JPS6036161B2 - Method for purifying anionic polysaccharides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying anionic polysaccharides.

Anionic polysaccharides are polysaccharides that produce anions in aqueous solution, such as xanthan gum, alginic acid,
Gum arabic, carrageenan, etc. exist, and are widely used in applications such as thickeners, food additives, oil well recovery agents, and pharmaceuticals. However, for example, xanthan gum is a polysaccharide produced by microorganisms, so it requires purification to remove microorganisms and culture medium components, and gum arabic is produced from resin liquid, so it requires purification to remove impure components. .
Furthermore, since alginic acid and carrageenan are produced from seaweed, they must be obtained through extraction and purification steps. Additionally, as a method for purifying anionic polysaccharides, a solution of anionic polysaccharides is diluted with 5 to 1 volume of water, centrifuged to remove bacterial cells, and then a non-solvent is added to the solution. A method in which an anionic polysaccharide is precipitated by adding methanol or isopropanol in an amount of 50 to 70% by weight of the solution, or a method in which a low molecular weight quaternary ammonium salt is mixed with an anionic polysaccharide to form a water-insoluble complex. There was a method of separating the complex and further decomposing the complex to recover the polysaccharide amount. However, the former method has the disadvantage of requiring a large amount of non-solvent such as methanol or isopropanol, and the latter method has the disadvantage that the strong toxicity of the quaternary ammonium salt makes it unusable as a food additive. The present invention was made with the aim of eliminating the above-mentioned conventional drawbacks, and provides a method for purifying anionic polysaccharides that does not require large amounts of expensive solvents and does not cause toxicity problems. The purpose is to provide.

The gist of the present invention is to obtain a water-insoluble polysaccharide from an anionic polysaccharide and a cationic polymer compound selected from the group consisting of nitrogen-containing water-soluble polymer compounds produced by microorganisms, water-soluble proteins, and nitrogen-containing water-soluble polysaccharides. A method for purifying an anionic polysaccharide, comprising: producing a complex, decomposing the complex with a liquid containing a lower alcohol and an inorganic salt, and separating anionic polysaccharides insoluble in the liquid. Exists.

Next, the method for purifying the anionic polysaccharide of the present invention will be explained in more detail. The anionic polysaccharide in the present invention is a polysaccharide that has a carboxyl group or a sulfone group and produces an anion in an aqueous solution, and includes, for example, acidic polysaccharides produced by microorganisms such as xanthan gum, alginic acid, carrageenan, gum arabic, and the like.

In particular, anionic polysaccharides which do not become insolubilized under acidic conditions are advantageously used. Xanthan gum is a rubbery anionic polyamide produced by microorganisms of the genus Xanthomonas. Alginic acid is an anionic polysaccharide extracted from brown algae such as kelp, kajime, and arame, and belongs to the polyuronic acid class.Alginic acid is usually used as a sodium salt, potassium salt, etc. to make it easier to dissolve in water. Therefore, in the present invention, these salts are also referred to as alginic acid. Carrageenan is an anionic polysaccharide extracted from a seaweed of the genus Aspergillus, which belongs to the red algae family, and is usually used as a salt of calcium, sodium, potassium, ammonium, magnesium, etc.; therefore, it is used in the present invention. including these salts is called carrageenan. Gum arabic is a gummy anionic polysaccharide secreted from the roots of plants of the genus Acacia. The cationic polymer compound is a polymer compound that generates cations in an aqueous solution, and naturally occurring compounds are preferable. As the cationic polymer compound, natural products such as nitrogen-containing water-soluble polymer compounds produced by microorganisms, water-soluble proteins, and nitrogen-containing water-soluble polysaccharides are used. The nitrogen-containing water-soluble polymer compound is a microorganism of the genus Arvergillus that can produce a nitrogen-containing water-soluble polymer substance that can form a complex with an anionic polysaccharide, such as Asbergus tamaris IF07465 1F043 spot: Asbergillus sojae 04252 , IF04239: Asbergillus ochraseu child 0s IF04072, ``043
46: Asbergillus flavus IF08558; Saccharomyces cerevisiae IFO0304, etc., in a medium containing a carbon source, a nitrogen source, an inorganic salt, and preferably essential nutrients, at a culture temperature of 20 to 40 qo, pH of 3 to 8, and a culture time. It can be obtained by culturing aerobically for 1 to 10 days.
Since this culture solution contains microbial cells, it can be obtained by removing the microbial cells by filtration or centrifugation, and then separating the culture solution from the culture solution. Separation from the culture solution can be carried out by adding a non-solvent such as acetone, adjusting the pH value to 7.0 to 7.8, gel filtration,
This may be carried out by ion exchange chromatography, salt folding method, or the like. The nitrogen-containing zero polymer compound obtained in this way contains protein and amino bran as constituent components, and contains water,
Soluble in alkalis, acids, lower alcohols, especially methanol, isopropanol, etc. The nitrogen-containing water-soluble polymer compound thus obtained is a polymer complex containing components such as protein, amino sugar, and 2-ketoglucotanic acid. Examples of water-soluble proteins used include albumin, protamine, and histones, and examples of nitrogen-containing water-soluble polysaccharides include chitosan.

