JPH04158794A - Extraction of polysaccharides - Google Patents

Abstract

PURPOSE:To extract a polysaccharide useful for various fluid friction reducing agents without damaging a molecule of the polysaccharide by fracturing cells of wet algae in a culture solution of algae, adding a specific enzyme and subjecting the cells to enzyme reaction. CONSTITUTION:A culture solution of algae is centrifuged to give wet algae, cells of the algae are fractured by heating and/or homogenizer, the fractured cells are subjected to enzyme reaction with a protease and/or a fatty acid esterase (10,000-50,000 U/100ml of the protease and 7,500-30,000 U/100ml of the fatty acid esterase) to carry out the objective extraction.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a method for extracting polysaccharide 111, and more specifically, to a method for extracting polysaccharides from microalgae without using strong acids or strong alkalis. Regarding the extraction method.

[Conventional technology]

Conventionally, methods using strong acids or strong alkalis have been known as methods for extracting polysaccharides from microalgae (Japanese Unexamined Patent Publication No.
8-201993). However, the amount of I! recovered with this method! There was a problem that some of the molecules were degraded by hydrolysis with strong acids and strong alkalis.

Furthermore, during alkali treatment, there was a risk that functional groups such as sulfate groups would be eliminated or anhydro bonds would be formed.

[Problem to be solved by the invention]

An object of the present invention is to solve the problems of the prior art described above and to provide a method for extracting polysaccharides from microalgae without using strong acids or strong alkalis.

[Means to solve the problem]

The present invention is characterized in that when extracting polysaccharides from wet algal bodies in an algae culture solution, after destroying the cells of the wet algal bodies, an enzymatic reaction is performed using a proteolytic enzyme and/or a fatty acid ester degrading enzyme. This invention relates to a method for extracting polymorphism II.

There are no particular restrictions on the microalgae used in the present invention, but the microalgae belonging to the red algae Porphyridium cruentum (Porphyridium cruentum) belong to the red algae.
phyridium cruentum), Porphyridium cruentum (marine), Porphyridium cruentum (marine)
iumaerugineum (freshwater) and the like are preferable because they contain a large amount of multi-vM species. These microalgal bodies are usually
It is used by culturing it by a known method.

Examples of the protease used in the present invention include papain and protease N "Amano" (trade name, manufactured by Ohno Pharmaceutical Co., Ltd.), which is a mixed enzyme agent of neutral or weakly alkaline endo-type or exo-type proteases. It will be done.

The fatty acid ester degrading enzyme used in the present invention includes:
Examples include lipase and baccreatin, which decompose fatty acids at the α- or β-position.

Polymer II can be extracted from the algae culture solution, for example, as follows.

First, the algae culture solution is separated into a supernatant and wet algae bodies by solid-liquid separation operations such as centrifugation and filtration.

The obtained wet algal bodies are placed in a sealed container, 5 to 7 times the volume of water is added thereto, and then the cells are disrupted by heating and/or using a homogenizer, ball mill, etc. The method of cell destruction is appropriately selected depending on the condition of the algae, but heating is preferable in order to denature the pigment proteins within the cells and facilitate the enzymatic reaction described below. However, if cell destruction is carried out at high temperatures for a long period of time, polysaccharides within the cells may be decomposed and become low molecular weight, so it is preferable to carry out cell destruction at a temperature of 70 to 120'C for about 10 to 60 minutes. Preferably 120°C
Heat at 70°C for 10 minutes or 30 minutes at 70°C. It is more preferable to thoroughly disrupt the material using a homogenizer, ball mill, etc. before and after heating.

Next, a proteolytic enzyme and/or fatty acid ester degrading enzyme is added to the destroyed cells to perform an enzymatic reaction.

