KR960007614B1 - Process for preparation of purified xanthan gum - Google Patents

Abstract

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Description

Process for producing purified chitantan rubber

The present invention provides a method for producing purified xanthan gum. More specifically, the present invention relates to a method for improving transparency of chitantan rubber fermentation broth and aqueous solution by enzyme treatment.

Xanthan rubber is obtained by well-known fermentation methods. That is, chitantan rubber is a bacterium belonging to the xanthomonas group that produces chitantan rubber, for example, chisantomonas campestris (the compound and its manufacturing method are described in US Pat. No. 3,659,026). It is produced by extracting and recovering using isopropanol in the fermentation liquor produced in the specification, 4th column).

As another method for producing chisantan rubber, instead of Chisantomonas campestris, other known Chisantomonas bacteria, i.e. Chisantomonas carotate. Chisantomonas incanae, chisantomonas begoniae, chisantomonas papavericola, chisangomonas translucens, chisantomonas bascules Chilumtan rubber fermentation broth can be produced using vasculorum, chisantomonas and heterae.

However, at the end of fermentation, in addition to about 2-5% by weight of chitan rubber, undissolved constituents such as nutrients and cell residues of fungi that have not yet been consumed are contained in 0.5-2% by weight of the fermentation broth. The transparency of chitantan rubber aqueous solution is very low. Therefore, there is a problem in the field where a transparent product is required, for example, the secondary recovery of food or cosmetics black or petroleum. As a method for purifying chitantan rubber. It is generally known to remove undissolved components in these enzyme solutions by centrifugation black silver cake filtration or the like. But,

These are because fermentation broth is highly viscous. By dilution with water followed by concentration. In terms of cost and operation, it is not a practical job. In these methods. In order to improve the filtration characteristics, it is also proposed to heat the fermentation broth. As an effective method, there is a method of solubilizing undissolved components in fermentation broth by enzyme treatment. Many methods have been proposed.

For example, U.S. Application No. 449875 (1974) (Japanese Patent Publication No. 50-1214939 and U.S. Application No. 513810. U.S. Patent 19763966618, 1977 4010071, U.S. Application 1070) No. 416525 suggests clarification using alkaline proteases and neutral proteases, but it is stated that the chitantan rubber solution is not necessarily crystal clear and somewhat cloudy. There is not enough to get enough transparency.

British Application No. 8122564 (Japanese Patent Laid-Open No. 58-81792) in 1981 proposes a method using an acidic protease and a neutral protease. In addition, U.S. Patent Application No. 797093 (Japanese Patent Publication No. 62-44918) in 1977 proposes to remove the cell body from an aqueous polymer solution by contacting the solution with a siliceous solid after protease treatment. French application No. 8210403 (Japanese Patent Laid-Open No. 57-202303) in 1981 discloses a fermentation treatment method using polysaccharase and protease, and US Patent No. 4431734 (Japanese Patent Laid-Open No. 63-287494) refers to poly A method using a combination of an enzyme having galactronase activity and an effect having protease activity, US Application No. 1980 Turn No. 147812 (Japanese Patent Laid-Open No. 57-5698) discloses β-1,3-glucanase activity. And protease treatment, such as a method using a complex enzyme having protease activity, have not been obtained.

Further, as a method using other enzymes, British Application No. 8431653 (Japanese Patent Laid-Open No. 61-146193) discloses a method using an enzyme having nuclease activity, French Application No. 8021395 ( Japanese Patent Application Laid-Open No. 57-91194) proposes a purification method using cellulase, but any effect is not sufficient. Japanese Patent No. 13185520 (Japanese Patent Laid-Open No. 60-44919) proposes a method of simultaneously adding and treating lysozyme and N-arithyl muramyl-L-alanine amidase and peptidase. have. Although they are known as cell wall lysing enzyme groups, they are difficult to act directly on Gram-negative bacteria and, despite the long-term treatment, they were surprisingly ineffective.

As described above, as a method of removing the water-insoluble microbial residue and the undissolved powder derived from the medium. Methods using centrifugation or cake filtration and the like by enzyme treatment for solubilizing undissolved powder are known. However, it was difficult to obtain high transparency of the chianthane rubber fermentation broth.

In view of these technical problems, an object of the present invention. It is to clarify with chisantan rubber fermentation broth.

That is, it is an object of the present invention that the fermentation broth is heat-treated under specific pH and temperature conditions, and subsequently enzymatically treated, thereby extracting and separating the solid from the fermentation broth using a specific organic solvent, for example, isopropyl alcohol. The light transmittance of 0.3% by weight aqueous solution of chitantan rubber is 80% or more, and to provide a method for producing chitantan rubber having excellent viscosity characteristics. According to the present invention, the fermentation broth of chitantan rubber is heat treated as a pretreatment for enzymatic treatment, followed by cooling, followed by continuous fermentation using alkaline protease followed by lysozyme, or alkaline protease followed by water insoluble microorganism cells. The solubilized residue from the residue and the medium component is sufficiently solubilized to obtain an aqueous solution of chitantan rubber having high transparency.

