HUT71705A - Purification of beta cyclodextrin - Google Patents

Abstract

The present invention relates to a process for the purification of non-branched β-cyclodextrin. According to the invention, an aqueous solution is formed and a precipitate is separated. Critical steps are repeated several times independently of each other.

Description

The present invention relates to a process for the purification of β-cyclodextrin, more particularly to a process for removing a branched β-cyclodextrin from an aqueous solution containing branched and non-branched β-cyclodextrin by multiple crystallization.

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- 2 Cyclodextrins, also known as Schardinger's dextrin-cycloamyl, cyclomaltose and cycloglucan, are cyclic anhydroglucose oligomers that are linked by α-1,4 bonds. If the cyclic oligosaccharide contains 6 monomers, it is called α-cyclodextrin. If the cyclic oligosaccharide contains 7 monomers, it is called β-cyclodextrin and finally when it is called γ-cyclodextrin containing 8 monomers. These 6-, 7-, and 8-membered rings are otherwise designated as cyclomaltohexaose, cyclomaltoheptaose, and cyclomaltooctaose.

Branched cyclodextrins have already been described by French et al., (1965) (see French et al., Archives of Biochem. And Biophys. Vol. III, pp. 153-160, 1965). Branched cyclodextrins contain α-1,4-anhydroglucose units to which one or more anhydroglucose monomers are attached through an α-1.6 bond. The branched chain may itself contain more than one anhydroglucose monomer, and thus the branched chain may be an oligomer or a polymer. The other anhydroglucose monomers which form the branched structure may be linked to each other by α-1,4 bonds. Branched β-cyclodextrin is relatively soluble in water compared to non-branched β-cyclodextrins. More particularly, the solubility of the non-branched β-cyclodextrin in water is about 10%. 2% by weight, while the solubility of the branched cyclodextrin in water is ca. 50% or more by weight.

The term "β-cyclodextrin" refers to cyclodextrins, which may be branched β-cyclodextrins and non-branched β-cyclodextrins, with or without "branched" modification. Non-branched and branched β-cyclodextrins were determined in each case.

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The preparation of β-cyclodextrins is carried out by reacting a suspension of starch with an enzyme. The enzyme used is cyclodextrin glycosyltransferase (CGT), which is used at the appropriate pH and temperature for a suitable period of time depending on the type of CGT chosen. The starch can be any vegetable starch. The CGT enzyme is obtained from (alkalophilic) microorganisms of Bacillus macerans, B, meqaterium, B. circulans, B. stearothermophilus and Bacillus sp, but other microorganisms are also used as sources. Depending on the CGT enzyme chosen or the starch chosen, the reaction conditions may vary and are described in detail in the literature. Generally, the starch is suspended in an aqueous solution for up to about. Up to 35% by weight. The suspension is then converted to gelatin or made liquid by enzymatic or acidic treatment having a dextrose equivalent value (DE) of ca. 1-5. A preferred enzyme for use in liquid conversion is bacterial α-amylase. Subsequently, a suitably selected CGT compound is added to the gelatinized or liquid suspension, and the pH, temperature and time are chosen so as to correspond to the nature of the enzyme chosen. Generally, the pH used is about. The temperature ranges from 4.5 to 8.5, and the temperature is from room temperature to approx. 75 ° C and reaction time approx. 10 hours to several days. The amount of β-cyclodextrin obtained will vary depending on the treatment conditions and the type of enzyme chosen. From the crude reaction mixture, β-cyclodextrin can be recovered by conventional techniques such as crystallization, precipitation or filtration.

In order to produce primarily β-cyclodextrin, the reaction between CGT and the gelatinized or liquid starch suspension is carried out in the presence of a solvent. Applicable solvents generally increase β-cyclodextrin production by promoting the precipitation of β-cyclodextrin as a precipitate and thus the equilibrium towards continuous cyclodextrin production

60,860 / SM • · · · · ♦ ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Suitable solvents include toluene and p-xylene. These solvents are also considered to be complexing agents or complexing agents for cyclodextrins, since they form a complex with cyclodextrin and also complex with a cyclodextrin precipitate consisting of cyclodextrin and a solvent complexing solvent. Typically, the solvent may be separated from β-cyclodextrin by refluxing an aqueous solution of the β-cyclodextrin complex. During boiling, the solvent is evaporated to give cyclodextrin as a residue. The cyclodextrin is then recovered from the solution by conventional means such as precipitation, crystallization or filtration.

