JPS63145293A - Production of l-rhamnose - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as pharmaceuticals and agricultural chemicals in high efficiency at a low cost, by passing a liquid containing rhamnan sulfate through a column packed with an H-type strongly acidic cation exchange resin and hydrolyzing the liquid under heating. CONSTITUTION:A liquid containing rhamnan sulfate (e.g. extracted liquid of algae of Montostroma family, Ulvaceae order) is passed through a column packed with an H-type strongly acid cation exchange resin. The passed liquid is hydrolyzed by heating preferably at 120-160 deg.C for 0.5-3hr to obtain the objective compound. The amount of the cation exchange resin is preferably 0.2-0.5pts.vol. per 1pt.vol. of the extraction liquid to be treated.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention provides a method for producing L-rhamnose. More specifically, the present invention relates to a method for producing L-rhamnose that can produce industrially useful L-rhamnose efficiently and inexpensively with simple operations.

L-rhamnose is a type of methylpentose, also called 6-dioxy-monomannose or L-mannomethylose, and crystals of α-type monohydrate are usually obtained from an aqueous solution. The crystal has a melting point of 88 to 92°C and is sublimable, and the specific rotation of the aqueous solution is levorotatory ([α].-
-7.7°), but about 1 hour after metarotation, it begins to show dextrorotation ([α]n = around +q°).

L-rhamnose exhibits a slightly bitter sweet taste, which is similar to the sweet taste of D-mannose.

It is not assimilated by normal yeast, and exists naturally as glycosides such as quercitrin in the bark of Fagaceae plants, xanthoramin in buckthorn fruits, hesveridin in mandarin oranges, and gum arabic and karaya gum. It is also known to exist in gums such as It is also known that L-rhamnose also exists in bacterial cell walls and is involved in antigen-antibody reactions.

Recently, the physiological activities of sugar chains have begun to attract attention, and their pharmaceutical and
As its use as a synthetic raw material for agricultural chemicals has become popular, L-rhamnose, which is one of the constituent sugars of such sugar chains, or its derivatives have also been used as a synthetic raw material for agricultural chemicals, etc. ! ! 7IIFIE Research is progressing in the fields of bacteriology, microbial engineering, genetic engineering, HH engineering, and immunology, and its use in medicine and agrochemicals is being expanded. Also, L-
Rhamnose undergoes a Maillard reaction with amino acids and produces a characteristic aroma, so it is also used as a raw material for reaction flavors.

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

Furthermore, as shown in JP-A-61-146200, a method using gum arabic as a raw material is also considered, but in order to separate L-rhamnose from the sugar solution after hydrolyzing the gum, a large amount of It required the use of organic solvents and chromatographic separation, and inexpensive L-rhamnose could not be obtained.

(Means for Solving the Problem) As a result of intensive studies to solve the problem, the present inventors discovered that Ulvales Monos tr.
omaceae) as a raw material, it is easy to obtain the raw material, and by hydrolyzing the rhamnan sulfate in the extract with an acid such as sulfuric acid, L- He discovered scales by producing rhamnose and filed a patent application (patent application 1459-1980).
No. 08, No. 60-263920).

As a result of further intensive studies, the present inventors have discovered that if the hydrolysis is carried out by passing a solution containing rhamnan sulfate through a column packed with an H-type strongly acidic cation exchange resin and then heating it, - The present invention was completed based on the discovery that the addition of acid necessary for the hydrolysis of m can be omitted, and that the entire process including the subsequent purification process can be made simpler.

That is, in the present invention, after passing a solution containing rhamnan sulfate through a column filled with an H-type strongly acidic cation exchange resin,
L characterized by hydrolysis by heating
- A method for producing rhamnose is provided.

The liquid containing rhamnan sulfate used in the present invention is not particularly limited as long as it contains rhamnan sulfate, but suitable examples include, for example, extracts of seaweed belonging to the order Ulva order and the family Aridaceae. Examples of seaweeds that belong to the order Ulva and the family Honostromidae include: Onostrom (scientific name: onostrom).
a nitidum Wittrock.