That is, the present invention. Heat treatment at an initial pH of 9 or more followed by enzymatic treatment. In the enzymatic treatment, a combination of alkali protease and lysozyme can be used to remove residues and media components of water-insoluble microbial cells without compromising the inherent viscosity characteristics of chitantan rubber. Provided is a method for producing fermented chitantan rubber exhibiting high transparency by solubilizing the resulting undissolved seafood.

The above heat treatment can be performed at an initial pH of 9-12.5, more preferably at pH 10-12, at a temperature of 45-7O < 0 > C, more preferably at 50-6O < 0 > C for 30 minutes or more. Below pH 9, the solubilization efficiency of bacteria may not be achieved well, which is not preferable. Above pH 12.5, the coloring of the fermentation broth or the viscosity of the product may be adversely affected, which is undesirable. During the treatment, the pH naturally decreases, but it is preferable to maintain the pH in the range of 8 or more. If the temperature is 45 ° C. or less, the solubilization efficiency of the bacteria in the enzyme treatment may not be satisfactorily achieved, which is not preferable. Above 70 ° C, the coloration of the fermentation broth and the viscous physical properties of the product may be adversely affected, which is not preferable. If it is 30 minutes or more, there exists a sufficient effect, but heat processing more than 2 hours may bring a fall of productivity, and is unpreferable. Heat treatment must be performed before enzyme treatment. Without this pretreatment, the effect of enzymatic treatment may not be exerted or a long time is required to exert the effect.

The enzyme treatment is carried out in two steps using an alkaline protease and lysozyme. The time for each enzyme treatment is preferably 20 minutes or more and less than 5 hours. If it is shorter than 20 minutes, the treatment effect may not be sufficiently expressed, which is not preferable. If the treatment is carried out for 5 hours or more, no further treatment effect is expressed, resulting in a decrease in productivity.

In the case of treatment with alkaline protease, pH 6.0-10.0, preferably pH 7.5-9.0. It is carried out by treating at least 10 minutes at a concentration of 10-500 ppm and a temperature of 40-65 ° C. Below 40 deg. C, the enzyme activity decreases, treatment is required for 6 hours or more, and is inappropriate in terms of productivity. In addition, the enzyme loses the activity above 65 ℃, there is no treatment effect.

Alkali proteases lose their activity in acidity below pH 6.0, and in pH above 10.0, the physical properties of the product may be deteriorated. In addition, when the concentration is 10ppm or less, the long-term treatment does not show sufficient effect. If the concentration is more than 500ppm, it is not preferable in terms of production cost, and even if added more, the effect is not improved.

In the case of treatment with lysozyme, the treatment is carried out at pH5.5 to 8.0, preferably at pH6.5-7.5, at a concentration of 100 ppm, for 20 minutes or more at a temperature of 25 to 45 ° C. Above 45 ° C, the activity of lysozyme may decrease, which is undesirable. Lysozyme does not exhibit activity at pH 8 or above, and at pH 5.5 or below, the physical properties of the product may be degraded, which is not preferable. If the concentration is 0.5 ppm or less, a long time treatment is required, and even if the treatment is performed for a long time, there may be insufficient effects. Above 100 ppm, it is not preferable in terms of cost. The order of two types of enzyme treatment may be made first.

Useful enzymes of embodiments of the present invention include N-acetylglucosamine and N-acetylglucosamine in bacterial cell walls and alkaline proteases produced by B, a microorganism in the genus Bacillus, Subtilis. lysozyme known as endo-β-1,4-N-acetylhexosamidase, which hydrolyzes β-1,4 bonds of acetylmuramic acid). Generally, in addition to B. subtilis, alkali proteases are those of B. licheniformis, B. amylaliquifaciens and B. pumilis origin. About lysozyme As animal lysozyme, chicken, duck, quail. Turkey or goose egg lysozyme, dog or rat spleen, lysozyme, human urine (leukemia patient), human milk, tear lysozyme. As plant lysozyme, it is known to exist in turnip, cabbage and papaya milk. However, the origin of the enzyme is not particularly important in the present invention.

Example

Using Examples and Comparative Examples. The present invention will be described in detail.

Example 1

The culture medium containing the composition of II was placed in a 30 L fermenter, and the culture solution of chisantomonas campestris, which is the composition of I after 24 hours of incubation, was cultured to obtain a pH of 6.5-7.0 and a temperature of 30. Aeration agitation for 2 days at ℃. A fermentation broth containing 30 g / L of chitantan rubber was obtained. The initial pHll. 90 minutes heat treatment at a temperature of 55 ℃. Thereafter, the mixture was prepared at pH8.5 as it was at 55 ° C, and 300 ppm of an alcatyprotease (Bioplase: manufactured by Nagase Seikagaku) was added and treated at 55 ° C for 2 hours with stirring. Next, the fermentation broth was cooled to 35 ° C, and 3 ppm of lysozyme (lysozyme thiioo: Taiyooka Kaguzen) was added and treated at 35 ° C for 1 hour while stirring.