In order to generate a hydrostatic complex from an anionic polysaccharide and a cationic polymer compound, the liquid containing the anionic polysaccharide is mixed with the liquid containing the cationic polymer compound, and the mixture is treated with hydrochloric acid, sulfuric acid, etc. Adjust the pH value to 6.5 or less with an aqueous solution.

The purpose of setting the pH value to 6.5 or lower is to increase the cationic nature of the cationic polymer compound and speed up the formation of a composite. An insoluble complex is formed. In order to separate the produced composite from the liquid, filtration, centrifugation, etc. are suitable. The complex thus obtained is decomposed by mixing it with a liquid containing a lower alcohol and an inorganic salt. In this decomposition, the composite separated by filtration, centrifugation, etc. is mixed with a liquid containing a lower alcohol and an inorganic salt without drying (in a water-containing state) at a temperature of 20 to 80°C and a pH value of 6. All you have to do is worship the lantern for at least 30 minutes under 5. As the lower alcohol, alcohols having 4 or less carbon atoms that dissolve cationic polymer compounds but do not dissolve anionic polysaccharides are suitable, such as methanol, isopropanol, and the like. The inorganic salt may be any inorganic salt as long as it dissolves in the lower alcohol and has an ionic strength to decompose the complex, and potassium chloride, sodium chloride, potassium nitrate, or sodium nitrate is particularly suitable.

When the complex is decomposed with a liquid containing a lower alcohol and an inorganic salt in this manner, the anionic polysaccharide as a decomposition product becomes an insoluble precipitate, and the cationic polymer compound dissolves in the liquid. Next, the anionic polysaccharide becomes an insoluble precipitate, and the anionic polysaccharide can be recovered by subjecting the liquid in which the cationic polymer compound is dissolved to a furnace or centrifugation.

According to the method for purifying anionic polysaccharides of the present invention, it is possible to obtain anionic polysaccharides from which microorganisms have been removed from polysaccharides produced by microorganisms such as xanthan gum, and also to obtain anionic polysaccharides from which microorganisms have been removed from polysaccharides produced by microorganisms such as xanthan gum. It is possible to obtain anionic polysaccharides from which impurities have been removed from the anionic polysaccharides that are produced, and it can also be applied to the purification of extracts from seaweed, such as alginic acid and carrageenan, to obtain highly pure anionic polysaccharides. It can be recovered in high yield.

Example 1 Xanthomonas campestris (ATCC-1395
Approximately 1.5% by weight of xanthan gum obtained by culturing 1)
A diluted culture solution was prepared by adding 9 days of distilled water to culture solution 1 containing .

Next, water 1 night, yeast extract 202, KH2P○41 night, Mta S04, 7 days 201 night, pH 6
．． A medium consisting of 2 was placed in a 3000 M Sakaguchi flask and sterilized by heating.

