JPH0699461B2 - Method for producing L-rhamnose - Google Patents

Description

The present invention relates to a method for producing L-rhamnose, more specifically,
Industrially useful L-rhamnose can be easily and efficiently operated,
The present invention also relates to a method for producing L-rhamnose that can be produced at low cost.

L-rhamnose is a kind of methyl pentose, and is also called 6-deoxy-L-mannose or L-mannometylose. Usually, α-type monohydrate crystals are obtained from an aqueous solution. The crystal has a melting point of 88-92 ° C and is sublimable, and the specific optical rotation of the aqueous solution is left-rotatory (▲ [α] ²⁰ _D ▼
= -7.7 °), but about 1 hour after rotatory rotation, it exhibits dextrorotation (▲ [α] ²⁰ _D ▼ = around + 9 °).

L-rhamnose exhibits a slightly bitter sweetness, which is similar to that of D-mannose. It is not assimilated by ordinary yeasts and naturally exists as glycosides such as xanthorhamin contained in berries of quercitrin and buckthorn in the bark of beech plants, gum hesperidin of mandarin, and gum arabic and karaya gum. It is also known to exist in gums such as. It is also known that L-rhamnose is also present in the bacterial cell wall and is involved in the antigen-antibody reaction.

The physiological activities of bacteria and sugar chains have begun to attract attention,
Since its use as a synthetic raw material for agricultural chemicals has become popular, L-rhamnose or one of its derivatives, which is one of the constituent sugars of such sugar chains, is also used in plant cytology, microbial engineering, genetic engineering,
Research in the fields of fermentation engineering and immunology has progressed, and its use in medicine and agricultural chemicals has been expanded. Further, L-rhamnose causes Maillard reaction with amino acids and emits a characteristic fragrance, and is therefore also used as a raw material for reaction flavors.

(Problems to be Solved by Prior Art and Invention) Conventionally, L-rhamnose has been industrially produced by hydrolyzing rutin. However, it is difficult to obtain rutin domestically, and it is necessary to rely on imports, and because it is expensive, L-rhamnose produced from rutin has become very expensive.

Further, as disclosed in JP-A-61-146200, a method of using gum arabic or the like as a raw material is also conceivable, but in order to separate L-rhamnose from the sugar solution after hydrolyzing the gum,
Requires the use of large amounts of organic solvents, chromatographic separation,
Inexpensive L-rhamnose could not be obtained.

(Means for Solving Problems) The inventors of the present invention have conducted diligent studies to solve such problems, and as a result, seaweeds of Ulvales Monostromaceae, which have been cultivated in large quantities in Japan for boiled nori seaweed. Is used as a raw material, the raw material can be easily obtained, and by hydrolyzing rhamnan-sulfuric acid in the extract with an acid such as sulfuric acid, a simple operation can be performed efficiently and at a low cost.
-It was discovered that rhamnose can be produced, and a patent application was previously filed (Japanese Patent Application Nos. 60-145908 and 60-263920).

As a result of further intensive studies, the present inventor has found that, if the hydrolysis is carried out by passing a liquid containing rhamnan sulfuric acid through a column packed with an H-type strongly acidic cation exchange resin and then heating, The present invention has been completed by finding that the addition of an acid necessary for the hydrolysis of can be omitted, and that the operation of all steps including the subsequent purification step can be further simplified.

That is, according to the present invention, a liquid containing rhamnan sulfate is passed through a column packed with a H-type strongly acidic cation exchange resin,
L characterized by being hydrolyzed by heating
-Providing a method for producing rhamnose.

