JPH05328988A - Production of high-molecular-weight pullulan - Google Patents

Abstract

PURPOSE:To provide a process for producing pullulan which can give pullulan of high molecular weight and prevent the culture medium from blackening due to production of black pigments. CONSTITUTION:In the production of pullulan by culturing a microorganism capable of producing pullulan, Aureobasidium pullulans, the culture mixture is heat-treated at 50 to 75 deg.C for 0.3 to 3hrs. The heat treatment inhibits the medium for producing pullulan from blackening.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing polymeric pullulan. More specifically, the present invention relates to a method for producing high molecular weight pullulan and a method for suppressing blackening of a pullulan production medium.

[0002]

2. Description of the Related Art Pullulan is a type of incomplete bacterium, Aureobasidium pullulans (Aureobasid).
It is a water-soluble polysaccharide produced in a culture solution when black yeast called ium pullulans) is cultivated under suitable conditions as a carbon source for monosaccharides, starch degradation products and the like.
Its chemical structure is maltotriose, which is a trimer of α-1,4 bond of glucose, and this trimer is α-
It is a linear polymer that is repeatedly linked by 1,6 bonds. Pullulan having a molecular weight of about 80,000 to 300,000 is industrially manufactured and sold, and excellent properties such as water solubility, adhesiveness and film forming property are widely used in the food industry and the chemical industry. It is also used in the field of chemistry as a standard substance for measuring the molecular weight of water-soluble polymers.

[0003] Here, it is known that the pullulan produced in the culture medium by culturing Aureobasidium pullulans is reduced in molecular weight by a pullulan-degrading enzyme centered on amylase secreted by the bacterium itself at the latter stage of the culture. Has been. As a method for suppressing the decrease in the molecular weight in such a culturing process, a method of adding an amylase activity inhibitor to the medium and culturing (Japanese Patent Publication No. 58-40479), a method of adjusting the pH and phosphate ion concentration of the medium ( Methods such as JP-B-51-42199) have been proposed.

[0004]

However, the above-mentioned conventional method does not fundamentally suppress the decrease in the molecular weight of pullulan and has not brought satisfactory results. Further, in the conventional method, there is a disadvantage that a black pigment is produced along with the production of pullulan, and the medium is blackened, and a countermeasure has been desired.

Therefore, an object of the present invention is to provide a method for producing pullulan capable of producing high-molecular weight pullulan and a method for inhibiting blackening of a pullulan production medium, which inhibits blackening of the medium due to the production of black pigment. It is in.

[0006]

Means for Solving the Problems As a result of intensive studies on a method for producing high molecular weight pullulan, the present inventors have found that, in addition to the reduction of the molecular weight in the culturing process, the culture solution is purified after the culturing is completed. The inventors have found that a remarkable decrease in the molecular weight and the production of a black pigment have occurred in the process of recovering pullulan, and have conceived the present invention.

To achieve the above object, the present invention according to claim 1 provides a method for producing pullulan by culturing a pullulan-producing bacterium, Aureobasidium pullulans, in which a culture solution after culturing is used. Temperature 50-7
It is characterized in that the heat treatment is performed under the condition of holding at 5 ° C. for 0.3 to 3 hours.

In order to achieve the above object, the gist of the invention of claim 2 is that the pullulan production medium comprises culturing the aureobasidium pullulans, which is a pullulan-producing bacterium, and then performing the heat treatment of claim 1. There is a blackening suppression method.

The pullulan-producing bacterium used in the present invention may be any strain belonging to Aureobasidium pullulans and may be a similar mutant strain. For example, IFO 6353, IFO 446 deposited at the Fermentation Research Institute
4 or ATCC 934 deposited internationally at ATCC (American Type Culture Collection)
8, ATCC 74100, ATCC 74101, A
TCC 74102, ATCC 74103, ATCC
74104, ATCC 74105 and the like and mutants thereof can be used. In particular, ATCC 74100, A
TCC 74101, ATCC 74102, ATCC
74103, ATCC 74104, ATCC 74
105 is suitable as a strain producing high molecular weight pullulan.

The medium and other conditions used in the present invention are not particularly limited, and commonly used conditions can be used.
That is, it is common to carry out culture under aerobic conditions using a water-soluble medium containing a carbon source, a nitrogen source, and an inorganic salt.

Examples of the carbon source include glucose, sucrose, maltose, starch hydrolyzate and the like.

As the nitrogen source, either inorganic or organic can be used. As the inorganic nitrogen source, for example, ammonium salts such as ammonium sulfate, ammonium nitrate or potassium nitrate or nitrates can be used. As the organic nitrogen source, for example, peptone, soybean powder, yeast extract, malt extract, meat extract, vegetable protein, casein, corn steep liquor and the like can be used.

