JPH06209783A - Method for purifying high molecular polysaccharides - Google Patents

Abstract

PURPOSE:To obtain a method for purifying high molecular polysaccharides produced by Alcaligenes.latus. CONSTITUTION:This method for purifying high molecular polysaccharides comprises adding a 1-4C aliphatic alcohol at a concentration so as to flocculate only a microbial cell but unflocculate the high molecular polysaccharides to a culture broth at ambient temperature of while heating the culture broth under atmospheric pressure, flocculating the microbial cell, then removing the microbial cell, further adding the alcohol to the culture broth removed from the microbial cell, precipitating and recovering the high molecular polysaccharides. Thereby, the removal of the microbial cell from the culture broth can simply be carried out with a high efficiency by specifying the kind and concentration of the alcohol used. As a result, the high-purity high molecular polysaccharides are obtained by the purifying treatment in a short period.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for purifying high molecular polysaccharides produced by microorganisms. Various microorganisms produce high molecular weight polysaccharides outside the cells. Among these microorganisms, an aqueous solution of a high molecular polysaccharide produced by Alcaligenes retus shows a pseudoplastic flow behavior and has a high viscosity even in a low concentration region. Such a property is used as an emulsion stabilizer in the emulsion field and the like. In addition, as other properties of this aqueous solution,
It has a high viscosity stability against acids, alkalis, various enzymes, heat, and salts, and is used in many fields as a thickener by utilizing these properties. For example, it is used as an additive for acidic foods such as dressings because of its stability against acids, and as a thickener for concrete because of its stability against alkalis and salts.

[0002]

2. Description of the Related Art In a culture broth of a microorganism that produces high molecular weight polysaccharides, salts and microorganisms contained in the culture solution are contained in addition to the high molecular weight polysaccharides of interest. Among these,
In particular, microorganisms are present wrapped in high molecular weight polysaccharides, and their properties are similar and the viscosity of the culture broth is very high, so it is difficult to remove them separately from the high molecular weight polysaccharides. Therefore, usually, the culture broth is diluted with a large amount of water to reduce the viscosity, and then the cells are preliminarily removed as a precipitate by centrifugation, and then a precipitant such as an aliphatic alcohol which is more than twice as much as the aqueous solution containing the high molecular polysaccharide is used. In addition, a method of aggregating and collecting high molecular polysaccharides has been taken.

[0003]

However, in the method of diluting the culture broth with a large amount of water and removing the bacteria by centrifugation for the purpose of removing the bacterial cells, the amount of the aqueous solution to be treated becomes large,
As a result, a large processing tank and a centrifuge are required, and there are great restrictions in industrial implementation. further,
In order to recover the high molecular polysaccharides from the aqueous solution after removing the bacterial cells, the amount of the precipitant such as alcohol used increases in proportion to the amount of the water used, which causes the disadvantage that the recovery efficiency of the high molecular polysaccharide itself deteriorates. It was In order to solve these problems, it has been strongly desired to develop a purification method for removing microorganisms with a low degree of dilution and efficiently.

[0004]

SUMMARY OF THE INVENTION The present inventors have arrived at the present invention as a result of earnestly examining an efficient purification method of a high molecular polysaccharide produced by Alcaligenes retus in view of the above situation. That is, the present invention includes: (1) Polymeric polysaccharides aggregated only in bacterial cultures of Alcaligenes latus, which produces macromolecular polysaccharides outside the bacterial cells, at room temperature or under heating at atmospheric pressure. Is an aliphatic alcohol having 1 to 4 carbon atoms at a concentration that does not agglomerate, aggregates the cells by a method such as stirring or standing, and filters the cells from the high molecular polysaccharide-containing culture broth by a method such as filtration with a filter. A method for purifying a high-molecular polysaccharide that is selectively removed.