The liquid containing rhamnan sulfate to be used in the present invention is not particularly limited as long as it contains rhamnan sulfate, and for example, a suitable extract is a seaweed extract of the family Rhacophoridae. Examples of seaweeds belonging to the order Lepidoptera include the human alga (Scientific name: Monostroma nitidum
Wittrock, hereinafter simply referred to as "Hitotegusa", Motukihitoe (scientific name: Monostroma zoste)
ricola Tilden), Ezohit Rusa (scientific name Monostroma an
gicava Kjellman), Hilohanohitohegusa (scientific name Monostr
oma latissimum Wittrock), Shiwahito Egusa (scientific name Mon)
ostroma pulchrum Farlow) and the like. For example, the human alga is a kind of green alga that is wild in the temperate sea area, and in Japan, it is cultivated from the Pacific Ocean to the Seto Inland Sea. The composition of the air-dried product is, for example, water of 16.9.
%, Protein 16.6%, lipid 1.0%, sugar 47.5%, fiber 5.6%,
The ash content is 12.4%. In addition, the sugar is about L-rhamnose
It is a polysaccharide containing 60%, and additionally contains uronic acid, D-xylose, D-glucose, D-mannose and the like. Most of this carbohydrate exists in the form of rhamnan sulfate.

The extraction of rhamnan sulfate contained in human Exa can be efficiently performed, for example, as follows. Dried human exfoliate is soaked in a large amount of water to swell it, and at the same time, washed to remove salt adhering to the alga. Subsequent studies revealed that the subsequent steps proceed without hindrance even if the salt is not removed.However, since the ion exchange load in the desalting step is significantly reduced by washing, it is recommended to use the washed one. preferable. The swollen alga body is heated together with the extraction solvent to extract the rhamnan sulfate contained in the alga body.
As the extraction solvent, a water-soluble solvent, for example, water, one kind of ethylene glycol, propylene glycol, glycerin, or the like, or a mixed solvent thereof can be used, but water is most preferable in consideration of the influence on the subsequent steps and the price. preferable.
Further, a preferred embodiment of the extraction method will be described below. When water is used as the extraction solvent, 5 to 20 parts by weight of water is preferably used to extract 1 part by weight of dried human exa, and more preferably 7 to 15 parts by weight. Extraction temperature is 90
Although the temperature is preferably 160 ° C, the extraction time may be short when the extraction temperature is high, and the extraction time may be long when the temperature is low. Extraction time is 90 ~ 100 ℃ for 6 ~ 96 hours, above 100 ℃ 120
20 minutes to 6 hours below ℃, preferably 1 minute to 1 hour above 120 ℃ and below 160 ℃, 24-96 hours at 100 ℃, 130
In the case of ° C, 10 to 40 minutes is particularly preferable. When extracting again,
Agitation of the whole helps to shorten the extraction time. Although the present inventors have described the case where dried human Exa is used, it goes without saying that the same extraction can be performed using undried human Exa immediately after harvesting. Alternatively, a method may be used in which the amount of water extracted once is reduced and the number of times of extraction is increased.

After that, the algal cells are removed by centrifugation or filtration to obtain an extract containing rhamnan sulfate. The pH of this solution is often 4 or more. The rhamnan-sulfuric acid contained in this liquid has a sulfuric acid group bonded to the polymer of L-rhamnose, and an alkaline metal such as potassium and an alkaline earth metal such as magnesium are bonded to the sulfuric acid group. According to the analysis of the generators,
This polymer has a sulfate group at a ratio of about 1 per molecule of L-rhamnose.

The liquid containing the rhamnan sulfate thus obtained is passed through a column packed with an H-type strongly acidic cation exchange resin. The cation exchange resin is not particularly limited as long as it is suitable for the purification treatment of sugars and can achieve the object of the present invention, but has a large ion exchange capacity, a high ion exchange rate, and is reusable. It is advantageous to choose the resin.

Examples of the cation exchange resin used for such purpose include, for example, Dowex 50W-X1 and Dowex 50W-X.
2, Dowex 50W-X8, Dowex 50W-X12 (or more,
(Dow Chemical Co., Ltd.), DIAION SK106, DIAION SK110, DIAION SK112, DIAION SK116, DIAION SK1B, DIAION PK208, DIAION PK2
12, DIAION PK216, DIAION PK220, DIAION PK228 (all manufactured by Mitsubishi Kasei Co., Ltd.), Amberlite IR-120B, Amberlite IR-121, Amberlite IR-122, Amberlite IR-124, Amber Light IR-2
52 (above, manufactured by Tokyo Organic Chemical Co., Ltd.) and the like.
Among them, the gel type resin is
It is more preferable because the adsorption of saccharides onto the resin is small and the amount of the dilute sugar solution flowing out at the end of the passage is small.

