JPS63233797A - Production of alginic acid at high ratio of guluronic acid - Google Patents

Abstract

PURPOSE:To produce alginic acid and/or salt thereof at a low ratio of mannuronic acid (M)/guluronic acid (G), by reacting alginic acid and/or salt thereof with polymannuronic acid-C5-epimerase and converting the M into G. CONSTITUTION:Polymannuronic acid-C5-epimerase is reacted with alginic acid and/or salt thereof to convert the mannuronic acid into guluronic acid and produce alginic acid and/or salt thereof at a high ratio of guluronic acid. More specifically, brown algae are dipped in a dilute acidic solution and then heated at pH10-11 and 50-80 deg.C, dissolved and then neutralized to pH4-9. Polymannuronic acid-C5-epimerase is subsequently mixed and reacted to dilute and filter the dissolved solution. The resultant filtrate is acidified to liberate and precipitate alginic acid, which is then dehydrated, dried and pulverized to afford the aimed alginic acid at a high ratio of guluronic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing alginic acid and/or alginate. Alginic acid is D-mannuronic acid (hereinafter M
It is a polyuronide polysaccharide in which L-guluronic acid (hereinafter referred to as G) and L-guluronic acid (hereinafter referred to as G) are combined in various proportions, and is filled between the cells of brown algae in general.

The M/G ratio differs between species, season, and part of the algal body. In addition, the bacteria Azotobacter vinelangii (A
Zotobacter vinelandii) e Pseudomonas
aeruginosa) and other bacteria of the genus Pseudomonas as exopolysaccharides in the culture solution. (For example, "Comprehensive Polysaccharide Science Volume" edited by Atsuya Harada and Asahi Misaki.
(December 1, 1972) Kodansha Co., Ltd. P150. P152
゜PH10-11, Japanese Patent Publication No. 52-31956, Japanese Unexamined Patent Publication No. 58-107174) Alginic acid and alginate salts are used in a wide range of fields, including foods, dyeing, dental materials, cosmetics, and as thickening agents and gelling agents. Used in welding rods, etc. In recent years, cadmium and strontium have also attracted attention in the medical field for their effects on inhibiting the absorption of cadmium and strontium in the body, lowering cholesterol levels, etc.

(For example, Fumio Kasahara, "Bessatsu Kagaku Kogyo 28-7 Newly Expanded Water-Soluble Polymer" (April 1, 1982) Kagaku Kogyo Co., Ltd. P3
3-36) (Prior art) Alginic acid or alginates have traditionally been produced by extracting, purifying, drying, and pulverizing brown algae. Separated. Brown algae are immersed in a dilute sulfuric acid solution to remove inorganic substances such as calcium and aluminum, mannitol, fucoidin, laminarin, and organic substances such as soluble proteins, and at the same time liberate alginic acid. Dissolve by heating to pH 10-11.50-80°C using soda carbonate, caustic alkali, etc. Dilute and filter the solution. After this, in pickling, dilute sulfuric acid is added to the filtrate to liberate and precipitate alginic acid. After the free alginic acid is dehydrated, it is again neutralized to produce alginate, which is then dried and ground into a product. In addition, in the calcium method,
A calcium chloride solution is added to the above filtrate to precipitate calcium alginate. After dehydrating calcium alginate,
Wash with dilute hydrochloric acid, dilute sulfuric acid, etc. to remove calcium.

Generates free alginic acid. Free alginic acid is again neutralized to form alginate, which is then dried and ground to form a product. (For example, see the above-mentioned "Bessatsu Kagaku Kogyo 28-7 Newly Expanded Water-Soluble Polymer JP 32)"Alginic acid produced by bacteria in the culture solution can be prepared either by adding dilute sulfuric acid to the culture filtrate and precipitating alginic acid, or by adding dilute sulfuric acid to the culture filtrate. Add calcium chloride solution,
Calcium alginate is precipitated and produced in the same manner as above.

(Problems to be solved by the invention) By the above method, alginic acid or alginate can be
However, the M/G ratio of the alginic acid or alginate to be manufactured is determined by the alginic acid produced in the culture solution by the raw material brown algae or bacteria.

(Means for Solving the Problems) In order to solve the above problems, the present inventors, as a result of intensive research, made polymannuronic acid-〇,-epimerase act on alginic acid and/or alginate to M+5-G. It was discovered that alginic acid and/or alginate with a small M/G ratio can be produced by conversion, and the present invention was completed.

Polymannuronic acid-C9-epimerase is produced in bacteria of the genus Pseudomonas and Azotobacter, as well as the seaweed Velvetia.
Pelvetia canali-cu
It is an enzyme produced by mannuronan-C9-epimerase, which is known to act on polymannuronic acid and convert M to G, and is also called mannuronan-C9-epimerase. (for example,
PIGOTT N H, 5UTHERLAND I
lit.

JARMAN T R (1981) Enzyme 1
evolved in the biosynthesis
s of alginate by Pseudomo
nasaeru'ginosa, Eur J Ap
pl Microbiol Biotechno1 However, a method for industrially producing alginic acid and/or alginate with a small M/G ratio by treating alginic acid and/or alginate with the above enzyme to convert M to G has not yet been invented. There wasn't.