(2) To the culture broth sterilized in (1) above, an aliphatic alcohol having 1 to 4 carbon atoms is further added to the concentration at which the high molecular polysaccharide is aggregated under atmospheric pressure at room temperature or under heating. Thus, a method for efficiently purifying a high molecular polysaccharide by precipitating and recovering the high molecular polysaccharide is provided.

Examples of the Alcaligenes lethas B-16 (FERM BP-20) used in the present invention for producing high molecular weight polysaccharides outside the cells include Alcaligenes latus B-16 (FERM BP-20)
15). Alcaligenes in the present invention
The lethas culture broth may be a stock solution of the culture broth after completion of the culture, or may be diluted with an arbitrary amount of water up to 10 times the stock solution amount. The amount of water used for dilution is not particularly limited, but the volume of the aqueous solution to be handled increases and the operability decreases, so it is preferable to use 0.5 to 3 times the amount of water relative to the stock culture broth solution. Dilute it. Further, the culture broth may be an aqueous solution in which the high molecular polysaccharide once recovered from the culture broth by another method is redissolved. The water used here may be so-called fresh water represented by industrial water, tap water, ion-exchanged water, distilled water, etc. in consideration of industrial implementation, and if necessary, a general buffer solution, physiological saline solution. It does not matter even if you use such as or mix it with fresh water.

The temperature of the culture broth can be carried out in any temperature range from 10 to 90 ° C. Heating from 30 to 90 ° C. is advantageous because the aqueous solution becomes homogeneous immediately after dilution. However, even at room temperature of 10 to 30 ° C., no inconvenience is given to the refining operation, and the heating operation and the apparatus are unnecessary. When cooled to 10 ° C. or less, it takes a lot of time and effort for cooling and is not suitable because it has no merit.
Further, at a high temperature of 90 ° C. or higher, evaporation of alcohol and water is extremely unfavorable.

The alcohol used is preferably an aliphatic alcohol having 1 to 4 carbon atoms. The alcohol may be monohydric or polyhydric, and the following can be exemplified. Methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, isobutyl alcohol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1 , 2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, glycerin and other alcohols.

These alcohols may be used alone or in admixture of two or more. Even if an alcohol having 5 or more carbon atoms such as n-pentyl alcohol is used together with the above alcohol, if the solubility in the culture broth is not hindered, the purpose is to improve the efficiency of cell aggregation and decrease the solution volume. Can be added to. Regarding the method for adding alcohol to the culture broth, the alcohol may be added directly to the culture broth, or it may be added after once diluted with water to an appropriate concentration. The alcohol may be added all at once, but it is more preferable to gradually add the alcohol by the increment method because the stirring time after the addition can be shortened.

The amount of alcohol added to the culture broth is not particularly limited as long as the cells to be removed efficiently aggregate and the high molecular polysaccharide does not aggregate. The optimum amount of alcohol changes depending on the type of alcohol. Generally, in order to obtain the aggregated state of the bacterial cells more efficiently without the aggregation of the high molecular polysaccharide, alcohol is added so that the final concentration (volume ratio) is 10 to 80%, and the bacterial cells are selectively aggregated. Can be presented. More preferably, 1
An alcohol concentration range of 5 to 65% is particularly preferred, 25 to 55% when the alcohol is isopropyl alcohol, 20 to 50% when the alcohol is n-butyl alcohol, and ethyl alcohol is the alcohol. In some cases, the range of 30 to 65% can be exemplified. At this time, regardless of which alcohol is used, if the alcohol concentration is lower than 10%, the bacterial cells will not sufficiently aggregate, and if it exceeds 80%, the polymeric polysaccharide will also aggregate, which is not preferable.

The aggregated cells thus obtained can be removed from the diluted culture broth solution by a conventionally known method, that is, a method such as centrifugation or filtration. When a filtration method is adopted, a method such as pressure filtration, vacuum filtration, or natural filtration can be used, but there is no particular restriction on the shape and material of the filtration material, but the filtration material is A range naturally arises in the aperture selection. When a mesh nylon filter, a metal filter, or the like is used as the filter material, a filter having a pore size of 2 to 500 μm, more preferably 5 to 200 μm can be used. If the pore size is smaller than 2 microns, the solution does not pass easily and the working efficiency is lowered, and if the pore size is larger than 500 microns, aggregated bacterial cells also pass and cannot be separated.
It is possible to empirically select and suitably use a filter having another shape such as a sintered filter or a membrane filter having another shape.

