JPH074266B2 - Extraction method of polysaccharides - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for extracting a polysaccharide, and more specifically, a polysaccharide capable of extracting a polysaccharide from a microalgae without using a strong acid or a strong alkali. Regarding the extraction method of.

[Conventional technology]

Conventionally, as a method for extracting a polysaccharide from a microalgae, a method using a strong acid or a strong alkali is known (JP-A-58).
-201993). However, there is a problem that the polysaccharide recovered by this method is hydrolyzed by a strong acid or a strong alkali to lower a part of the molecule. Further, during the alkali treatment, there is a risk that functional groups such as sulfate groups may be eliminated or that anhydro bonds may be formed.

[Problems to be Solved by the Invention]

An object of the present invention is to solve the above-mentioned problems of the prior art and to provide a method for extracting a polysaccharide that can extract a polysaccharide from a microalgae without using a strong acid or a strong alkali.

[Means for Solving the Problems]

The present invention is characterized in that, when a polysaccharide is extracted from a wet algal body in an algal culture solution, after destroying the cells of the wet algal body, an enzymatic reaction is carried out with a proteolytic enzyme and / or a fatty acid ester degrading enzyme. The present invention relates to a method for extracting a polysaccharide.

The microalgae used in the present invention are not particularly limited, but Porphyridium kruentum (Porphy
ridium cruentum marine), Porphyridium aerugineum (freshwater) and the like are preferable because they contain a large amount of polysaccharides. These microalgae are usually used by culturing by a known method.

Examples of the proteolytic enzyme used in the present invention include papain, protease N “Amano” (trade name of Amano Pharmaceutical Co., Ltd.), which is a mixed enzyme agent of neutral or weakly alkaline endo-type or exo-type protease. To be

Examples of the fatty acid ester-degrading enzyme used in the present invention include
Examples thereof include lipases and pacreatins that decompose fatty acids at the α-position or β-position.

The polysaccharide can be extracted from the algae culture solution, for example, as follows.

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

The obtained wet algal cells are placed in a closed container, 5 to 7 volumes of water are added, and then cells are heated and / or disrupted by a homogenizer, a ball mill or the like. The method of cell disruption is appropriately selected depending on the state of the algal cells, but heating is preferable in order to denature the intracellular pigment protein and facilitate the enzymatic reaction described later. However, when the cells are destroyed at a high temperature for a long time, the intracellular polysaccharides may be decomposed to lower the molecular weight, so it is preferable to perform the treatment at 70 to 120 ° C. for about 10 to 60 minutes, and more preferably At 120 ° C for 10 minutes or 70
Heat at ℃ for 30 minutes. Before and after heating, it is more preferable to sufficiently destroy with a homogenizer, a ball mill or the like.

Next, a proteolytic enzyme and / or a fatty acid ester degrading enzyme is added to the destroyed cells to carry out an enzymatic reaction.
The proteolytic enzyme and the fatty acid ester degrading enzyme may be used alone or in combination. When used in combination, they may be added separately or simultaneously. The amount added to 100 ml of wet algal cells is usually 10,000-
It is 50,000 U (unit), and it is usually 7,500 to 30,000 U for fatty acid ester degrading enzyme. Enzyme reaction is usually 30 ~
It is carried out at a temperature of 80 ° C., more preferably 40 to 60 ° C., with stirring or shaking for about 30 minutes to 24 hours.

After the completion of the enzymatic reaction, a water-miscible organic solvent is added until the volume becomes 2 to 3 times that of the reaction mixture, and the polysaccharide is precipitated as a suspension.
As the water-miscible organic solvent, alcohols such as ethanol and isopropanol are used. The suspended matter is collected by means such as filtration. After recovering the suspended matter, the process of redissolving in water and the step of recovering the suspended matter using a water-miscible organic solvent are repeated several times to purify the polysaccharide. After desalting by dialysis, etc., if necessary,
dry. Drying is performed by a usual method such as vacuum drying, spray drying or freeze drying.

The polysaccharide obtained by the method of the present invention does not use a strong acid and a strong alkali, so that the molecule of the polysaccharide is not broken and contains a large amount of high molecular weight polysaccharide. Polysaccharides containing a large amount of high molecular weight polysaccharides are excellent in reducing the resistance to fluid friction,
It is useful as various fluid friction modifiers.

〔Example〕

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

Example 1 A microalgae (Polyphyridium kruentum) was irradiated with 5 to 10 klx of light using ASW as a medium and a halogen lamp as a light source, and CO ₂ 5 at 25 ° C. for about 2 weeks. Culturing was carried out while aerating air containing 10% to obtain an alga culture solution.

The obtained alga culture solution was centrifuged to separate into a supernatant and a wet algal body. Distilled water 5 was added to 1 kg of wet alga body (solid content 13% by weight), and the mixture was homogenized at 10,000 rpm for 5 minutes. Next, this is heated at 80 ° C for 10 minutes, then cooled to 40 ° C, and 13 g of papain (manufactured by Amano Pharmaceutical Co., Ltd.), which is a protease,
In addition, the enzyme reaction was carried out at 50 ° C for 24 hours. After the reaction was completed, ethanol was added in an equal amount, and the floating polysaccharide was filtered with a nylon mesh to remove water and ethanol. The obtained polysaccharide was again dissolved in distilled water 5, 13 g of papain was added, and the enzyme reaction was sufficiently carried out at 50 ° C. for 24 hours. Then, ethanol was added in an equal amount to collect the floating polysaccharides. The polysaccharide was thoroughly washed with ethanol and dried by a vacuum drying method.