In the present invention, brown algae are immersed in an acidic solution such as dilute sulfuric acid, hydrochloric acid, or nitric acid, and then purified using soda carbonate, caustic alkali, etc.
H101-11, heated to 50-80℃, dissolved, P
Neutralize to H4-9, mix with polymannuronic acid-05-epimerase, allow to act, dilute the lysate, filter, make the filtrate acidic with sulfuric acid etc., liberate and precipitate alginic acid, dehydrate, dry, Either pulverize it, or neutralize it again with caustic soda etc. in alcohol to form an alginate salt such as sodium alginate, then dry and pulverize it, or
Add a calcium solution to the filtrate to precipitate calcium alginate, dehydrate the calcium alginate, wash with an acidic solution such as dilute sulfuric acid to remove calcium, produce free alginic acid, and dry.

This is a method for producing alginic acid and/or alginates with a small M/G ratio by pulverizing or neutralizing in alcohol again with caustic soda etc. to form an alginate such as sodium alginate, drying and pulverizing. . Alternatively, a method for producing alginic acid and/or alginate with a small M/G ratio by mixing the above-mentioned enzyme with a culture solution of bacteria capable of producing alginic acid, diluting it, filtering it, and performing the same operations as above. It is.

Polymannuronic acid-C9-epimerase (hereinafter referred to as enzyme) can be obtained from a culture solution of bacteria capable of producing the enzyme, such as Pseudomonas aeruginosa or Azotobacter vinelangii. Although the enzyme may be used as it is, the culture solution may be used as it is, but the filtrate obtained by centrifuging the cells from the culture solution may be subjected to salting out with ammonium a-acid, precipitation with a solvent such as alcohol, ion-exchange chromatography, or gel. Those separated and purified by filtration can also be used. Alternatively, the cells of seaweed, such as Verveteia canariculata, may be disrupted using a homogenizer or the like, centrifuged, and the filtrate may be used as an enzyme, or the cells may be purified and used in the same manner as described above.

The operating conditions for the enzyme are preferably pH 4 to 9, and temperature preferably 20 to 50'C. Below this, the reaction rate becomes slow, and above this, the enzyme is inactivated. In addition, it is preferable that the calcium ion concentration in the reaction solution is 0.1 to 1.2 mM; if it is less than this, the reaction rate will be slow;
If it exceeds this range, the alginic acid will gel and become difficult to operate. In addition, the concentration of alginic acid is 0.1 to 5%, preferably 0.5 to 2.0%. If the concentration is lower than this, the efficiency will be poor, and if the concentration is higher than this, the viscosity will increase and the operation will be difficult. It becomes difficult. Enzyme concentration and reaction time are determined by the required M/
It is necessary to change it depending on the G ratio.

(effect) The present invention is configured as described above.

Regardless of the raw material, alginic acid or alginate with a small M/G ratio can be produced by converting M in alginic acid or alginate to G using an enzyme. Moreover, when alginic acid or alginate with a higher M/G ratio than the required M/G ratio is used as a raw material, alginic acid or alginate with the required M/G ratio can be produced.

(Example) Example 1 Ecklonia kurome dry weight 1
After immersing 00g in IN dilute hydrochloric acid for 3 hours, filtering
of water, adjust the pH to 10 with 50% caustic soda, and adjust the pH to 7.
The mixture was heated to 0°C for 5 hours, cooled to 30°C, 1.5 mfi of 1M aqueous calcium chloride solution was added, neutralized to pH 7 with IN diluted hydrochloric acid, and 0.1 g of enzyme was added.

The reaction was carried out at 30°C for 20 hours. This enzyme was prepared using Pseudomonas aeruginosa IF012689 (obtained from Fermentation Research Institute (Yodoyo Ward, Osaka City)) with 20 g of gluconic acid.
, glutamic acid 5g, disodium phosphate 1.5g
, monopotassium phosphate 1.5g, sodium chloride 1.0g
, magnesium sulfate heptahydrate 0.2g, calcium chloride 0.02g, ferrous sulfate heptahydrate 0.005g, manganese sulfate 0.09B, barium borate 2.9fl1g,
Dissolve 1.2 mg of cobalt chloride, 0.1 mg of copper sulfate, and 1.2 mg of zinc sulfate in water IQ, dispense 100 mQ each into a 500 mQ Erlenmeyer flask, and inoculate 1 platinum loop into a medium that has been sterilized at 120°C for 20 minutes. The culture solution was then cultured with shaking at 30° C. for 24 hours, and the culture solution was centrifuged at 14,000 g for 20 minutes. The 20-50% ammonium sulfate salt fraction of the filtrate was dialyzed and freeze-dried. After reaction.

After diluting 10 times with water and filtering, a 5M aqueous calcium chloride solution was added to precipitate calcium alginate. After dehydration,
The mixture was washed with IN hydrochloric acid, neutralized with 50% caustic soda in methanol, filtered, dried, and ground to obtain 30 g of sodium alginate.

As Comparative Example 1, sodium alginate was obtained in the same manner as in Example 1, except that no enzyme was added.