By further adding alcohol to the diluted culture broth solution thus sterilized, the high molecular polysaccharide can be insolubilized and aggregated in the solution and recovered as a precipitate. The optimum amount of alcohol to be added depends on the type of alcohol to be added, but as an alcohol concentration that causes the high molecular polysaccharide to aggregate sufficiently efficiently, a final concentration (volume ratio) of 50 to 90% can be exemplified. A more preferable alcohol concentration range is 60 to 80% when the alcohol is isopropyl alcohol, and 55 to 7 when the alcohol is n-butyl alcohol.
5%, 70 if the alcohol is ethyl alcohol
The range of from 85% to 85% can be exemplified. Here, for all alcohols, the final concentration of alcohol is 50%
If it is lower, the high molecular polysaccharides do not aggregate sufficiently, and if it is higher than 90%, the salts, nutrients and waste products generated by microorganisms contained in the culture medium components are also precipitated and the purity of the recovered high molecular polysaccharides is reduced. It becomes a cause and is not preferable.

The alcohol concentration at which the high molecular polysaccharide causes coagulation and precipitation depends on the type of the high molecular polysaccharide, the molecular weight, the molecular weight distribution,
It is determined by the combination of the type of alcohol, etc. As a matter of course, when the alcohol is the same type, the concentration of alcohol that causes cell aggregation should be set lower than the concentration that causes high-molecular polysaccharides to aggregate and precipitate. . The alcohol used here and the addition method can be carried out according to the one used for the aggregation of the bacterial cells and the conditions, but it is not particularly necessary to use the same alcohol.
However, in consideration of recovery and reuse, it is industrially preferable to use the same kind.

The thus-obtained high molecular polysaccharide aggregate can be finally separated from the alcohol solution by a known method such as a filter. The polymer polysaccharide thus obtained may be used as it is in the next process, or the polymer polysaccharide having higher purity can be obtained by repeating the above-mentioned aggregation and purification. The high-molecular polysaccharide once coagulated and collected is dissolved in, for example, 10 to 80% aqueous alcohol solution by stirring, and then the alcohol concentration is increased from 50 to 90% to re-coagulate and precipitate to achieve the purpose of re-purification. be able to.

Alcohol can be recovered and reused by a known method such as distillation from the alcohol solution remaining after separating the high molecular polysaccharides, which is convenient for industrial implementation. Hereinafter, the present invention will be specifically described according to examples, but the present invention is not limited to these examples. In the examples,% is based on volume.

Example 1 At room temperature, Alcaligenes latus B-16 (Alcaligenes latu)
s B-16) (FERM BP-2015) culture broth stock solution (5 L) was mixed with water (5 L) with stirring. To this, 5 L of isopropyl alcohol was added and mixed with stirring to adjust the isopropyl alcohol concentration to 33%. This solution was filtered through a nylon filter having a pore size of 50 microns to remove bacteria. While stirring, add 1 part of isopropyl alcohol to the passing solution so that the isopropyl alcohol concentration becomes 66%.
5 L was added. The polymer polysaccharide began to aggregate from the time when about 12 L of isopropyl alcohol was added, and completely aggregated when 15 L had been added. The aggregated polymeric polysaccharide was collected on a nylon filter having a pore size of 470 microns. The recovered substance was vacuum-dried at 60 ° C. and pulverized to obtain a polymer polysaccharide powder.

The results of measuring the nitrogen content and the ash content for the purpose of determining the purity of the obtained high molecular polysaccharide are shown in Table 1.
Table 2 shows the amount of isopropyl alcohol and the maximum working volume required for treating 1 L of the culture broth stock solution.
The nitrogen content is an index of the remaining amount of the bacterial cells, and the ash content is an index of the inorganic salt remaining in the high molecular polysaccharide. The lower the values of the two types, the higher the purity of the recovered polymeric polysaccharide.