The separation of the unbranched β-cyclodextrin from the β-cyclodextrin-containing solution is extremely difficult. A known process for this separation, or purification process for branched β-cyclodextrins, which provides separation from non-branched β-cyclodextrins, is the chromatographic method disclosed in U.S. Patent No. 5,007,967, issued April 16, 1991. juice.

Another procedure is described in U.S. Patent Application No. 4,840,679, filed June 20, 1989. The process utilized a β-cyclodextrin complexing reagent to separate branched β-cyclodextrin from non-branched β-cyclodextrin. However, this process requires further processing of the complexed non-branched β-cyclodextrin, wherein the complexing reagent is separated from the unbranched cyclodextrin. It is necessary to develop a simple, inexpensive process to purify the non-branched β-cyclodextrin and to separate the branched cyclodextrin.

Crystallization is a known process for purifying materials on an industrial scale. Crystallization is essentially the separation of solids from solutions. In many cases, evaporation is also used in combination with crystallization for the purification process.

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The problem with the crystallization process is that not all substances form a pure crystalline precipitate. In some cases, it is known that mixed crystals are formed.

The present invention relates to a novel, non-branched β-cyclodextrin purification process comprising first forming an aqueous solution containing the first or initial β-cyclodextrin; subsequently forming a first precipitate and forming a first mother liquor from the first solution by crystallization; then recovering the first precipitate from the first solution; then forming a second aqueous solution from the first precipitate; forming a second precipitate or second mother liquor from this solution by crystallization; recovering the second precipitate; and finally, the non-branched β-cyclodextrin is recovered from the second precipitate.

Preferably, in order to increase the purity of the non-branched β-cyclodextrin, rather than recovering the non-branched β-cyclodextrin from the second precipitate, a third aqueous solution is formed and a third crystallization step is carried out in the third solution to form a third precipitate. and producing a third mother liquor; and recovering the third precipitate and finally isolating the non-branched β-cyclodextrin from this third precipitate.

In the process of the invention, the crystallization step is carried out without the addition of a complexing aid or solvent. It should be noted that it is possible to incorporate a residual solvent which is either used in the production process or in the production of cyclodextrin during the starch processing or in the starch hydrolyzate using CGT. The above residual solvent is also removed from the non-branched β-cyclodextrin by the purification process of the present invention. In addition, certain acyclic dextrins are also present in the cyclodextrin directly recovered from the crude degraded product. These acyclic dextrins can also be considered as impurities and can be removed from the product by the purification process of the present invention.

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The process of the present invention and its features will be described in detail with reference to the following accompanying drawings:

Figure 1: General description of the process of the present invention pertaining to non-branched β-cyclodextrin; and

Figure 2 illustrates a further third solution and third precipitate forming method in accordance with the present invention.

As shown in Figure 1, in the process, Step 8, an aqueous solution or first solution of β-cyclodextrin is obtained, which cyclodextrin is obtained from the crude decomposed material. The crude cleavage material is prepared by a conventional method by cleaving a starch slurry with CGT using the appropriate pH and temperature for a period appropriate to the CGT enzyme. The mixture is then treated to isolate the β-cyclodextrin by conventional crystallization or filtration. The β-cyclodextrin recovered from the crude digested mixture is generally ca. 85% by weight -

90% by weight of non-branched β-cyclodextrin; and approx. 2% by weight -

9% by weight of branched β-cyclodextrin and ca. It contains from 6% to 13% by weight of acyclic material.

The stock solution or the first solution used in the process of the invention is characterized in that it contains water and β-cyclodextrin directly recovered from the crude digested mixture and has a solids content of ca. 24% to 40% by weight, more preferably approx. 25% to 30% by weight. This mixture is heated and mixed in a conventional manner using a suitable apparatus commonly used to form the first solution.

