JPS606704A - Manufacture of cyclooctaamylose - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides the production of cyclooctaamylose (γ-
(also called cyclodextrin).

γ-Cyclodextrin is relatively water soluble and has a hydrophobic donut shape approximately 1 ON in diameter within which guest molecules can be trapped.

Due to this property, γ-7 clodextrin is a highly sought-after insert in the field of medicine, agrochemicals, cosmetics or the food industry, inter alia.

The prior art according to European Patent Application No. 4.5464 is to separate .gamma.-cyclodextrin from starch hydrolysis products by precipitation with, for example, brompenzole. Furthermore, it is known from the above-mentioned publications that r-cyclodextrin can be obtained from starch by enzymatic decomposition and isolated from the reaction product using the chromatogelane method.

In general, it has been confirmed that known production methods for γ-cyclodextrin are expensive and, therefore, γ-cyclodextrin cannot be widely used industrially due to its properties. Reduce.

It is an object of the present invention to provide a process for the preparation of clooctaamylose which gives an improved and reduced yield.

The object of the present invention is to produce cyclooctaamylose by enzymatically decomposing starch and subsequently separating all the decomposition products by: a) adding cyclodextrin glycosyltransferase to an aqueous preparation of starch; b) at least: −
A process for the preparation of cyclooctaamylose, which is then characterized in that brompenzole is added to the preparation according to a) after the amount of cyclodextrin formed no longer increases.

In principle, various starches can be used according to the invention, including natural starches or partially hydrolyzed starches. Examples are potato starch, corn starch, kubioka starch, etc.

As aqueous preparations of starch it is possible to use all aqueous preparations which have not previously been used for enzymatic degradation of starch. The solution is in particular a 5-30% by weight aqueous solution of gelled starch. These are obtained in the simplest case by boiling a considerable amount of starch in water.

The above-mentioned preparations Ww <For enzyme stabilization, most contain small amounts of calcium chloride, in particular about 5 mmol/l calcium chloride.

Advantageously, the starch preparations to be subjected to the enzymatic degradation reaction according to the invention additionally contain all alkali salts of short-chain alkanoic acids. The amount is preferably 5 to 3 per ton of starch preparation.
00 mmol. Examples of the above-mentioned alkali salts are sodium formate, potassium formate, sodium acetate, potassium acetate, sodium zolopionate, potassium propionate, etc., especially sodium acetate. By this means, the formation of cyclooctaamylose is promoted.

Therefore, in the above-mentioned starch preparation, the enzymes shifrobukis and phosphoryl glycosyltransferase (Oycl), which are known per se, are added to the starch preparation.
odextring lykosyltransfera
se). As sources of this enzyme, Bacillus macerans (J), Bacillus megaterium (Bacillus megaterium),
rium), Pasillus cillans (Bacillus
s circulans), Bacillus stearo-thermophilus (Bacillus stearo-11+e
rmopl+ylus), Micrococcus (Mic-r
ococcus) S P P , alkalophilic bacteria C such as Bacillus Nr, 38-2 or 1
7-1), Klebsiella pneumoniae jKIebs
iella pneun].

The enzyme is preferably used in a 5 weight ratio of enzyme to starch φ; 1:20
00-1:10000, especially 1:4000-1:500
Add in an amount such that 0.

The decomposition reaction is carried out at a temperature of 30 DEG to 55 DEG C., in particular 40 DEG to 45 DEG C., with stirring. The pH value of the starch preparation is 4.5
~7.9.

Advantageously, a portion of the amount of enzyme, from about 10 to about 10 of the total amount of enzyme used
40 weight scale, especially 25-35 weight scale, already 60-70 weight scale
Addition at reaction mixture temperature of °C. Thereafter, the reaction mixture is cooled to a temperature of 30-55° C. under sonic stirring and finally the remaining amount of enzyme is metered in.

The progress of the enzymatic degradation reaction can be monitored, for example, by sampling and chromatographic analysis. As a criterion for this, the amount of cyclodextrin subsequently formed is used.

This 21. The predominant amount is α- or β-cyclodextrin, depending on the source of the cyclodextrin lycosyltransferase. For example, the cyclodextrin lycodyltransferases of Pacillus macerans and Klebsiella pneumoniae form α-cyclodextrin in a predominant amount, and the cyclodextrin lycodyltransferases of alkalophilic bacteria primarily form β-cyclodextrin.

Finally, the enzymatic decomposition reaction brings about a situation in which the amount of this subsequently formed cyclodextrin no longer increases in the reaction mixture. At least after this stage is reached, brompenzole is added according to the invention. Typical culture time to reach this stage is 5-9 hours. Preferably, the addition of brompenzole is carried out only when more than 50% by weight, especially more than 70% by weight of the subsequently formed cyclodextrin is present in the reaction mixture.