The pH of the effluent treated with such an H-type strongly acidic cation exchange resin decreases to about 1 because cations contained in the liquid containing rhamnan sulfate exchange with hydrogen ions. The amount of the H-type strongly acidic cation exchange resin is preferably 0.1 to 1 times the volume of the extract to be treated, particularly preferably 0.2.
Good results can be obtained by using ~ 0.5 volume. The liquid whose pH is lowered in this way is heated in the pressure vessel as it is without adding an acid to be hydrolyzed.

The hydrolysis temperature is preferably 120 to 160 ° C. The heating time varies depending on the heating temperature, and for example, at 140 ° C., 0.5 to 3 hours is suitable. In the hydrolysis, if necessary, a trace amount of acid can be added to the effluent within the range that does not impair the effects of the present invention.

Then, for example, a neutralizing agent such as a hydroxide or carbonate of calcium or barium is added to the thus obtained hydrolysis to adjust the pH to 3 to 6, and the generated precipitate is filtered. Further, this filtrate is purified according to a conventional method.

The purification treatment step includes decolorization, desalting for removing salts remaining in the filtrate, crystallization step for obtaining crystalline L-rhamnose, and the like.

Desalting can be performed by an ordinary method using, for example, an ion exchange resin. As an example of the method, for example, the filtration is performed by passing the filtrate decolorized with activated carbon or the like from the upper part of a column packed with an ion exchange resin. In this operation, in order to improve the yield of L-rhamnose, it is necessary to collect the effluent having a low concentration of L-rhamnose, and the effluent has a lower concentration than the feed liquid (the filtrate), The capacity becomes large. When the amount of salt in the total feed solution is larger than the ion exchange capacity of the ion-exchange resin to be passed through, as in the case of using the rhamnan-sulfuric acid of the present invention as a raw material, a single flow-through is required. Since desalting cannot be completed, the liquid passing is stopped in the middle of the treatment, the ion exchange resin is regenerated, and the liquid passing is repeated. As a result, the concentration of the effluent further decreases, and the burden of the subsequent concentration operation increases,
In addition, in terms of operation complexity, it is unavoidable that it will be industrially disadvantageous. Therefore, in other words, it is desirable to reduce the residual amount of salt in the filtrate as much as possible and reduce the load on the ion exchange resin before performing the desalting treatment.

The amount of salt in the filtrate obtained by filtering the precipitate generated after the pH adjustment is about 1/2 as compared with the case where the H-type strongly acidic ion exchange resin treatment of the present invention is not carried out, and the subsequent removal of salt is performed. Very advantageous for salt treatment.

To obtain L-rhamnose crystals from the desalted solution, the solution is concentrated to a predetermined concentration by a means such as vacuum concentration,
A method of cooling and precipitating L-rhamnose crystals, or, if necessary, adding an organic solvent such as ethanol to the liquid after concentration and cooling to accelerate L-rhamnose crystal growth may be used.

Now, the present inventors have studied a method for further increasing the yield of L-rhamnose in the above-mentioned series of production steps, and have found that the following method is used to obtain more preferable results. That is, it focuses on the fact that L-rhamnose is not assimilated by general yeasts (for example, commercially available baker's yeast that can be obtained at a low price). By treating a sugar solution containing L-rhamnose with yeast, , L-rhamnose is utilized to increase the content of L-rhamnose in the sugar solution. As a result, the crystallization yield of L-rhamnose can be dramatically increased, and the present invention has been further enhanced.

Next, the method will be described.

The yeast treatment is preferably performed in the desalting step and the crystallization step. In particular, the sugar solution from which L-rhamnose crystals have been removed in the crystallization step has a high content of sugars that are subjected to yeast assimilation, and thus yeast treatment of this solution further increases the yield of L-rhamnose. Gives good results.