(Embodiment 2) Embodiment 1. The high-molecular polysaccharide obtained in (1) was dissolved in 15 L of 33% isopropyl alcohol aqueous solution and passed through a nylon filter having a pore size of 50 μm. To the passing liquid, while stirring, add 15% of isopropyl alcohol so that the isopropyl alcohol concentration becomes 66%.
L was added. The high-molecular-weight polysaccharide began to aggregate from the time point when about 13 L of isoplopyrrole alcohol was added, and completely aggregated after the completion of addition of 15 L. The aggregated polymeric polysaccharide was collected on a nylon filter having a pore size of 470 microns.
These operations were performed at room temperature. The recovered substance was vacuum-dried at 60 ° C. and pulverized to obtain a polymer polysaccharide powder. Table 1 shows the results of measuring the nitrogen content and the ash content for the purpose of determining the purity of the obtained high molecular polysaccharide. Table 2 shows the amount of isopropyl alcohol and the maximum working volume required for treating 1 L of the culture broth stock solution.

(Embodiment 3) Alcaligenes lettus B
5 L of water was added to 5 L of the stock solution of culture broth of -16, and the solution was heated to 50 ° C. and mixed by stirring. To this, 7.2 L of n-butyl alcohol was added and mixed with stirring to adjust the concentration of n-butyl alcohol to 42%. The solution was filtered and sterilized by passing through a stainless steel filter having a pore size of 125 microns. N-butyl alcohol concentration is 74% in the passing liquid while stirring
21.6 L of n-butyl alcohol was added so that The polymeric polysaccharide started to aggregate from the time when about 14.5 L of n-butyl alcohol was added, and completely aggregated when 21.6 L had been added. The aggregated polymeric polysaccharide was collected on a nylon filter having a pore size of 410 microns. The recovered substance was vacuum-dried at 60 ° C. and pulverized to obtain a polymer polysaccharide powder. Table 1 shows the results of measuring the nitrogen content and the ash content for the purpose of determining the purity of the obtained high molecular polysaccharide. In addition, Table 2 shows the amount of n-butyl alcohol and the maximum working volume required for treating 1 L of the culture broth stock solution.

(Example 4) At room temperature, 5.9 L of isopropyl alcohol was added to 10 L of the stock culture broth of Alcaligenes lethas B-16, and the mixture was stirred and mixed to adjust the isopropyl alcohol concentration to 37%. This solution is used for pore size 5
The bacteria were removed by filtration through a 0 micron nylon filter. 1% isopropyl alcohol was added to the passing solution while stirring to adjust the isopropyl alcohol concentration to 70%.
7.4 L was added. 11.5L of isopropyl alcohol
The high molecular weight polysaccharide started to aggregate from the time of addition,
By the time the L addition was finished, it completely coagulated. The aggregated polymeric polysaccharide was collected on a nylon filter having a pore size of 470 microns. The recovered substance was vacuum-dried at 60 ° C. and pulverized to obtain a polymer polysaccharide powder. The results of measuring the nitrogen content and the ash content for the purpose of determining the purity of the obtained high molecular polysaccharide are shown in Table 1. Table 2 shows the amount of isopropyl alcohol and the maximum working volume required for treating 1 L of the culture broth stock solution.