Example 2

The fermentation broth as in Example 1 was subjected to the enzyme treatment in the reverse order to the same example. After cooling, it was prepared at pH 7.0, 3 ppm of lysozyme (Lysozyme Taiyo: manufactured by Taiyooka Furniture Co., Ltd.) was added, and the treatment was performed at 35 ° C for 2 hours while stirring. Next, the fermentation broth was heated up to 55 ° C, and 300 ppm of alkali protease (Bioplase: Nigase Seika Furniture Co., Ltd.) was added thereto and treated at 55 ° C for 1 hour while stirring.

For analysis, the fermentation broth after each fermentation after the end of fermentation, after the heat treatment, after the first step and after the second step of the enzymatic treatment was taken out, and chitantan rubber was extracted and separated using 1.6 wt. Times of isopropyl alcohol. Blow dry. Thereafter, a 0.3 wt% aqueous solution of the obtained solid chilate rubber was prepared, and the light transmittance and viscosity (Brookfield viscometer, 30 rpm) were measured for each sample. The results are shown in Table 1.

Examples 3 and 4

As in Example 1, the concentration of lysozyme used was changed. The results are shown in Table 2.

Example 5

It fermented like Example 1 and obtained the fermentation broth containing 30 g / L chitantan rubber. The fermentation broth was heat-treated for 90 minutes at an initial pH of 7.0 and a temperature of 80 ° C while stirring, and then cooled to 55 ° C. After cooling, the mixture was prepared at pH 8.5, and 300 ppm of an alkaline protease (Bioplase: Nagase Seika Furniture Co., Ltd.) was added thereto and treated at 55 ° C. for 2 hours with stirring.

Next, the fermentation broth was cooled to 35 degreeC, 3 ppm of lysozyme (Lysozyme Taiyo: Taiyooka furniture) was added, and it processed at 35 degreeC for 1 hour, stirring.

For analysis, the fermentation broth after each fermentation after the end of fermentation, after the heat treatment, after the first step of the fermentation, and after the second step is taken, and the chitantan rubber is extracted using 1.6 wt. Times of isopropyl alcohol. Separated and blow-dried. Thereafter, a 0.3 wt% aqueous solution of the obtained solid chianthane rubber was prepared, and the light transmittance and viscosity (Brookfield panometer, 30 rpm) were measured for each sample. The results are shown in Table 3.

After fermentation and heat treatment as in Example 1, either treatment with lysozyme or alkaline protease was performed alone. Treatment conditions of the two enzymes are the same as in Example 1. Each time, 1.6 weight times of isopropyl alcohol was used to extract and separate chianthane rubber, followed by air drying. Then, the light transmittance and viscosity of the 0.3 wt% aqueous solution of chitantan rubber obtained were measured.

As shown in Table 4, high transparency obtained by the method according to the present invention could not be achieved even by a long time treatment.

Comparative Example 3

After fermentation and heat treatment as in Example 1, it was combined treatment with lysozyme and alkaline protease. The reaction conditions were all performed at pH 7.5 and 45 degreeC of activity, and 300 ppm of bioplase and 3 ppm of lysozyme were added, and it was made to react for 6 hours. Transmittance and viscosity were measured every hour as in Comparative Example 1. The results are shown in Table 5.

Comparative Example 4

After fermentation was carried out as in Example 1, the two-step enzyme treatment shown in Example 1 was carried out without heat treatment, and the light transmittance and viscosity were measured for each sample. The results are shown in Table 6. It can be seen that the effect of the enzyme is lowered in the unheated fermentation broth.

According to the method of the present invention, complicated operations such as dilution, cake filtration, and concentration that the conventional method for removing undissolved components are required can be omitted, which is advantageous in view of ease of operation and economical efficiency. Moreover, the high transparency which cannot be achieved by the method using the conventional enzyme can be obtained.

Claims (4)

After the heat treatment of the xanthan rubber fermentation broth, it is successively treated with alkaline protease first and then with lysozyme or initially with lysozyme and subsequently with alkali protease, followed by recovery of the xanthan rubber. The manufacturing method of refined fine carbonate rubber. The process of claim 1 wherein the heat treatment comprises an initial pH of 9 to 12.5. 30 minutes or more at a temperature of 45-70 ℃. The treatment with alkaline protease is carried out for 30 minutes or more at 40-65 ° C. and pH 6.0-10.0 with the addition amount of alkali protease being 10 to 500 ppm with respect to the chitantan rubber fermentation broth. Manufacturing method characterized in that. The treatment with lysozyme according to claim 1 or 2, wherein the amount of lysozyme is added to the chitantan rubber fermentation broth at 0.5-100 ppm, and the temperature is performed at 25-45 ° C. and pH5.5-8.0 for at least 30 minutes. Characterized in the manufacturing method.