The M/G ratio of both was determined by Haug's method (A, llaug,
B.

Larsen Acta Chew 5cand...
16.1908 (1960)). Sodium alginate was hydrolyzed with sulfuric acid, and the resulting acid hydrolyzate was adsorbed onto an anion exchange resin (DowxIX8).
M and G are separated using 5-2N acetic acid as an eluent,
It was quantified by the 0rcinol method. As a result, the M/G ratio of Example 1 was 0°2, and the M/G ratio of Comparative Example 1 was 1.
．． It was 5.

Example 2 The same operation as in Example 1 was carried out using Ecklonia cava as a raw material. However, the enzymes used were Azotobacter vinelangii-UFO13581 (obtained from Fermentation Research Institute), 20 g of mannitol, 0.2 g of monopotassium phosphate, 0.8 g of dipotassium phosphate, and 0.2 g of magnesium sulfate heptahydrate.

Dissolve 0.1 g of calcium sulfate dihydrate, 15B of ferrous sulfate heptahydrate, and 2.5 g of sodium molybdate in water IQ, dispense 100IIQ each into a 500-a Erlenmeyer flask, and heat at 120℃ for 20 minutes. One platinum loop was inoculated into a medium that had been sterilized for minutes, and the enzyme was incubated at 30° C. for 48 hours and treated in the same manner as in Example 1. After one reaction, the 10-fold diluted filtrate was poured into an IN sulfuric acid bath to gel the alginic acid, which was separated, dehydrated, and dried to obtain 25 g of alginic acid.

As Comparative Example 2, alginic acid was obtained in the same manner as in Example 2, but without adding enzyme.
I investigated in the same way. The M/G ratio of Example 2 was 0.3, and the M/G ratio of Comparative Example 2 was 1.7.

Example 3 Azotobacter vinelangii-NCIB9068 (N
ational Co11ection of Iud
ustrial Bacteria 135 Abb
ey Road, Aberdeen, 5cotla
nd) with 20 g of sucrose and 0.0 g of monopotassium phosphate.
Oosg, dipotassium phosphate 0.032 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0°2 g,
Calcium chloride 0.064g, sodium molybdate leg1. Dissolve 1 g of ferrous sulfate heptahydrate 3I in 111 water and dispense 100 mfi into a 500 cm Erlenmeyer flask.
One platinum loop was inoculated into a medium that had been sterilized at 20°C for 20 minutes, and 30
The cells were cultured at ℃ for 96 hours.

A 1 on fermenter containing the above medium 6Q was heated to 120°C.

Sterilize for 20 minutes and inoculate with the above culture solution 200IIQ.

Culture was carried out at 30° C. for 96 hours with aeration and stirring. This culture solution 5Q
Add 5 parts of 1M calcium chloride solution to the solution.

0.2g of the enzyme of Example 1 was added and reacted at 30°C for 30 hours. 15 g of sodium alginate was obtained in the same manner as in Example 1.

As Comparative Example 3, sodium alginate was obtained in the same manner as in Example 3, except that no enzyme was added.

The M/G ratio of both was investigated in the same manner as in Example 1.

The M/G ratio of Example 3 was 1.2, and the M/G ratio of Comparative Example 3
The ratio was 4.0.

(Effects of the Invention) Since the present invention is configured as described above, as is clear from the examples, alginic acid with a small M/G ratio and alginic acid with a low M/G ratio and /or alginates can be produced. M/G
Alginic acid or alginate with a small M/G ratio is said to have a greater effect on inhibiting the absorption of cadmium strontium in the body than one with a large M/G ratio. (For example, “
Bessatsu Kagaku Kogyo 28-7 Newly expanded water-soluble polymer JP33-3
6) It is also said that alginic acid and/or alginate with a small 1M/G ratio have a stronger binding force with calcium and a stronger gelling power than those with a large ratio.

Furthermore, G-rich alginic acid and/or alginate are useful as a raw material for G by hydrolysis.

Claims (3)

[Claims] (1) A method for producing alginic acid and/or alginate with a high guluronic acid ratio, which comprises converting mannuronic acid into guluronic acid by allowing polymannuronic acid-C_S-epimerase to act on alginic acid and/or alginate. (2) After immersing brown algae in a dilute acidic solution, PH10
~11.Heat to 50-80℃, dissolve, neutralize to pH 4-9, mix and act with polymannuronic acid-C_S-epimerase, dilute the solution, filter, make the filtrate acidic, alginic acid Either free precipitate, dehydrate, dry and grind, or neutralize again in alcohol to form alginate, dry and grind, or add an aqueous solution of calcium to the filtrate to precipitate calcium alginate. After dehydration, the alginic acid is washed with an acidic solution to remove calcium to produce free alginic acid, which is then dried and ground, or neutralized again in alcohol to form an alginate, which is then dried and ground. A manufacturing method according to claim 1, characterized in that: (3) A method for producing alginic acid and/or alginate with a high guluronic acid ratio, which comprises mixing polymannuronic acid-C_S-epimerase with a culture solution of bacteria capable of producing alginic acid and allowing it to act.