Hereinafter, when we simply refer to "Hitoegusa", we will refer to this species), Motsuki Hitoe (scientific name: Monostromazos)
Tericola Ti1den), Monostroma angicava Kj
ellman), Hirohanohitoegusa (scientific name: Mono
Stroma latissimum Wittroc
k), Monostroma p.
ulchrum Farlow). For example, Hitoegusa is a type of green algae that grows wild in temperate waters, 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, 16.9% moisture, 16.6% protein, 1.0% lipid, 47.5% carbohydrate, and 5% fiber.
．． 6%, ash content 12.4%. The carbohydrate is a polysaccharide containing about 60% L-rhamnose, and also contains uronic acid, D-xylose, D-glucose, D-mannose, etc. Most of this carbohydrate exists in the form of rhamnan sulfate.

Rhamnan sulfate contained in human extract can be efficiently extracted, for example, as follows. Dry human egusa is soaked in a large amount of water to swell it and at the same time is washed to remove salt adhering to the algae. Later research revealed that the subsequent processes proceed without any problems even without removing the salt, but washing greatly reduces the ion exchange load in the desalting process, so it is better to use washed ones. preferable. The swollen algal bodies are heated together with an extraction solvent to extract rhamnan sulfate contained within the algal bodies.

As the extraction solvent, water-soluble solvents such as water, ethylene glycol, propylene glycol, Bresselin, etc., or a mixture of these solvents can be used, but considering the impact on subsequent steps and cost, water is the most preferred. preferable.

Further, preferred embodiments of the extraction method are as follows. When water is used as an extraction solvent, the amount of water used to extract 1 part by weight of dried human extract is preferably 5 to 20 parts by weight, more preferably 7 to 15 parts by weight. The extraction temperature is preferably 90 to 160°C, but when the extraction temperature is high, the extraction time may be short, and when the extraction temperature is low, it takes a long time.

Extraction time is 6 to 96 hours at 90°C to 100°C, 20 minutes to 6 hours at over 100°C to 120'C, 1 minute to 1 hour at over 120°C to 160°C. Preferably, at 100°C, for 24 to 96 hours, 13
In the case of 0°C, 10 to 40 minutes is particularly preferable. Also, stirring the whole thing during extraction helps shorten the extraction time. In the above, the inventors have explained the case of using dried human extract, but it goes without saying that extraction can be similarly performed using undried human extract immediately after harvest. Alternatively, a method of increasing the number of extractions by decreasing the amount of extraction water in the second extraction may also be used.

Thereafter, the algae are removed by centrifugation or filtration to obtain an extract containing rhamnan sulfate. The pl+ of this liquid is often 4
That's all. In the rhamnan sulfate contained in this liquid, a sulfate group is bonded to a polymer of L-rhamnose, and an alkali metal such as potassium or an alkaline earth metal such as magnesium is bonded to the sulfate group. According to analysis by the inventors, this polymer contains approximately 1 sulfate group per molecule of L-rhamnose.

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

Examples of cation exchange resins used for this purpose include DOWEX 50W-Xi and DOWEX 5.
QW-X2, DOWEX 50W-X8, DOWEX 50W-X12 (manufactured by Dow Chemical), DIAION 5K106, DIAION 5KILO, DIAION 5K112, DOWEX 5K116, DIAION 5KIB.

Diaion PK208, Diamondion PK212, Diamondion PK216, Diamondion PK220, Diamondion PK228 (all manufactured by Mitsubishi Chemical Corporation),
Amberlight IR-120B, Amberlight ■R-
121, Amberlite IR-122, Amberlite IR-124, Amberlite TR-252 (manufactured by Tokyo Yuten Kagaku ■), and the like. Among these, gel-type resins are more preferable than porous-type resins because tFillN is less adsorbed to the resin and the amount of dilute trisaccharide solution that flows out at the end of liquid passage is reduced.