Proteolytic enzymes and fatty acid ester degrading enzymes may be used alone or in combination. When used in combination, they may be added separately or at the same time. Wet algae 100-
For proteolytic enzymes, the amount added is usually 10,
000 to 50,000 OOU (units), and for fatty acid ester degrading enzymes, it is usually 7,500 to 30,000 U. The enzyme reaction is usually carried out at a temperature of 30 to 80°C, preferably 40 to 60°C, for about 30 minutes to 24 hours while stirring or shaking.

After the enzymatic reaction is completed, a water-miscible organic solvent is added to the volume up to 2 to 3 times the volume of the reactant to precipitate polyMs as a suspended substance.

As the water-miscible organic solvent, alcohols such as ethanol and isopropanol are used. Floating substances are collected by means such as filtration. After collecting the suspended matter, the process of redissolving it in water and collecting the suspended matter with a water-miscible organic solvent is repeated several times as necessary to purify the polysaccharide.

Further, if necessary, the product is desalted by dialysis or the like, and then dried. Drying is carried out by ordinary methods such as vacuum drying, spray drying, freeze drying, etc.

Since the polysaccharide obtained by the method of the present invention does not use strong acid or strong alkali, the polysaccharide molecules are not broken, and the polysaccharide contains a large amount of high molecular weight polysaccharide.

Polysaccharides containing a large amount of high molecular weight polysaccharides have an excellent effect of reducing fluid friction resistance and are useful as various fluid friction reducing agents.

〔Example〕

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 Microalgae (Polyphyridium tarentum) were grown at 5 to 10 kp using ASW as a medium and a halogen lamp as a light source! , x light and cultured at 25°C for about 2 weeks while aerating air containing 5% CO□ to obtain an algae culture solution.

The obtained algal culture solution was centrifuged to separate the supernatant and wet algal bodies. Add 5 liters of distilled water to 1 l of wet algae (solid content 13% by weight) and mix 10. Homogenized for 5 minutes at OOOrpm. Next, after heating this at 80°C for 10 minutes,
Cool to 0°C and add the proteolytic enzyme papain (
(manufactured by Ohno Pharmaceutical Co., Ltd.) was added, and an enzymatic reaction was performed at 50°C for 24 hours.

After the reaction was completed, an equal amount of ethanol was added, and the floating Ws were filtered through a nylon mesh to remove water and ethanol. The obtained polyIII was dissolved again in distilled water 51,
13 gjJO of papain was added, and the enzymatic reaction was carried out sufficiently at 50°C for 24 hours. Thereafter, an equal amount of ethanol was added to collect the floating tungsten.

This multi-#M was thoroughly washed with ethanol and dried using a vacuum drying method.

The molecular weight distribution of the obtained poly II was analyzed by GPC (gelper
The results are shown in FIG. 1.

Comparative Example I Add NaOH to a culture solution containing algae to pH 10-12
and heated at 100°C for 1 to 2 hours.

After cooling this to room temperature, add HCI to pH 2-4.
2 to 3 times the volume of ethanol was added, and the suspended polysaccharides were collected. The recovered polysaccharide was dissolved in distilled water containing 2M CaCl2. Then, stir at 90°C until the polysaccharide is completely dissolved, then cool to 35°C.
Add 2 to 3 times the volume of ethanol to this and add more! The materials were collected and dried under vacuum at 40°C.

The molecular weight distribution of the obtained polysaccharide was measured in the same manner as in Example 1, and the results are shown in FIG.

A comparison between FIG. 1 and FIG. 3 revealed that the polysaccharides obtained by the method of the present invention contain more high molecular weight polysaccharides than those obtained by acid/alkali treatment.

Example 2 The algae culture solution used in Example 1 was separated into a supernatant and wet alga bodies by centrifugation. Distilled water (3β) was added to 1 kg of the obtained wet algal bodies, and the mixture was heated at 100°C for 30 minutes. After heating, add distilled water and then protease N "Amano".
(Product name manufactured by Ohno Pharmaceutical Co., Ltd.) Add 25g and heat at 40°C for 2 hours.
Enzyme reaction was carried out for 4 hours.