The addition of brompenzole precipitates β-cyclodextrin and γ-cyclodextrin as poorly soluble clathrates. The amount of brompenzole used is at least equimolar to the amount of cyclodextrin present and formed in the reaction mixture.

In particular, however, a 1.5- to 10-fold, in particular a 2- to 3-fold molar excess of brompenzole is added relative to the abovementioned amount of cyclodextrin produced.

Thereafter, the reaction mixture was cooled to 30-50°C, especially 40-45°C.
The mixture is stirred further until no increase in the desired product (cyclooctaamylose) can be observed. Reaction control can be carried out, for example, by sample collection and chromatographic analysis. Typical total incubation time to the end of the reaction is 24-48 hours.

For working up the reaction mixture, the solids are first separated, for example by decanting, filtration, centrifugation or the like. The desired product is
As a solid (bromobenzo-leuclate slate), β
The bromopenzole clathrate of the cyclodextrin is present in a mixture with long-chain and short-chain acyclic decomposition products, which are also formed as by-products.

For separation of the mixture, all of the cyclodextrin is redissolved by destruction of the corresponding bromobenzo-luclathrate. A suitable method for this process is to treat the solid mixture described above with hot or boiling water or steam, in which case the dibromopenzole is removed from the mixture in the form of steam distillation. Finally, the long-chain acyclic decomposition products can be separated from the brompenzole-free aqueous solution by precipitation with an alcohol, for example methanol.

The major amount of β-cyclodextrin is separated from the target product by crystallization. For this purpose, the aqueous solution is preferably concentrated and the main amount of the resulting β-cyclodextrin is concentrated at 2-10°C.
, especially at a temperature of about 4°C. As supernatant, an aqueous solution of γ-cyclodextrin (target) with a small amount of β-cyclodextrin and short-chain acyclic decomposition products remains. Often, the removal of short-chain acyclic decomposition products is This product is often present in small amounts and is therefore not necessary. If separation is desired, it can be isolated by forming the brombenthol-clathrate of β-hydrogen γ-cyclodextrin using the same procedure as previously described. In this case,
Short chain acyclic decomposition products remain in solution. after that,
The cyclodextrin is redissolved by expelling the brompenzole from the mixture.

When the obtained solution was evaporated to dryness, '3. Or, by spray drying or the like, crude cyclooctaamylose having a purity of >90% is obtained as the target product.

Crude cyclooctaamylose if pure product is desired? Treatment with pyridine is carried out at a temperature of 20 DEG to 60 DEG C., in particular about 50 DEG C. Upon addition of the pyridine suspension K7 alcohol, for example methanol, cyclooctaamylose forms as a readily precipitable precipitate. Another purification step is the reprecipitation of cyclooctaamylose from an aqueous solution using, for example, ethanol.

The process according to the invention makes it possible to produce cyclooctaamylose starting from starch with strongly improved yields as well as high purity. The objects are inter alia agrochemicals, pharmaceuticals, cosmetics - 1: It is used as an ingredient in bamboo food.

The invention will now be explained in more detail with reference to examples.

Example 1 Potato starch 1502 and sodium acetate 2
The suspension was suspended in water containing 00 mmol of calcium chloride and 5 mmol of calcium chloride, and glued by heating at 120'CK for 30 minutes.

The pH value l'i was 6.9. After cooling the mixture to 70'C, with stirring and further cooling, add a 20 mmol triethanolamine hydrochloride buffer (pH 7.2;
10 g of Klebsiella pneumoniae cyclodextrin-glycosyltransferase dissolved in 5 mmol of calcium chloride was added. It was then cooled to 40° C. with stirring, and then 20 ~ of Klebsiella pneumoniae cyclodextrin-glycosyltransferase in the same preparation as described above was added.

After an incubation period of 7 hours at 40° C., an increase in the primarily formed α-cyclodextrin was no longer confirmed by chromatography analysis.

265"i of brompenzole was added to the reaction mixture. After a further 15 hours of incubation time at 40° C. with agitation, 61.4 parts by weight of starch were degraded to cyclodextrin.

α-/β-/γ-cyclodextrin ratio 1d 1 :
4.1: 2.23.

The poorly soluble components of the reaction mixture were separated by centrifugation, and water J
suspended in water and heated to boiling, in which case brompenzole distills out as an azeotrope with water.

Obtained 21. The slightly cloudy liquid was cooled to room temperature and 1.2 t of methanol was added. After separating the precipitate that has not formed,
All methanol and alcohol were distilled off, and the aqueous solution was concentrated until the concentration of γ-cyclodextrin became 20% by weight. Continuing,
The resulting concentrate was stored at 4°C for 12 hours.