The yeast treatment may be carried out in the hydrolyzed solution as it is, but it is preferable to apply the hydrolyzed solution to a pH-adjusted one with a hydroxide or carbonate of an alkali metal or an alkaline earth metal because the assimilation rate is faster. Here, the use of calcium or barium hydroxide or carbonate as the pH adjuster produces an insoluble salt and enables filtration and separation, and thus gives good results in the preparation step after yeast treatment. The insoluble salt produced by adjusting the pH may be filtered before the yeast treatment. More preferably, the yeast treatment is performed at pH 3-8. In addition, the pH-adjusted solution may be treated with activated carbon, or may be subjected to ion-exchange treatment, and treated with yeast.

An example of yeast treatment will be specifically described.

To the sugar solution shown above, baker's yeast is usually added in an amount of 0.5 to 5% by weight based on the sugar solid content contained in the sugar solution, and the temperature is adjusted to 25 to 45 ° C.
After stirring or standing for about 2 to 48 hours to utilize sugars other than L-rhamnose, yeast is removed by centrifugation or filtration. By this operation, D-glucose, D contained in the sugar solution
-Because mannose and the like are changed to carbon dioxide gas or ethanol, the purity of L-rhamnose with respect to the sugar solid content is increased. After the liquid in which the purity of L-rhamnose is increased in this way is purified by the desalting purification method described above,
When concentrated and crystallized L-rhamnose, the yield of L-rhamnose is dramatically improved as compared with the case where this method is not adopted.

(Function) The H-type strongly oxidizing cation exchange resin used before the hydrolysis of rhamnan sulfate in the present invention causes the pH of the liquid containing rhamnan sulfate to be extremely lowered at the same time as desalting the liquid containing rhamnan sulfate. can do. Therefore, the effluent from the column packed with the H-type strongly acidic cation exchange resin can be hydrolyzed by heating without adding an acid.

(Examples) Next, examples and comparative examples will be described.

Example 1 12 kg of human Exa (containing 17% of water) and 155 l of water were placed in a 300 l stainless steel tank, equipped with a thermometer, a cooler and a stirrer and kept at 96 to 100 ° C for 72 hours while stirring. After cooling, the supernatant of the contents was filtered to obtain an extract (concentration 5%).
The total cations in this extract were 6,600 mg as CaCO ₃ / l.

145 l of the extract obtained in step 1 was passed through the column (inner diameter 20 cm, length 1 m) filled with 30 l of H-type strongly acidic cation exchange resin Diaion SK-1B (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.), and then water was flowed. did. The concentration became constant (3.9%) when 42 liters were discharged from the bottom. Further, 110 l of the liquid that had flowed out was decomposed.

The liquid obtained in 1. was heated as it was at 140 ° C. for 1 hour for hydrolysis. At this time, the amount of L-rhamnose produced was 19.5 mg.
It was / ml. After cooling, p with calcium hydroxide (1.38 kg)
After adjusting to H5, insoluble calcium sulfate (water content: 4.2 kg) was removed by filtration. The total cation of this filtrate is 5,600 mg
as CaCO ₃ / l and the total anion was 7,000 mg as CaCO ₃ / l.

Activated carbon (28 g) was added to the solution obtained in step 1, and the mixture was stirred at 50 ° C for 1 hour, filtered, desalted with an ion exchange resin, concentrated under reduced pressure to a concentration of 80%, and 1.4 kg of ethanol was added to crystallize L. 1.22 kg of crystals of rhamnose monohydrate were obtained.

When the crystals were measured by liquid chromatography, the purity of L-rhamnose was 98.8%. Further, the melting point of this crystal was 89.5 ° C., and the specific optical rotation of the aqueous solution was ▲ [α] ²⁰ _D ▼ = 9.1 ° (calculated as anhydrous) when measured 1 hour after preparation.

Comparative Example 1 To the extract obtained in the same process as in Example 1, 12.5% concentrated sulfuric acid of the solid content of the extract was added, and heated at 140 ° C. for 1 hour for hydrolysis. The content of L-rhamnose in this solution was 19.7 mg / ml, which was comparable to that in Example 1.
After cooling, calcium hydroxide (0.94 k
The pH was adjusted to 5 with g) and calcium sulfate (water containing 2.3 kg) was filtered. The total cations in this filtrate are 12,800 mg as CaCO ₃ / l
And the total anion is 13,000 mg as CaCO ₃ / l,
Compared with the filtrate obtained in Example 1, the cation and the anion were contained in about twice each amount. Therefore, the amount of L-rhamnose liquid after desalting with the ion exchange resin was about twice the amount in the case of Example 1, the concentration amount for the subsequent crystallization was increased, and concentration was difficult.