If β-cyclodextrin is recovered from the crude digested mixture in the form of complex β-cyclodextrin, i.e., a solvent is used in the process, distillation of the first solution is carried out during the preparation of the first solution in 8 steps to obtain the first crystallization step from the solution. before

60.860 / SM evaporate the solvent. The distillation is carried out using standard techniques and equipment. Occasionally, in the conventional distillation process, the total amount of solvent is not evaporated and traces of solvent remain in the cyclodextrin. The subsequent crystallization step of the process of the invention also removes the above trace solvent.

Using the above aqueous stock solution during the first crystallization in step 10, a first precipitate and a first mother liquor are formed. The first crystallization step 10 is carried out so that the first solution is ca. After cooling to a temperature below 20 ° C, the first solution is cooled to ca. 4 ° C to approx. Store at 26 ° C for an appropriate period of time. Typically, the temperature of the stock solution or the first solution is about 20 ° C. 95 ° C and then ca. It is cooled to 20 ° C, but the exact temperature of the stock solution is not critical as long as the solid used to form the first solution remains in solution.

During the cooling of the first or the stock solution, the solution is cooled for approx. 1 - approx. Allow to stand for 7 days to allow first precipitation to occur. One skilled in the art will recognize that the first precipitate does not form immediately but develops over a period of time. We found that approx. Within 3 days most of the first precipitate precipitated as a solid. Most preferably, the time taken to form the precipitate is about 1 hour. 2 days or longer.

The apparatus used in the first crystallization step may be any crystallization apparatus such as a crystallization tank, a mixed crystallization vessel or a Swenson-Walker crystallizer. The process preferably utilizes a container crystallizer in the process of the present invention which is provided with a cooling element, such as a cooling cover, which is located on the outer surface of the container. In the case of a mixed vessel, and when using a Swenson-Walker crystallizer, the mixing should be sufficient to suspend the crystallized form of cyclodextrin.

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In the procedure, it was found that the use of seed crystals was necessary.

Recovery of the first precipitate in Step 12 may be carried out by conventional means using conventional equipment. Filtration or centrifugation, preferably filtration, may be used. In the process, the first mother liquor is obtained as a by-product.

In step 14, a second solution is formed by mixing water with the first precipitate in a conventional manner using a conventional apparatus. Preferably, the precipitate is dissolved in water with heating and stirring, so that the precipitate is dissolved in water for approx. 2% by weight - approx. 35% by weight solids, more preferably ca. 25% by weight - approx. A solution containing 30% by weight of solids was formed. In our experiments, it has been found that the amount of heat required to completely dissolve the first precipitate is sufficient to bring the second solution to boiling point.

In the second crystallization step 16, a second precipitate and mother liquor are formed. The second crystallization step is carried out in the same manner as the first crystallization step. The second solution is diluted with ca. After cooling to a temperature below 20 ° C, the reaction mixture is cooled to ca. 4 ° C to 26 ° C, more preferably approx. The temperature is maintained between 15 ° C and 20 ° C until a second precipitate forms. Preferably, the formation time of the second precipitate is approx. 2 days or longer. The process employs a conventional crystallization process and a conventional method.

In step 18, the second precipitate is recovered from the second solution and this step is carried out using a conventional apparatus according to a conventional procedure. Recovery can be by centrifugation or filtration. Preferably, the second precipitate is filtered off. Subsequently, in Step 20, the non-branched β-cyclodextrin is recovered from the second precipitate.

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If it is advantageous to produce a higher purity non-branched β-cyclodextrin, before recovering the unbranched β-cyclodextrin from the second precipitate, in step 22, a third aqueous solution is formed from the second precipitate by mixing the second precipitate with water as 2. A third precipitate and a third mother liquor are then formed using a third crystallization step 24. Then, in step 26, the third precipitate is recovered and the unbranched β-cyclodextrin is isolated from the third precipitate in step 28.

The third solution of the second precipitate and water is ca. 2% by weight - approx. 35 wt.% Solids, more preferably ca. 25% by weight - approx. Contains 30% by weight of solids. The third solution is prepared by heating and stirring.

The third crystallization step, wherein the third precipitate and the third mother liquor are prepared from the third solution, is carried out in the same manner as the second crystallization step. The third solution is prepared by heating the solution, and thereafter approx. Cool to below 20 ° C. The cooling temperature of the third solution is kept cool during the formation of the third precipitate, and the mother liquor is formed. This cooled value is approx. 4 ° C to approx. 26 ° C, more preferably ca. 15 ° C to approx. Temperature between 20 ° C. Preferably, the duration of the third precipitation is about 10 minutes. 2 days or longer. The third crystallization step from the third solution is carried out by conventional means using conventional equipment.