The molecular weight distribution of the obtained polysaccharide was analyzed by GPC (gel permeation
chromatohraphy) and the results are shown in FIG.

Comparative Example 1 pH was adjusted to 10 to 12 by adding NaOH to a culture solution containing algal cells,
Heat at 0 ° C. for 1-2 hours. After cooling it to room temperature, add HCl to adjust the pH to 2-4 and add ethanol to 2-3.
Double the volume was added and the floating polysaccharides were collected. 2M of recovered polysaccharide
Was dissolved in distilled water containing CaCl ₂ . Then, the mixture was stirred at 90 ° C. until the polysaccharide was completely dissolved, and when it was dissolved, it was cooled to 35 ° C., and ethanol was added to this in an amount of 2 to 3 times to recover the polysaccharide, and vacuum dried 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.

From the comparison between FIG. 1 and FIG. 3, it was found that the polysaccharide obtained by the method of the present invention contained a larger amount of high molecular weight polysaccharide than that obtained by the acid / alkali treatment.

Example 2 The alga culture solution used in Example 1 was centrifuged to separate into a supernatant and a wet algal cell. 3 kg of distilled water for 1 kg of the obtained wet alga
Was added and heated at 100 ° C. for 30 minutes. After heating, 3 g of distilled water was added, 25 g of Protease N “Amano” (trade name of Amano Pharmaceutical Co., Ltd.) was further added, and the enzyme reaction was carried out at 40 ° C. for 24 hours. After the reaction, ethanol 7 was added and the floating polysaccharides were collected by filtration. This polysaccharide was dissolved again in distilled water, 25 g of protease N "Amano" was added, and the enzyme reaction was sufficiently carried out for 24 hours. Then, ethanol was added to suspend the polysaccharide, and the polysaccharide was 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 to Comparative Example 1.
It was found that it contained a large amount of high-molecular-weight polysaccharides as compared with.

Example 3 The alga culture solution used in Example 1 was centrifuged to separate it into a supernatant and wet algal cells. Distilled water 5 was added to 1 kg of wet alga and homogenized at 10,000 rpm for 5 minutes, and then 1 hour at 80 ° C.
Heated for 0 minutes. After cooling this to 20 ° C., 35 g of lipase (manufactured by Amano Pharmaceutical Co., Ltd.) was added and reacted at 30 ° C. for 24 hours. After completion of the reaction, an equal amount of ethanol was added and the floating polysaccharide was filtered through a nylon mesh to remove water and ethanol. The obtained polysaccharide was sufficiently dissolved again in water 5, 13 g of papain was added, the enzyme reaction was sufficiently carried out at 50 ° C. for 24 hours, and ethanol was added in an equal amount to collect the floating polysaccharide. 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 result is shown in FIG. 2. The polysaccharide obtained by the method of the present invention has a higher molecular weight than that of Comparative Example 1. It was found to contain a large amount of polysaccharides.

Example 4 The alga culture solution used in Example 1 was centrifuged to separate into a supernatant and wet algal cells. Distilled water 2 was added to 1 kg of wet alga and heated at 80 ° C. for 20 minutes. After that, 5 at 7000 rpm
Homogenize for 1 minute, add distilled water 2 and stir. 30
After cooling to ℃, pancreatin (Amano Pharmaceutical Co., Ltd.) 50
g was added and the reaction was carried out at 40 ° C. for 24 hours. After the reaction was completed, an equal amount of ethanol was added and the floating polysaccharides were filtered through a nylon mesh to remove water and ethanol. Distilled water 5 was further added to the recovered polysaccharide to dissolve it, 50 g of pancreatin was added, and the reaction was carried out at 40 ° C. for 24 hours. Equal amount of ethanol is added again to recover the polysaccharide, and dialyzed against water at 4 ° C.
After 48 hours, it was freeze-dried.

The obtained polysaccharide was measured for molecular weight distribution 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 contained a large amount of high-molecular-weight polysaccharides as compared with.

〔The invention's effect〕

According to the extraction method of the present invention, since a strong acid or a strong alkali is not used, it is possible to recover a polysaccharide containing a large amount of high molecular weight polysaccharide without damaging the molecule of the polysaccharide. This polysaccharide containing a large amount of high molecular weight polysaccharides is excellent in the effect of reducing fluid friction and is therefore useful as various fluid friction reducing agents.

[Brief description of drawings]

FIG. 1 is a molecular weight distribution curve of a polysaccharide obtained by enzymatic treatment with protease, and FIG. 2 is a molecular weight distribution curve of a polysaccharide obtained by enzymatic treatment with lipase and protease.
The figure is a molecular weight distribution curve of a polysaccharide obtained by acid and alkali treatment.

Claims (1)

[Claims] 1. When extracting a polysaccharide from a wet alga body in an alga culture solution, after destroying the cells of the wet alga body, an enzymatic reaction is carried out with a protein degrading enzyme and / or a fatty acid ester degrading enzyme. Extraction method of polysaccharides.