TWI461437B - Rapid extration method for fucoidan - Google Patents

Description

Method for rapidly extracting fucoidan

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.

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.

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.

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.

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.

In one aspect, the invention provides a method for rapidly extracting fucoidan from algae, comprising the steps of:

(1) taking a small amount of algae raw material dissolved in water or an aqueous solution to obtain an aqueous algae solution;

(2) treating the aqueous solution of the algae with ribonuclease;

(3) further adding phenol/guanidine/chloroform reagent for a period of time;

(4) After adding chloroform and mixing rapidly and vigorously, the supernatant is taken by high speed centrifugation;

(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) Adding a second alcohol aqueous solution extract, and centrifuging the supernatant to obtain fucoidan.

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.

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.

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.

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.

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.

In another aspect, the present invention provides a method for rapidly detecting the content of fucoidan from algae, comprising the steps of:

(1) taking a small amount of algae raw material dissolved in water or an aqueous solution to obtain an aqueous algae solution;

(2) treating the aqueous solution of the algae with ribonuclease;

(3) further adding phenol / hydrazine / chloroform reagent for a period of time extraction;

(4) After adding chloroform and mixing rapidly and vigorously, the supernatant is taken by high speed centrifugation;

(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) adding a second alcohol aqueous solution extract, and centrifuging the supernatant to obtain fucoidan;

(7) The obtained fucoidan extract is dissolved in water or an aqueous solution to detect the sugar component and content thereof.

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.

Example 1 Extraction of Haiyun

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.

Example 2 Analysis of extract components Polysaccharide analysis

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.

Sulfate content analysis

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.

Monosaccharide (fucose) analysis

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.

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.

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.

Claims (10)

A method for rapidly extracting fucoidan from algae, comprising the steps of: (1) taking a small amount of algae raw material dissolved in water or an aqueous solution to obtain an aqueous algae solution; (2) treating the algae aqueous solution with ribonuclease; (3) Add phenol / guanidine / chloroform reagent to mix evenly; (4) add chloroform and mix well, then centrifuge at high speed to remove the supernatant; (5) add equal amount of the first alcohol to mix evenly Then, the bottom extract is taken by high speed centrifugation to remove the supernatant; (6) the second alcohol aqueous solution extract is added, and the supernatant is centrifuged to obtain fucoidan. According to the method of claim 1, wherein the algae raw material is used in an amount of about 0.1 mg to 10 mg. According to the method of claim 1, the required processing time is only about 1 hour. The method of claim 1, wherein the algae material is brown algae. The method of claim 1, wherein the algae raw material is dissolved using distilled water or double distilled water. The method according to item 1 patent range, wherein the phenol / guanidine / chloroform TRIzol ^® reagent system reagent. The method of claim 1, wherein the first alcohol is 2-propanol. The method of claim 1, wherein the second alcohol aqueous solution is 75% ethanol. A method for rapidly extracting fucoidan from algae, comprising the following steps: (1) grinding the algae raw material into a powder, taking 5 mg of seaweed powder, dissolving in 100 μl of distilled water; (2) adding 1 μl of RNase A to hydrolyze; (3) Add 1ml of TRIzol ^® reagent and mix until uniform; (4) Add 200μl chloroform, mix well, centrifuge at high speed to remove the supernatant; (5) Add equal amount of 2-propanol to mix evenly, centrifuge at high speed to take the bottom extract (6) Add 1 ml of 75% ethanol, centrifuge to remove the supernatant, and obtain fucoidan. A method for rapidly detecting the content of fucoidan from algae comprises the following steps: (1) taking a small amount of algae raw material dissolved in water or an aqueous solution to obtain an aqueous algae solution; (2) treating the algae aqueous solution with ribonuclease; (3) Add phenol/guanidine/chloroform reagent evenly; (4) add chloroform and mix well, then centrifuge at high speed; (5) add equal amount of first alcohol Mix evenly, then centrifuge the bottom extract to remove the supernatant; (6) add the second alcohol aqueous extract, centrifuge to remove the supernatant to obtain fucoidan; (7) the obtained fucoidan extract It is dissolved in water or an aqueous solution to detect the content and content of sugars.