(Comparative Example 1) (A high molecular polysaccharide is aggregated with alcohol and recovered without sterilization) 5 L of a culture broth stock solution of Alcaligenes retus B-16 was added with 5 L of water, and the solution was heated to 50 ° C. The mixture was heated to and mixed with stirring. To this, 45.5 L of isopropyl alcohol was added and mixed with stirring to adjust the isopropyl alcohol concentration to 82%. The high-molecular-weight polysaccharide began to aggregate from the time when about 17 L of isopropyl alcohol was added, and completely aggregated when 45.5 L had been added. This aggregated polymeric polysaccharide has a pore size of 4
Recovered by passing through a 70 micron nylon filter. The recovered substance was vacuum-dried at 60 ° C. and pulverized to obtain a polymer polysaccharide powder.
Table 1 shows the results of measuring the nitrogen content and the ash content for the purpose of determining the purity of the obtained high molecular polysaccharide. Table 2 shows the amount of isopropyl alcohol and the maximum working volume required for treating 1 L of the culture broth stock solution.

(Comparative Example 2) (Centrion sterilization of a bacterium by a conventional method to collect a high-molecular polysaccharide with alcohol and recovery) At room temperature, 1 L of culture broth of Alcaligenes lethas B-16 was mixed with 9 L of water. The mixture was added and mixed with stirring. this,
The cells were sterilized by dividing each 2.5 L into about 4 times and centrifuging at about 40,000 g for 1 hour. 20 L of isopropyl alcohol was added to the supernatant liquid after sterilization and mixed with stirring to adjust the isopropyl alcohol concentration to 66%. 16.5 isopropyl alcohol
Polymeric polysaccharide started to aggregate from the time of adding about L
By the time the addition was completed, it completely coagulated. The aggregated polymeric polysaccharide was collected on a nylon filter having a pore size of 470 microns. The recovered substance was vacuum-dried at 60 ° C. and pulverized to obtain a polymer polysaccharide powder. Table 1 shows the results of measuring the nitrogen content and the ash content for the purpose of determining the purity of the obtained high molecular polysaccharide. Table 2 shows the amount of isopropyl alcohol and the maximum working volume required for treating 1 L of the culture broth stock solution.

[0023]

[Table 1]

[0024]

[Table 2]

[0025]

EFFECTS OF THE INVENTION According to the present invention, the removal of bacterial cells from the culture broth can be carried out with surprisingly high efficiency and simply by defining the type of alcohol and the concentration used, and the amount of treatment solution can be reduced. It is possible to significantly reduce the number of days required for the reduction and purification of the polymeric polysaccharide, and it is possible to obtain a highly purified polymeric polysaccharide, which is extremely useful in terms of industrial implementation.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ryuichiro Kurane 1-3, Higashi, Tsukuba, Ibaraki Prefectural Institute of Microbial Technology, Industrial Technology Institute (72) Noboru Tomizuka 1-3, Higashi, Tsukuba, Ibaraki (72) Inventor Hajime Mita 2-11-24 Tsukiji, Chuo-ku, Tokyo Inside Nippon Synthetic Rubber Co., Ltd. (72) Inventor, Yusho 2-chome Tsukiji, Chuo-ku, Tokyo 11-24 No. 24 within Japan Synthetic Rubber Co., Ltd. (72) Kenji Yasuda 2-chome Tsukiji, Chuo-ku, Tokyo No. 11-24 within Synthetic Rubber Co., Ltd. Japan (72) Yasuhiro Nobata Yokkaichi, Mie Prefecture 6-chome 6-9 Hakuto Co., Ltd. Yokkaichi Research Center

Claims (2)

[Claims] 1. Alcaligenes lat producing extracellular high molecular polysaccharide
(us) to a culture broth at atmospheric pressure at room temperature or under heating,
A polymeric polysaccharide characterized by selectively removing bacterial cells from the culture broth containing the polymeric polysaccharide by adding an aliphatic alcohol having 1 to 4 carbon atoms to a concentration at which only the bacterial cells aggregate and the polymeric polysaccharide does not aggregate. Purification method of sugars. 2. The culture broth sterilized in claim 1 is further supplemented with an aliphatic alcohol having 1 to 4 carbon atoms at a concentration at which the high molecular polysaccharide is aggregated under atmospheric pressure at room temperature or under heating.
A method for purifying a high molecular polysaccharide, which comprises precipitating and recovering the high molecular polysaccharide.