JPS6379893A - Production of l-rhamnose - Google Patents

Abstract

PURPOSE:To obtain the titled substance from algae in high yield and purity with simple and inexpensive procedure, by extracting a water-soluble component containing rhamnan sulfate from algae, hydrolyzing and neutralizing the extract and treating the obtained liquid with yeast. CONSTITUTION:An algae belonging to Monostroma family, Ulvaceae order, Chlorophyta is extracted with hot water to obtain a water-soluble component containing rhamnan sulfate. The component is hydrolyzed with an inorganic acid, etc., and neutralized with calcium hydroxide, etc., and insoluble calcium sulfate, etc., are removed from the product. The obtained liquid is decolorized with activated carbon, etc., and treated with yeast (e.g. baker's yeast) to obtain the objective L-rhamnose.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention involves extracting water-soluble components, mainly containing rhamnan sulfate, from seaweed belonging to the order Ulva order and family Hypophyllidae using hot water, and extracting the water-soluble components mainly composed of rhamnan sulfate using inorganic acids. After hydrolyzing and neutralizing with an alkali such as calcium hydroxide, it can be used as is or after decolorization.
A method for producing L-rhamnose in which sugars other than L-rhamnose are assimilated by yeast after purification such as desalting, and a method for producing L-rhamnose in which yeast is applied to a filtrate from which L-rhamnose crystals have been removed. .

(Technical background) L-rhamnose is a type of methylpentose, also called 6-dioxy-monomannose or L-mannomethylose, and crystals of the alpha monohydrate are usually obtained from an aqueous solution. The crystal has a melting point of 105 to 106°C and is sublimable, and the optical rotation of the aqueous solution is initially levorotatory ([α]. = −
7,7°), but about 1 hour after the metarotation, [α]
. = around +9°.

L-rhamnose has a slightly bitter taste similar to the sweetness of D-mannose, which is not assimilated by normal yeast. Naturally, quercitrin is found in the bark of Fagaceae plants, and xanthoramnin is found in buckthorn fruits.
It exists as a glycoside such as hesveridin in mandarin oranges, and is also known to exist in gums such as gum arabic and gum karaya. It is also known that L-rhamnose is also contained in bacterial cell walls and is involved in antigen-antibody reactions.

Recently, the physiological activity of sugar chains has begun to attract attention, and their use as synthetic raw materials for medicines, agricultural chemicals, etc. has become popular.
One such sugar, L-rhamnose or its derivatives, has been desired for use in medicines and agricultural chemicals in fields such as plant cytology, microbiology, genetic engineering, fermentation engineering, and immunology.

(Prior Art) Conventionally, L-rhamnose has been industrially produced by hydrolyzing rutin. Rutin is also used as a medicine and contains about 25% L-rhamnose.
However, it is difficult to produce rutin domestically and we have to rely on imports, which are expensive, so L- produced from rutin has the advantage of being easy to purify.
Rhamnose is extremely expensive.

There are also methods filed by the same applicant (Japanese Patent Application No. 145908/1982 and Patent Application No. 26392/1983).
No. 0), the method was to obtain L-rhamnose from rhamnan sulfate in human extract.

(Problem to be solved by the invention) As stated in the application specification of the same applicant,
The composition of air-dried products of human egusa varies depending on the place of production, the time of collection, the tidal conditions of the year, etc., but in general, the composition of air-dried products is
6%, protein 17%, lipid processing%, fiber 6%, ash 12%
, tM quality is said to be 48%. Also, among the carbohydrates,
Approximately 60% is L-rhamnose, and uronic acid and D-
Contains xylose, D-glucose, D-mannose,
It exists in the form of rhamnan sulfate.

This rhamnan sulfate is obtained by extracting human extract with hot water, and this extract is a viscous liquid.

When this extract is hydrolyzed by adding an inorganic acid and heating, L-
A rhamnose-containing liquid is obtained.

Normally, the method for purifying the hydrolyzed solution of natural products is to neutralize the hydrolyzed solution with calcium hydroxide or barium hydroxide, remove it as insoluble calcium sulfate or barium sulfate, decolorize it with activated carbon, etc., and then use ion exchange resin to remove the hydrolyzed solution. Desalination methods are being used.

In addition, desalting from the hydrolyzed solution can also be achieved by ion exclusion methods, but no matter which method is used, the ratio of L-rhamnose to carbohydrates is at most 60 to 75%.
% range, and because other sugars present interfere with the crystallization of L-rhamnose, normal concentration and crystallization operations satisfy both the demands of "high yield" and "high purity". I couldn't do it. Furthermore, L is further crystallized from the filtrate.
-Recovering rhamnose has been extremely difficult because the purity of rhamnose in the sugar solution decreases.