Examples of the inorganic salts include salts of metal ions such as magnesium, iron, calcium, sodium and potassium. Specifically, for example, magnesium sulfate, ferrous sulfate, calcium carbonate,
Examples thereof include sodium chloride, potassium phosphate, manganese sulfate, potassium chloride, zinc sulfate, cobalt chloride and ammonium molybdate.

An initial pH of about 6.0 to 8.0 is suitable, but it is not always necessary to adjust it to this range during culturing, and it is general that the pH decreases as the culturing progresses. .. The temperature is appropriately 25 to 30 ° C., and the culturing time is 2 to 7
It is about a day. Furthermore, the conditions under which the cells grow in a yeast-like form are more preferable.

After completion of the culture, the culture solution is heated to a temperature of 50 to 75.
C., preferably 55 to 70.degree. C. is maintained. By this heat treatment, the bacterial cells in the culture broth are killed, and the amylase degrading enzyme centering on amylase is deactivated.
At this time, when the treatment temperature is 50 ° C. or lower, the killing of the cells and the inactivation of the enzyme become insufficient, and the molecular weight of pullulan is reduced and the black pigment is produced in the subsequent pullulan recovery step,
Not preferable. Further, if the treatment temperature is 75 ° C. or higher, the molecular weight of pullulan is lowered by heating and the culture solution is colored, which is not preferable.

The processing time is preferably 0.3 to 3 hours. When the treatment time is shorter than 0.3 hours, the treatment effect is insufficient, and when it is longer than 3 hours, the molecular weight of pullulan may be decreased by heating and the culture solution may be colored, which is not preferable. The culture solution after the heat treatment is sterilized by filtration or centrifugation, and if necessary, decolorized, desalted, and concentrated. Then, it is mixed with water such as hydrophilic alcohols such as isopropanol, ethanol and methanol or hydrophilic ketones such as acetone, and is mixed with a solvent having a property that does not dissolve pullulan. By this mixing operation, pullulan is precipitated and collected, and then, pullulan is collected by drying or by directly drying.

[0017]

According to the method for producing polymeric pullulan of claim 1, the culture is carried out by performing a heat treatment on condition that the culture solution after the culture is kept at a temperature of 50 to 75 ° C. for 0.3 to 3 hours. The bacterial cells in the liquid die, and the amylase degrading enzyme, mainly amylase, is deactivated. As a result, it is possible to prevent a decrease in the molecular weight in the step of purifying the culture medium after the culture is completed and recovering pullulan, and it is possible to produce a high-molecular weight pullulan.

According to the method for suppressing blackening of the medium for producing polymeric pullulan according to claim 2, the aureobasidium pullulans which is a pullulan-producing bacterium is cultured, and then the heat treatment according to claim 1 is performed. The cells in the culture broth die,
The amylase degrading enzyme centering on amylase is deactivated. As a result, it is possible to suppress the production of black pigment in the step of purifying the culture solution and collecting pullulan after the completion of the culture, and it is possible to suppress the blackening of the medium.

[0019]

EXAMPLES Examples of the present invention will be given below. In the following Examples and Comparative Examples, ATCC 74105 was used as Aureobasidium pullulans.

Example 1 (1) Pre-culture medium component composition Sucrose 10 g / L Yeast extract 2 g / L (NH ₄ ) ₂ SO ₄ 0.5 g / L K ₂ HPO ₄ 3 g / L MgSO ₄ / 7H ₂ O 0 _{.2g / L FeSO 4 · 7H 2} O 0.01g / L MnSO 4 · 4~5H 2 O 0.01g / L ZnSO 4 · 7H 2 O 0.01g / L water 2.5L pH 7.0 (2) the present culture medium component composition sucrose _{50g / L (NH 4) HPO} 4 1.0g / L K 2 HPO 4 2.0g / L FeSO 4 · 7H 2 O 0.01g / L MnSO 4 · 4~5H 2 O 0 _{.01g / L ZnSO 4 · 7H 2} O 0.01g / L water 16.2L pH 7.0

30 L of a medium solution having the composition of (2) above
The aureobasidium pullulans preculture liquid consisting of the composition of (1) above after 24 hours of culture in a fermenter was inoculated, initial pH 7.0, aeration 9 L / min, stirring speed 300-
Aeration culture was performed at 600 rpm for 2 days, and pullulan 2 was used.
A pale reddish cloudy viscous liquid containing 0 g / L was obtained.

This culture solution was heat-treated for 30 minutes at 55-60 ° C. with stirring. After this treatment, the liquid lost the pink color and became milky white.