Recovery of the third precipitate from the third solution is carried out by conventional means and using conventional equipment such as centrifugation or filtration.

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The process of the present invention is illustrated in detail by the following examples. The examples are not to be construed as limiting the scope of the invention.

Example 1

The crude solid was cooled from 95 ° C to 20 ° C for 24 hours for 250 g of a 30% solid carbohydrate. Cyclodextrin is then directly obtained from the crude digested material. The solution is then filtered through a 18.5 cm filter paper (paper) on a Buchner funnel under vacuum. About 90 g of cyclodextrin are obtained after drying of the filtered material in an oven at 100 ° C. The analysis of the material is shown in Table 1 below. The analysis can be performed by HPLC (high performance liquid chromatography) column.

Table 1

component % By weight / solid All grams acyclic components 0.66 .594 non-branched β-cyclodextrin 89.97 89.07 branched β-cyclodextrin 0.37 0.333

The filter cake obtained is then mixed with 450 ml of distilled water and then stirred with a magnetic stirrer for ca. Stir for 30 minutes and heat to 95 ° C. A first solution was formed, which was then cooled to 20 ° C and allowed to stand at 20 ° C for 16 hours. The solid precipitate was collected by filtration and the filter cake dried in an oven. This precipitate is designated I in Table 2 below. Subsequently, from precipitate I, a second solution is formed in the same manner as the first solution. Then, like the first precipitate, a second precipitate is formed. The second precipitate is shown in Table 2 below. .

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Table 2

1 precipitation Precipitation II component % Gram % Gram acyclic 0.11 .0946 0 0 all branched β-cyclodextrins 0.15 0,129 0 0 total non-branched β-cyclodextrin 99.74 85.78 100 85 all solids 100 86 100 85

As shown in Table 2 above, 100% of the non-branched β-cyclodextrin-related impurities were successfully removed by the two-step crystallization process of the present invention.

Of course, the foregoing description of a preferred embodiment of the invention is illustrative and includes all changes and modifications which do not affect the spirit of the invention.

Claims (7)

PATENT CLAIMS A process for purifying a non-branched β-cyclodextrin comprising the steps of: (a) forming a first precipitate and a first mother liquor from a first solution during the first crystallization step; (b) isolating said first precipitate; (c) forming a second solution by mixing water with said first precipitate; (d) forming a second precipitate and a second mother liquor from said second solution in a second crystallization step; (e) isolating said second precipitate; and (f) recovering the purified non-branched β-cyclodextrin from said second precipitate. The process according to claim 1, wherein the first crystallization step is carried out such that the first solution is ca. 10 ° C to approx. After cooling to 25 ° C, the temperature is maintained at ca. For a period of 3 days or longer. The process according to claim 1, wherein the first and second crystallization steps are carried out by cooling the first and second solutions to a temperature below 20 ° C, and then the first and second solutions have a temperature of about 20 ° C. 4 ° C to approx. Keep at 26 ° C for approx. 3 days or longer. The process of claim 1, wherein the isolation process of steps (b) and (e) is carried out by filtration. A process for purifying a non-branched β-cyclodextrin comprising the steps of: 60.860 / SM (a) forming a first precipitate and a first mother liquor from a first solution during the first crystallization step; (b) isolating said first precipitate; (c) forming a second solution by mixing water with said first precipitate; (d) forming a second precipitate and a second mother liquor from the second solution in a second crystallization process; (e) isolating said second precipitate; (f) forming a third solution of said second precipitate; (g) forming a third precipitate and a third mother liquor from said third solution in a third crystallization step; (h) isolating said third precipitate; and (i) recovering the purified non-branched β-cyclodextrin from the above third precipitate. The process according to claim 5, wherein the first, second and third crystallization steps are carried out by cooling the first, second and third solutions to a temperature below 20 ° C, and then the first, second and third solutions are cooled to ca. . 4 ° C to approx. The temperature is maintained at 26 ° C for approx. 3 days. The process of claim 5, wherein the isolation (b), (e) and (h) is isolated. steps are performed by filtration.