The PL of the effluent treated with such H-type strongly acidic cation exchange resin decreases to almost 1 because the cations contained in the liquid containing rhamnan sulfate are exchanged with hydrogen ions.H-type strongly acidic cation exchange resin Good results are obtained when the amount of resin is preferably 0.1 to 1 volume, particularly preferably 0.2 to 0.5 volume, of the extract to be treated.

The liquid whose pH has been lowered in this way is heated and hydrolyzed in a pressurized container without adding any acid.

The hydrolysis temperature is preferably 120 to 160°C.

Heating time varies depending on the heating temperature, for example 11°C
In this case, 0.5 to 3 hours is suitable. In addition, in the hydrolysis, if necessary, a trace amount of acid can be added to the effluent within a range that does not impair the effects of the present invention.

Next, a neutralizing agent such as a hydroxide such as calcium or barium or a carbonate is added to the hydrolyzed solution thus obtained to adjust the pH to 3 to 6, and the resulting precipitate is filtered. Further, this filtrate is purified according to a conventional method.

The purification process includes decolorization, desalting to remove salts remaining in the filtrate, and crystallization process to obtain crystalline L-rhamnose.

Desalting can be carried out by a conventional method using, for example, an ion exchange resin. One example of this method is, for example, by passing the filtrate decolorized with activated carbon or the like from the top of a column filled with an ion exchange resin. In this operation, in order to improve the yield of L-rhamnose, it is necessary to also collect the effluent with a low concentration of L-rhamnose, and the concentration of the effluent is lower than that of the feed liquid (the filtrate). The capacity is large (in addition, when the salted fish in the total feed liquid is larger than the ion exchange capacity of the ion exchange resin to be passed through the liquid, as when the rhamnan sulfate of the present invention is used as a raw material, −
Desalting cannot be completed by passing the liquid through the process once, and the process of stopping the liquid passage midway through the process, regenerating the ion exchange resin, and repeating the process of passing the liquid through the process is necessary. As a result, the concentration of the effluent further decreases, the burden of subsequent concentration operations increases, and the operations become more complicated, which inevitably leads to industrial disadvantages. Therefore, in other words, it is desirable to reduce the amount of salt remaining in the filtrate as much as possible and reduce the load on the ion exchange resin before performing the desalting treatment.

The amount of salted fish in the filtrate obtained by filtering the precipitate generated after the above pl (adjustment) is about 2 compared to the case where the H-type strong acid ion exchange resin treatment of the present invention is not performed, and the subsequent desalination treatment This is extremely advantageous.

To obtain crystals of L-rhamnose from the desalted liquid, concentrate the cough liquid to a predetermined concentration by means such as vacuum concentration.
The solution may be cooled to precipitate L-rhamnose crystals, or, if necessary, an organic solvent such as ethanol may be added to the concentrated and cooled solution to promote L-rhamnose crystal growth.

The inventors investigated a method for further increasing the yield of L-rhamnose in the series of production steps described above, and found that more favorable results could be obtained by employing the following method. That is, it focused on the point that L-rhamnose is not assimilated by general yeast (for example, commercially available baker's yeast that is available at a low price), and it is necessary to yeast-treat +M-rhamnose containing L-rhamnose. This is a method of assimilating sugars other than L-rhamnose and increasing the content of L-rhamnose in the sugar solution. As a result, the crystallization yield of L-rhamnose could be dramatically increased, and the present invention was further enriched.

Next, the method will be explained.

The gamma mother treatment is preferably carried out in the desalting step and the crystallization step. In particular, the sugar solution from which L-rhamnose crystals have been removed during the crystallization process has a high content of sugars that can be assimilated by yeast.
If this liquid is treated with yeast, more favorable results can be obtained in terms of the yield of L-rhamnose.