After cooling, the medium was inoculated with Asbergil Stamari IF07465, heated to 30 CO, and cultured for 3 days. culture solution p
The H value rose to 8.5. The bacterial cells were removed from the culture solution by centrifugation, and the culture solution was used to produce a water-insoluble complex with xanthan gum. The amount of nitrogen-containing polymer compounds contained in this culture solution can be determined by adding 4 N hydrochloric acid aqueous solution to 5 volumes of the culture solution from which the bacterial cells have been removed until the pH value reaches 7.0, and removing the insoluble matter produced. It was determined by centrifuging, dissolving the insoluble matter in a 0.5 N aqueous solution of caustic soda, adding 7.5 mm of acetone, centrifuging the formed insoluble matter, and drying it at 80°C. . The weight of this dry product was 1&9. Next, the former diluted culture solution 50' containing xanthan gum 75:9 and the latter culture solution 75' containing nitrogen-containing polymer compound 27:9 were mixed, and 4N hydrochloric acid aqueous solution was added. When the pH value was adjusted to 2, a complex of xanthan gum and a nitrogen-containing polymer compound precipitated and precipitated within 3 minutes.

When this complex was centrifuged, a complex containing water for about 2.7 hours was obtained. To this complex, 30 parts of methanol and 0.06 parts of potassium chloride were added and heated at 55:00 for 2 hours, and then centrifuged to obtain a mixture containing 0.2% by weight of potassium chloride. The washing with methanol and centrifugation in step 5 were repeated twice. The precipitate thus obtained was xanthan gum and had a weight of 80% and a weight of 70% after drying. The xanthan gum after drying was completely dissolved in 5' of water. Example 2 A diluted culture solution 50' containing 0.15% by weight of xanthan gum obtained in the same manner as in Example 1 and 0.5% by weight of xanthan gum were added.
When 15 parts of a 5% by weight aqueous egg albumin solution was mixed and a 4N hydrochloric acid aqueous solution was added to adjust the pH to 4, a complex of xanthan gum and egg albumin precipitated within 3 minutes.

When this composite was centrifuged, the composite containing water for about 2.2 hours was obtained. Add methanol 30% to this complex,
After adding 0.06% of potassium chloride, the mixture was heated at 60 qo for 1 hour to decompose the complex, and the resulting precipitate was centrifuged. It was washed with methanol containing 5 upstream, further centrifuged, and this washing and centrifugation were repeated once again. The precipitate thus obtained was xasotane gum, and the weight after drying for 8,000 yen was 9 of 71. After drying, the xanthan gum was completely dissolved in five volumes of water. Example 3 A diluted culture solution 66 containing 0.15% by weight of xanthan gum obtained in the same manner as in Example 1 was diluted with 0.1% by weight of xanthan gum.
A 2% by weight chitosan aqueous solution 6' was mixed and mixed, and a 4N hydrochloric acid aqueous solution was added to adjust the pH to 2.5, and a complex precipitated within 3 minutes.

When this composite was centrifuged, the aforementioned composite containing water for about 3 hours was obtained. To this complex, 40% methanol and 0.089% potassium chloride were added and heated at 60°C for 2 hours to decompose the complex, followed by centrifugation. The precipitate was washed with 6 methanol containing 0.2% by volume of potassium chloride, further centrifuged, and the washing and centrifugation were repeated once again. The precipitate thus obtained was xanthan gum, and the weight after drying at 8000 was 94.9. The xanthan gum after drying was completely dissolved in 8' of water. Example 4 To 15 parts of an aqueous solution containing 0.5% by weight of alginic acid (sodium salt), 75 birds were added to a culture solution containing 27 parts of the nitrogen-containing polymer compound obtained in the same manner as in Example 1. When the mixture was mixed and the pH value was adjusted to 6.5 by adding 4N hydrochloric acid aqueous solution, the complex was precipitated within 3 minutes.

Claims (1)

[Scope of Claims] 1. A water-insoluble polysaccharide made of an anionic polysaccharide and a cationic polymer compound selected from the group consisting of a nitrogen-containing water-soluble polymer compound produced by a microorganism, a water-soluble protein, and a nitrogen-containing water-soluble polysaccharide. A complex is produced, the complex is decomposed by a liquid containing a lower alcohol and an inorganic salt, and an anionic polysaccharide insoluble in the liquid is separated.
Method for purifying anionic polysaccharides. 2. The method for purifying an anionic polysaccharide according to claim 1, wherein the anionic polysaccharide is xanthan gum, alginic acid, carrageenan, or gum arabic. 3. The method for purifying an anionic polysaccharide according to any one of claims 1 to 2, wherein the lower alcohol is methanol or isopropanol. 4. The method for purifying an anionic polysaccharide according to any one of claims 1 to 3, wherein the inorganic salt is potassium chloride, potassium nitrate, or sodium nitrate.