Therefore, L-rhamnose obtained from human extract has L-
A sugar solution containing 1 to 5% arabinose, 3 to 10% of D-galactose and D-mannose, 5 to 20% of D-glucose, 2 to 10% of D-xylose, or crystallized from these to produce L-rhamnose. There has been a strong desire to develop a method for extracting high yield and high purity L-rhamnose from the filtrate after removing crystals using simple and inexpensive operations.

(Means for Solving the Problems) In order to solve these problems, the present inventors have developed a method for recovering L-rhamnose from a hydrolyzed solution of human extract, a purified solution thereof, or a filtrate from which L-rhamnose crystals are removed. We conducted various studies and focused on the sugar composition. In other words, after neutralizing the extracted/hydrolyzed liquid obtained from the human extract with an alkali metal or alkaline earth metal hydroxide or carbonate, the liquid can be made as it is, or the liquid can be decolorized and desalted.
Or a sugar that can improve the relative purity of L-rhamnose in carbohydrates by adding yeast to the liquid after removing the crystals and assimilating sugars other than L-rhamnose in carbohydrates. We found that the composition was different and conducted various studies.

As a result, they succeeded in producing high-yield, high-purity L-rhamnose from human explants using an inexpensive operation on the surface side, thereby completing the invention.

Among the gist of the invention, yeast is added to a solution containing a high concentration of inorganic salts (hydrolyzed/neutralized solution after extraction) to assimilate sugars other than L-rhamnose, thereby increasing the relative content of L-rhamnose. It was completely unexpected that the purity could be improved.

Moreover, when this method is adopted, the yeast bacteria that have completed the reaction are also removed at the same time as the activated carbon is filtered, so the operation is extremely simple.

The present invention will be explained in more detail below.

First, the amount of water required to extract 1 part by weight of human extract is 5 to 20 parts by weight, and the more preferable amount of water to extract is 7 to 15 parts by weight.
Parts by weight. Extraction temperature and time are 90-150℃, 0
．． Extraction is possible within the range of 5 to 96 hours, but at 100℃
24-96 hours in case of 1-3 hours in case of 130℃
Time is an appropriate extraction condition. Another effective extraction method is to reduce the amount of extraction water and increase the number of extractions.

Thereafter, the algal bodies and the extract are separated by centrifugation or filtration to obtain an extract containing rhamnan sulfate, and an inorganic acid such as sulfuric acid or hydrochloric acid is added in an amount of 3 to 30% by weight based on the liquid solid content.
Hydrolyze by heating at 20-140°C. Although the heating time varies depending on the heating temperature, for example, when 12% sulfuric acid is added to the solid content of rhamnan sulfuric acid and heated at 140°C, hydrolysis is completed in 1 to 2 hours.

Furthermore, the resulting hydrolyzed solution is neutralized to pH 3 to 7 by adding hydroxides of alkali metals or alkaline earth metals, or it is filtered by adding 1 to 5% activated carbon (to the liquid). Desalted by ion exchange resin, each ti
Yeast is added in an amount of 0.5 to 10% by weight based on the solid content, and carbohydrates other than L-lanose are assimilated at a temperature of 25 to 45°C for 5 to 48 hours.

As a result, the L-rhamnose purity in carbohydrates was improved from 60 to 75% before yeast treatment to 70 to 90%,
The crystal purity and crystal yield after crystallization were significantly improved.

Furthermore, when comparing cases where the yeast substrate has been bleached/desalted and cases where it has not, it is found that when yeast is added to the hydrolyzed solution before bleaching/desalting, the assimilation reaction progresses more quickly and the sugar assimilation reaction progresses more rapidly. The purity of L-rhamnose in the sample was increased, and the yield of the final crystals was also increased.

On the other hand, as filtrates accumulate as industrial production progresses, allowing yeast to act on these sugar solutions also contributes to the recovery of L-rhamnose with high purity and high yield.

The general reaction conditions using yeast and bacteria are a clean substrate with the necessary amount of dilute nutrient salt added, but the reaction at a high salt concentration in the present invention is a breakthrough that departs from common sense. This is a typical method. Furthermore, the remarkable improvement in purity and yield achieved with such a simple operation far exceeded initial expectations.

(Examples) Examples are given below to explain specific embodiments of the present invention in more detail, but these are not intended to limit the scope of the present invention.

Example-1 ■ 12 kg of human extract (product containing 19% water) and 20 kg of water
01 in a stainless steel tank of 3001, attached a thermometer, cooler, and stirrer, and heated to 96 to 10 while stirring.
It was kept at 0°C for 72 hours. After cooling, the contents were taken out and filtered to obtain an extract (solid content: 8.1 kg).