Yeast treatment may be carried out with the hydrolyzed solution as it is, but the assimilation rate will be faster if the hydrolyzed solution is pretreated with an alkali metal or alkaline earth metal hydroxide or carbonate. Fast and preferable. Here, the use of calcium or barium hydroxide or carbonate as a pH adjuster produces insoluble salts that can be separated by filtration, resulting in good results in the purification process after yeast treatment. . Insoluble salts produced by pH adjustment may be filtered before yeast treatment.

It is more preferable to perform the yeast treatment at p113-8.

In addition, pH3JR liquid preparation treated with activated carbon or further subjected to ion exchange treatment may be treated with yeast.

An example of yeast treatment will be specifically explained.

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 shown above, and the temperature is 25 to 40%.
After stirring or leaving for about 12 to 48 hours at 5°C to assimilate sugars other than L-rhamnose, yeast is removed by centrifugation or filtration. This operation converts D=nibruose, D-mannose, etc. contained in the sugar solution into carbon dioxide or ethanol, so that the purity of L-rhamnose relative to sugar solids increases. After the liquid in which the purity of L-rhamnose has been increased in this way is purified by the desalting and purification method described above, it is subjected to layering and crystallization of +--rhamnose, resulting in a yield of L-rhamnose. is dramatically improved compared to the case without this method.

(Function) In the present invention, the H-type strongly acidic cation exchange resin used before calohydrolysis of rhamnan sulfuric acid, simultaneously desalinates the liquid containing rhamnan sulfuric acid, extremely lowers the PL of the acid liquid,
can be around 1. Therefore, the effluent from the column packed with the H-type strongly acidic cation exchange resin can be hydrolyzed only by heating without adding any particular acid.

(Example) Next, examples and comparative examples will be given and explained.

Example 1 ■ 12 kg of human extract (product containing 17% water) and 15 kg of water
5j2 was placed in a 30ON stainless steel tank, equipped with a thermometer, cooler, and stirrer, and kept at 96-100"C for 72 hours while stirring. After cooling, the supernatant liquid of the contents was filtered.
An extract (residency: 5%) was obtained. The total cations for this extraction night were 6+600 mg as CaC0z/l.

■ Column (inner diameter 20 cm, length 1 m) packed with extract 1452 obtained in ■ 1 = ] type strongly acidic cation exchange resin Diaion 5K-IB (manufactured by Mitsubishi Chemical Corporation) 30a
A liquid was passed from the top, and then water was poured.

3. The concentration remains constant when 42β is allowed to flow out from the bottom.
9%). Furthermore, 110 ρ of the liquid that flowed out was collected.

(2) The liquid obtained in (2) was directly heated at 140° C. for 1 hour to be hydrolyzed. At this time, the amount of L-rhamnose produced was 1
It was 9.5■/mE. After cooling, the pt (pt) was adjusted to 5 with calcium hydroxide (1,38 kg), and insoluble calcium sulfate (water content 4.2 kg) was removed by filtration. The total cation of this filtrate was 5.600 mgs CaC0
z/l and the total anion is 7,000 mg as
It was CaCO3/β.

28g of activated carbon was added to the liquid obtained in step 2, stirred at 50°C for 1 hour, filtered, and soaked in an ion exchange resin for desalting.
under reduced pressure/concentrate to 80% ethanol], 4
kir was added and crystallized to obtain 1.22 kg of crystals of L-rhamnose 1.

When this crystal was determined by liquid chromatography, the purity of L-rhamnose was 98.8%.

The melting point of this crystal is 89.5''c, and the specific optical rotation of the aqueous solution was measured 1 hour after preparation [α)o=
+9.1° (calculated as anhydrous).

Comparative Example 1 To the extract obtained in the same process as in Example 1 (2), scrap sulfuric acid (12.5% of the solid content of the extract) was added, and the mixture was heated at 140°C for 1 hour for hydrolysis. The L-rhamnose content of this liquid was 19.7■, which was comparable to that of Example 1.
/me was. After cooling, the pH was adjusted to 5 with calcium hydroxide (0.94 kg) in the same manner as in Example 1, and calcium sulfate (water content 2.3 kg) was filtered. The total cations in this filtrate are 12.80011+g asCaCO,J/
(2, and the total anion is 13,000 mg asC
aCO:I/β, and contained approximately twice the amount of cations and anions as compared to the filtrate obtained in Example 1 (2). For this reason, the amount of L-rhamnose liquid after desalination using the ion exchange resin is approximately twice that of Example 1, and the layer line exposure for subsequent crystallization becomes thicker, making it difficult to concentrate. Met.