Colorless crystals of β-cyclodextrin were obtained and separated from the solution and washed once with 200 g of ice-cold water.

7.22 g of brompenzole was added to the combined supernatant and stirred at room temperature for 6 hours. The resulting colorless precipitate was subsequently centrifuged, poured into 150 ml of water and dissolved by boiling with azeotropic distillation of dibrompenzole.

Finally, the obtained aqueous solution was freeze-dried. β-cyclodextrin 2.42 and r-cyclodextrin 27.6
A mixture 30y obtained from F was obtained.

Therefore, the yield of γ-cyclodextrin was 98.4% based on the γ-cyclodextrin content of the conversion mixture.

For further purification, the mixture was dried at 105°C for 2 hours, then suspended in pyridine 100- and stirred at 50°C for 2 hours. Subsequently, 200 g of methanol was added.

The resulting colorless precipitate was suspended in 150 rnl of water and dissolved by heating. The γ-cycloOT' kistrin was precipitated with 450% ethanol, reprecipitated with ethanol, and then vacuum-dried.

γ-Cyclodext with a purity of 98.2%! J
y 27.2 y was obtained. The yield based on the r-cyclodextrin content of the conversion mixture was 95.2%.

By repeating the described purification method, γ with a purity of 99.8% was obtained.
-25.6 f of cyclodextrin was obtained.

The yield of pure product was 17% based on the starch used. :,.

Example 2 Tapioca starch 2QQf was suspended in water It additionally containing 200 mmol of sodium acetate and 5 mmol of calcium chloride and was gelatinized by heating to 120° C. for 30 minutes. The 911 value of the reaction mixture is 6.
It was 9. After cooling to 70 DEG C., cyclodextrin-glycosyltrans7-erase of Klebsiella zonoimoniae was added in the same preparation as described in Example 1, with stirring and cooling to 4 degrees. While stirring further, cool to 43℃, and then refrigerate.
27 μl of Pneumoniae cyclodextrin-glycosyltransferase was added in the same preparation as described above.

After an incubation period of 8 hours at 43 DEG C., the increase in α-cyclodextrin initially formed was no longer evident by chromatographic analysis.

Thereafter, a total of 32.62 ml of brompenzole was added to the reaction mixture. After a further 40 hours of incubation at 43°C, 59% by weight of the starch was degraded to cyclodextrin. α−
/β-/γ-cyclodextrin) The ratio of IJn is 1:l
O,6: It was 3.92.

The insoluble components of the conversion mixture were separated by centrifugation and diluted with water.
I took it during t. The suspension was heated to boiling, in which case the brompenzole was distilled off as an azeotrope with water.

The resulting slightly cloudy liquid was concentrated until the concentration of γ-cyclodextrin reached 20% by weight. The resulting concentrate was stored at 4°C for 12 hours.

Crude β-cyclodextrin 742 was obtained.
It was separated from the two solutions and washed once with ice-cold water.

- The combined liquid was worked up as described in Example 1.

29.217' of crude γ-cyclodextrin (98.6 based on the r-cyclodextrin content of the conversion mixture)
% yield), 99.7% pure γ-cyclodextrin 27 y
(Wako in a yield of 13.5% based on the n-starch used) was obtained.

Example 3 The procedure according to Example 2 was repeated, with the exception that gelling was carried out in the presence of 200 mmol of sodium formate instead of 200 mmol of sodium acetate.

After a total incubation time of 48 hours, 57% of the starch was degraded to cyclodextrin. The proportion of γ-cyclodextrin was 14% by weight.

Example 4 Except that gelation occurs in the presence of 200 mmol of potassium propionate instead of 200 mmol of sodium acetate.
The working method according to Example 2 was repeated.

After a total incubation time of 48 hours, 56.7 weight percent of starch was degraded to cyclodextrin. The proportion of γ-cyclodextrin was 13.8% by weight.

Claims (1)

[Claims] ■ A process for producing cyclooctaamylose by enzymatically decomposing starch and subsequently separating the decomposition products, comprising: a) adding cyclodextrin lycosyltransferase to an aqueous preparation of starch; b) at least , - subsequently formed siflovkis)
A process for the preparation of 7-octaamylose, characterized in that brompenzole is added to the preparation according to a) after the amount of IJ is no longer increasing. 2. The method of claim 1, wherein an alkali salt of a short-chain alkanoic acid is added to the aqueous preparation of starch prior to the addition of the cyclodextrin glycosyltransferase. 3. The method according to claim 1 or 2, wherein cyclooctaamylose is obtained from the pyridine suspension by adding alcohol during separation of the decomposition products.