(2) 2.9 kg of 35% hydrochloric acid was added to this extract and heated at 130°C for 60 minutes in an acid-resistant container. Furthermore, after cooling, the hydrolyzed solution was taken out and plied with 10% sodium hydroxide.
Neutralized to H4.

This neutralized solution contained 3.56 kg of carbohydrates, and the composition of the carbohydrates was as follows.

L-rhamnose 67.5% D-glucose 15.9 D-mannose and D-galactose 7.5 D-xylose 5.0 L-arabinose 3.3 Others 0.8 ■ Add to the neutralized solution obtained in (■) above 34 g of commercially available baker's yeast (approximately 1% quality per l) was added, and the assimilation reaction was carried out at 35° C. for 24 hours to obtain a reaction solution.

This reaction solution contained 2.75-carbohydrates, and the composition of the carbohydrates was as follows.

L-rhamnose 87.3% D-glucose 0.6 D-mannose and D-galactose 0.3 D-xylose 6.5 L-arabinose 4.3 Others 1.0 ■ Add activated charcoal to the sugar solution obtained in step (■) above. Add 2 kg and make 50
After stirring for 1 hour at
．． 3 kg of L-rhamnose was added and crystallized to obtain 2.12 kg of crystals of L-rhamnose 1 (yield: 17.7%). The purity of this crystal was measured by liquid chromatography, and the purity of L-rhamnose was 99. , 2%. Further, the melting point of this product was 106°C, and the specific optical rotation was measured 1 hour after preparation with a 10% concentration aqueous solution [α)o=+9.1°.

■ For comparison, when the above operation (■) was performed on a product that omitted the step (■) above, the crystal fit was 1.28 kg (yield: 10
．． 7%), and the purity of the crystals was 98.6%.

Example-2 ■ After the same operation as in Example-1-■, the pH of the operation in Example-1-0 was adjusted to 6, and 2 kg of activated carbon was added to the resulting neutralized liquid.
The sugar solution containing L-rhamnose was then stirred at 50°C for 1 hour, filtered, and desalted using an ion exchange resin.
0 kg was obtained at a concentration of 1%.

(2) 30 g of commercially available baker's yeast was added to the sugar solution obtained in (1) above, and an assimilation reaction was carried out at 39°C for 18 hours. This reaction solution contained 2.39 kg of carbohydrates.

■ The sugar solution obtained in step (■) above was filtered through a ceramic filter to remove bacterial cells, desalted using an ion exchange resin, concentrated to a concentration of 81% under reduced pressure, and then mixed with ethanol at a pressure of 12 kr. Glue ↓ catty! ,■-Crystal of a monomer sum of ra-9nose 1.
78 kg was obtained (purity 99.4%).

The sugar composition of each liquid was as follows.

Example-3 ■ 300 kg of sugar solution obtained in Example-2-■ (concentration 1%)
was concentrated to 84%, crystallized by adding 1.7 kg of ethanol, and the crystals were filtered to give 1.03 kg of crystals (purity 98.
4.2 kg of liquid was obtained. Concentrate this filtrate,
Water from which ethanol had been removed was added to make a total weight of 19.7 kg. The concentration at that time was 10%.

■ Add 20g of yeast to the filtrate obtained in step ■ above and
The assimilation reaction was carried out at ℃ for 24 hours. Then activated carbon 0.3k
g was added, filtered to remove bacterial cells and activated carbon, and desalted using an ion exchange resin.

(2) The sugar solution obtained in (1) above was concentrated to a temperature of 85%, and 0.7 kg of ethanol was added to cool and crystallize to obtain 0.77 kg of L-rhamnose crystals (purity 98.8%).

(2) For comparison, the filtrate obtained in (1) above was directly concentrated and crystallized in the same manner as (2) above, but no crystals of L-rhamnose were obtained.

The sugar compositions of items 1 to 2 above are shown in the table below.

Sugar Example-3-■ Example-3-■Others 1.0 1.5 (Effects of the invention) As explained above, according to the present invention, seaweed can be produced in high yield by simple and inexpensive operations. High purity L-rhamnose can be obtained.

Claims (1)

[Claims] 1. Water-soluble components including rhamnan sulfate are extracted from seaweed, hydrolyzed and neutralized, and then yeast is applied to the liquid or to the liquid after purification treatment such as decolorization and desalting. A method for producing L-rhamnose, which comprises: 2 Water-soluble components including rhamnan sulfate are extracted from seaweed, hydrolyzed and neutralized, subjected to purification treatments such as decolorization and desalting, concentrated, crystallized and filtered to remove L-rhamnose crystals. After treating the obtained filtrate with yeast bacteria, L-
A method for producing L-rhamnose, which comprises obtaining crystals of rhamnose.