Example 2 ■ Add 12kg of human extract (product containing 17% water) to 50% of water.
01 to remove salts adhering to the algal bodies. The amount of rhamnan sulfuric acid eluted by this operation is only a trace.
On the other hand, the amount of base removed was 2.26 kg. After washing, the human extractor 132 containing water +r was placed in a 300β stainless steel tank and kept at 96 to 100° C. for 48 hours while stirring. After cooling, the contents were filtered to obtain an extract (concentration 4%). The total cations in this extract are 2,100 w as C
aCO3/It.

■ Extract 856 obtained in ■
A liquid was passed through the top of the column (inner diameter 15 cm, length 1.2 m) packed with 1, followed by water.

The concentration is constant (3,
2%). Furthermore, the liquid 60A that flowed out was collected.

(2) The liquid obtained in (2) was directly heated at 140° C. for 1 hour to be hydrolyzed. The amount of L-rhamnose produced at this time was 20
．． It was 5 .mu./ml. After cooling, add calcium hydroxide (0
, 82 kg) to adjust the pH to 5.8, and insoluble calcium sulfate (water content 3.15 kg) was filtered out. The total cations of this filtrate are 2+800 mg as CaCO3/It, and the total anions are 2,900 mg as CaC
It was 0z/l.

24 g of activated carbon was added to the liquid obtained in step 2, stirred at 50°C for 1 hour, filtered, desalted with an ion exchange resin, concentrated under reduced pressure to a concentration of 80%, added 1 kg of ethanol and crystallized. - Rhamnose 1 permanent crystal 1.03 k
I got g. The purity of this crystal was measured by liquid chromatography, and the purity of L-rhamnose was 98.8.
%Met. In addition, the melting point of this crystal is 89.5°C,
The specific rotation of the aqueous solution was measured 1 hour after preparation [
α], = +9.1° (calculated on anhydrous basis).

(Effects of the Invention) As described above, the present invention hydrolyzes a liquid containing rhamnan sulfate by passing it through a column packed with an H-type strongly acidic cation exchange resin and then heating it. L- is produced efficiently and inexpensively from rhamnan sulfate using simple operations.
Rhamnose can be produced.

Furthermore, the present invention has the following effects.

That is, since rhamnan sulfate used as a raw material in the method of the present invention is obtained as a salt of -1'lQ with sodium, potassium, magnesium, etc., after its hydrolysis, calcium sulfate produced by pH adjustment is produced. After removing insoluble salts such as, the pH 8Il adjustment liquid (hereinafter simply referred to as pH adjustment liquid) contains highly soluble sulfates such as sodium sulfate, potassium sulfate, magnesium sulfate, etc. As an effective method for removing such salts, a desalting method using an ion exchange resin can be adopted. The operation must be repeated. Therefore, as a result of water contamination during the regeneration process of the ion exchange resin and the need to collect low-concentration liquid in order to increase the yield of L-rhamnose, the amount of L-rhamnose increases and the amount of liquid required for crystallization increases. The burden of concentration operation increases. On the other hand, according to the method of the present invention, as is clear from a comparison between Example 1 and Comparative Example 1, the salt content in the pH 311 liquid preparation is reduced to about half compared to the above method, so ion exchange Desalination using a resin and concentrating treatment for crystallization also become easier.

Claims (1)

[Claims] A method for producing L-rhamnose, which comprises passing a liquid containing rhamnan sulfate through a column packed with an H-type strongly acidic cation exchange resin, and then hydrolyzing the column by heating.