After the reaction, ethanol 71 was added and floating polysaccharides were collected by filtration. This polysaccharide was again dissolved in distilled water, 25 g of protease N "Amano" was added, and the enzymatic reaction was carried out sufficiently for 24 hours. Thereafter, ethanol was added to suspend the polysaccharide, which was then collected by filtration. This polysaccharide was redissolved in distilled water, dialyzed against water at 4°C for 48 hours, and then freeze-dried.

The molecular weight distribution of the obtained polysaccharide was measured in the same manner as in Example 1, but the polysaccharide obtained by the method of the present invention was compared with Comparative Example 1.
It was found that it contains a large amount of high molecular weight polysaccharide compared to .

Example 3 The algae culture solution used in Example 1 was separated into a supernatant and wet alga bodies by centrifugation. 5 r of distilled water per 1 k, g of wet algae
Add 10. Homogenize for 5 minutes at OOOrpm,
Then, it was heated at 80"C for 10 minutes. This was heated at 20°C.
After cooling to 30°C, 35g of lipase (manufactured by Amano Pharmaceutical Co., Ltd.) was added and reacted for 24 hours at 30°C. After the reaction was completed, an equal amount of ethanol was added, and the suspended 1m was filtered through a nylon mesh. and ethanol were removed. After sufficiently dissolving the obtained polysaccharide in 5 r of water, 13 g of papain was added.
was added and the enzymatic reaction was carried out sufficiently at 50''C for 24 hours, and an equal amount of ethanol was added to recover floating polysaccharides.This was thoroughly washed with ethanol and vacuum-dried at room temperature.

The molecular weight distribution of the obtained polysaccharide was measured in the same manner as in Example 1, and the results are shown in FIG. It was found that it contains a lot of polysaccharide.

Example 4 The algae culture solution used in Example 1 was separated into a supernatant and wet alga bodies by centrifugation. 21 parts of distilled water was added to 1 kg of wet algal bodies, and the mixture was heated at 80°C for 20 minutes.

Thereafter, the mixture was homogenized at 7000 rpm for 5 minutes, and 2 p of distilled water was added and stirred. After cooling to 30°C, add 50g of Vancreatin (manufactured by Amano Pharmaceutical Co., Ltd.) and cool to 40°C.
The reaction was carried out for 24 hours. After the reaction is completed, an equal amount of ethanol is added and the floating multi-tlT is separated using a nylon mesh.
．． water and ethanol were removed. Recovered polysaccharide Q
Furthermore, add 5 r of T-distilled water and dissolve, pancreatin 5
The reaction was carried out at 40°C for 24 hours. The polysaccharide was recovered by adding an equal amount of ethanol again, dialyzed against water at 4°C for 48 hours, and then freeze-dried.

The molecular weight distribution of the obtained polysaccharide was measured in the same manner as in Example 1, but the polysaccharide obtained by the method of the present invention was compared to Comparative Example 1.
It was found that it contains a large amount of high molecular weight polysaccharide compared to .

[Effects of the Invention] According to the extraction method of the present invention, since strong acids and strong alkalis are not used, polysaccharides containing a large amount of high molecular weight polysaccharides can be recovered without damaging poly II molecules. Since the polysaccharides containing a large amount of high molecular weight polysaccharides have excellent fluid friction reducing effects, they are useful as various fluid friction reducing agents.

[Brief explanation of the drawing]

Figure 1 shows multi-IR obtained by enzymatic treatment with protease.
Figure 2 is the molecular weight distribution curve of polysaccharides obtained by enzymatic treatment with lipase and protease. Figure 3 is the molecular weight distribution curve of poly-IRs obtained by acid and alkali treatment. It is.

Claims (1)

[Claims] (1) When extracting polysaccharides from wet alga bodies in an algae culture solution, after destroying the cells of the wet alga bodies, an enzymatic reaction is performed using a proteolytic enzyme and/or a fatty acid ester degrading enzyme. Extraction method of polysaccharides.