HCL(Kanto Chemical CO.,INC)及0.5g BaCl2 (Sigma),混合均勻。每次使用前須再震盪混合製備反應呈色劑。使用分光光度計(Amersham Ultrospec3100 pro)判讀波長360nm,以MgSO4 (Fluka)作為標準品,檢測所得萃取物中之硫酸根含量。The sulfate content in the extract was analyzed with reference to previous literature (J Biol Chem 1968; 243: 1536). A 10 μl sample was taken, 840 μl of double distilled water was added, and the mixture was placed in a 37 ° C water bath for 24 hours. 150 μl of Trifluoroacetic Acid was added and the solution was placed in a 37 ° C water bath for 4 hours. 300 μl of the completely hydrolyzed sample was taken, 700 μl of the reaction coloring agent was added, and the mixture was uniformly mixed, and allowed to stand at room temperature for 10 minutes. 0.5 g of Gelatin (Sigma Co, USA) was dissolved in 100 ml of secondary water and completely dissolved in a 60% water bath, and 1.48 ml of HCL (Kanto Chemical CO., INC) and 0.5 g of BaCl 2 (Sigma) were added. well mixed. The reaction color former should be prepared by shaking and mixing before each use. Using a spectrophotometer (Amersham Ultrospec 3100 pro) The wavelength of 360 nm was interpreted, and the sulfate content in the obtained extract was measured using MgSO 4 (Fluka) as a standard.
取上一步驟水解後的樣品400μl,以真空冷凍乾燥後,回溶於1ml三次水,並以0.22μm過濾磨過濾,使用醣類分析儀分析(Sugar Analysis System Dionex DX-500),分析其中單糖成分(岩藻糖)及含量。Take 400 μl of the hydrolyzed sample in the previous step, freeze-dry in vacuum, dissolve it back into 1 ml of water, filter it with a 0.22 μm filter mill, and analyze it with a sugar analyzer (Sugar Analysis System Dionex DX-500). Sugar component (fucose) and content.
以本發明方法自海蘊中萃取褐藻糖膠,四重複後所得多醣體的含量平均為36.67%,其中含有1.2%硫酸根,以及1.08%岩藻糖(Fucose)。並以醣類分析儀分析得光譜圖如圖一所示。由此可知,此萃取物最主要的單糖成分為岩藻糖。The fucoidan was extracted from the seaweed by the method of the present invention, and the content of the obtained polysaccharide after the four repetitions was 36.67% on average, which contained 1.2% sulfate, and 1.08% fucose. The spectrum obtained by the analysis of the sugar analyzer is shown in Fig. 1. It can be seen that the most important monosaccharide component of this extract is fucose.
圖一為以本發明方法萃取褐藻糖膠所得醣類分析儀之光譜圖,顯示此萃取物最主要的單糖成分是岩藻糖。Figure 1 is a spectrum diagram of a saccharide analyzer obtained by extracting fucoidan by the method of the present invention, showing that the most important monosaccharide component of the extract is fucose.
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EP1327448A1 (en) * | 2000-09-13 | 2003-07-16 | Takara Bio Inc. | Homeostasis-maintaining agents |
US20040219525A1 (en) * | 2000-08-25 | 2004-11-04 | Heiko Haertel | Plant polynucleotides encoding novel prenyl proteases |
JP2005247757A (en) * | 2004-03-04 | 2005-09-15 | Nikken Sohonsha Corp | Antiviral agent |
JP2006176552A (en) * | 2004-12-21 | 2006-07-06 | Akizuki Shizuka | Method for collecting slimy fucoidan-containing extract from brown alga sargassum horneri |
JP2006233099A (en) * | 2005-02-25 | 2006-09-07 | Univ Of Ryukyus | Fucoidan polysaccharide originating from discoidal phase of brown alga and its use |
CN101098894A (en) * | 2005-03-01 | 2008-01-02 | 宇部兴产株式会社 | High-molecular fucoidan, method of producing the same and cosmetic composition |
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US20040219525A1 (en) * | 2000-08-25 | 2004-11-04 | Heiko Haertel | Plant polynucleotides encoding novel prenyl proteases |
EP1327448A1 (en) * | 2000-09-13 | 2003-07-16 | Takara Bio Inc. | Homeostasis-maintaining agents |
JP2005247757A (en) * | 2004-03-04 | 2005-09-15 | Nikken Sohonsha Corp | Antiviral agent |
JP2006176552A (en) * | 2004-12-21 | 2006-07-06 | Akizuki Shizuka | Method for collecting slimy fucoidan-containing extract from brown alga sargassum horneri |
JP2006233099A (en) * | 2005-02-25 | 2006-09-07 | Univ Of Ryukyus | Fucoidan polysaccharide originating from discoidal phase of brown alga and its use |
CN101098894A (en) * | 2005-03-01 | 2008-01-02 | 宇部兴产株式会社 | High-molecular fucoidan, method of producing the same and cosmetic composition |
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