Example 2 12 kg of human Exa (17% water content product) was washed with 500 l of water to remove salts adhering to algae. The amount of rhamnan sulfate that was eluted by this operation was a trace amount, while the amount of salt removed was 2.26 kg. Human Exa with water after washing
132kg is put in a 300l stainless steel tank and stirred for 96 to 100
Hold at 48 ° C for 48 hours. After cooling, the content was filtered to obtain an extract (concentration 4%). The total cations in this extract are 2,100 m
It was g as CaCO ₃ / l.

85 l of the extract obtained in step 1 was passed through the column (inner diameter 15 cm, length 1.2 m) filled with 20 l of H-type strongly acidic cation exchange resin Dowex 50W-X8 (manufactured by Dow Chemical Co.), and then water was flowed. . The concentration became constant (3.2%) when 10 l was discharged from the bottom. Further, 60 l of the liquid that had flowed out was decomposed.

The liquid obtained in 1. was heated as it was at 140 ° C. for 1 hour for hydrolysis. The amount of L-rhamnose produced at this time was 20.5 mg / m
It was l. After cooling, pH with calcium hydroxide (0.82 kg)
Adjusted to 5.8, insoluble calcium sulfate (water content 3.15kg)
Was filtered. The total cations in this filtrate are 2,800 mg as CaCO ₃
/ l and the total anion was 2,900 mg as CaCO ₃ / l.

Activated carbon (24 g) was added to the solution obtained in step 1, and the mixture was stirred at 50 ° C for 1 hour, filtered, desalted with an ion exchange resin, concentrated under reduced pressure to a concentration of 80%, and added with 1 kg of ethanol to crystallize L
-1.03 kg of crystals of rhamnose monohydrate were obtained. When the purity of this crystal was measured by liquid chromatography, the L-rhamnose purity was 98.8%. The melting point of this crystal is 89.5 ° C, and the specific optical rotation of the aqueous solution is 1 after preparation.
When measured at the time, ▲ [α] ²⁰ _D ▼ = + 9.1 ° (calculated as anhydrous).

(Effects of the Invention) As described above, according to the present invention, a liquid containing lannam sulfate is passed through a column filled with an H-type strongly acidic cation exchange resin and then hydrolyzed by heating.
Rhamnan Sulfuric Acid L
-Rhamnose can be produced.

Further, the present invention has the following effects.

That is, the rannam sulfuric acid used as a raw material in the method of the present invention is generally obtained as a salt of sodium, potassium, magnesium, etc., and therefore, after hydrolysis thereof, an insoluble salt such as calcium sulfate generated by pH adjustment is generated. PH adjustment liquid after removal (hereinafter simply referred to as pH adjustment liquid)
In the solution, sulfates having high solubility such as sodium sulfate, potassium sulfate and magnesium sulfate remain. As an effective method for removing such salts, a desalting method using an ion exchange resin can be adopted.
-145908), the amount of residual salt is large, so it is necessary to repeat desalting with an ion exchange resin. Therefore, when the ion exchange resin is regenerated, it is necessary to collect water at a low concentration in order to increase the yield of L-rhamnose and the mixing of water. As a result, the amount of L-rhamnose is increased and the amount of L-rhamnose is increased. The burden of concentration operation increases. On the other hand, according to the method of the present invention, as is clear from the comparison between Example 1 and Comparative Example 1, the salt content in the pH adjusting solution is reduced to about half that in the above method, so that the ion exchange is performed. Desalting with a resin and, consequently, concentration treatment for crystallization becomes easier.

Claims (1)

[Claims] 1. A method for producing L-rhamnose, characterized in that a liquid containing rhamnan sulfate is passed through a column packed with an H-type strongly acidic cation exchange resin and then heated to carry out hydrolysis.