The treated liquid was filtered with a filter medium containing diatomaceous earth to obtain a pale yellow transparent viscous liquid. White pullulan was then recovered by mixing with 1.5 volumes of 86 wt% isopropanol.

The intrinsic viscosity of this pullulan is 6.4 dl / g
And the relational expression of intrinsic viscosity and molecular weight of Bulga et al.

[0025]

[Equation 1]

The molecular weight calculated by the above was 6.4 × 10 ⁶ . In addition, the culture solution after this heat treatment was heated at 25 ° C. for 6 hours.
After storing for one day, pullulan was recovered by the same treatment. No coloration was observed on pullulan and the molecular weight was 6.4 x 1
0 ^6, did not change the molecular weight after storage for 6 days.

Comparative Example 1 The same treatment as in Example 1 was carried out except that the culture solution obtained in Example 1 was not heat-treated. The intrinsic viscosity of the obtained pullulan was 4.6 dl / g and the molecular weight was 3.8 × 10 ⁶ , which was 60% lower than that of Example 1. Further, the culture solution which was not subjected to this heat treatment was stored for 6 days in the same manner as in Example 1, and the molecular weight after that was measured. The molecular weight is 1.9x
It was 10 ⁶ and was significantly reduced.

Comparative Example 2 The same treatment as in Example 1 was carried out except that the culture solution obtained in Example 1 was heat-treated at 80 ° C. for 1 hour. The pullulan thus obtained has an intrinsic viscosity of 6.1 dl / g and a molecular weight of 5.8 × 10.
It was ⁶ .

This heat treatment liquid was used in the same manner as in Example 1
It was stored for a day, and its molecular weight was measured thereafter. The culture medium after storage was blackened, and the obtained pullulan was blackened.
The molecular weight was 5.4 × 10 ⁶ , which slightly decreased.

The results of Example 1 and Comparative Examples 1 and 2 are summarized in Table 1. In addition, the culture broth was 20 ° C. and the B-type viscometer was 30 rpm.
The viscosities measured in Table 1 are also shown in the table.

[0031]

[Table 1]

Example 2 The same medium composition as in Example 1 except that the sucrose content of the main culture medium was 100 g / L, and the initial pH was 7.0.
Aeration culture was carried out for 5 days at an aeration rate of 9 L / min and a stirring rate of 300 to 600 rpm. As a result, pullulan 58g / L
A pale reddish white turbid viscous liquid containing was obtained. This culture solution was heat-treated at 60 to 65 ° C. for 30 minutes while stirring. After this treatment, the liquid lost the pink color and became milky white.

The treated solution was centrifuged to remove bacteria, and then mixed with 1.5 volumes of 86 wt% isopropanol to obtain white pullulan. The pullulan had an intrinsic viscosity of 3.6 dl / g and a molecular weight of 2.6 × 10 ⁶ .

Comparative Example 3 The same treatment as in Example 2 was carried out except that the culture solution of Example 2 was not heat-treated. The pullulan thus obtained had a black color, its intrinsic viscosity was 2.5 dl / g, and its molecular weight was 1.5 × 10 ⁶ .

[0035]

As apparent from the above, according to the present invention, a method for producing pullulan capable of producing high-molecular weight pullulan and a pullulan production medium for suppressing the blackening of the medium due to the production of black pigment It is possible to provide a method for suppressing blackening.

[Procedure amendment]

[Submission date] July 13, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0015

[Correction method] Change

[Correction content]

After completion of the culture, the culture solution is heated to a temperature of 50 to 75.
C., preferably 55 to 70.degree. C. is maintained. By this heat treatment, the bacterial cells in the culture solution are killed, and the pullulan-degrading enzyme centering on amylase is deactivated. At this time, if the treatment temperature is 50 ° C. or lower, the killing of the bacterial cells and the inactivation of the enzyme become insufficient, and the molecular weight of pullulan decreases and the production of black pigment occurs in the subsequent pullulan recovery step, which is not preferable. Further, if the treatment temperature is 75 ° C. or higher, the molecular weight of pullulan is lowered by heating and the culture solution is colored, which is not preferable.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

The processing time is preferably 0.3 to 3 hours. When the treatment time is shorter than 0.3 hours, the treatment effect is insufficient, and when it is longer than 3 hours, the molecular weight of pullulan may be lowered by heating and the culture solution may be colored, which is not preferable. The culture broth after the heat treatment is sterilized by filtration or centrifugation, and if necessary, decolorized, desalted, and concentrated. Then, it is mixed with a solvent having a property of being miscible with water and not dissolving pullulan, such as hydrophilic alcohols such as isopropanol, ethanol and methanol or hydrophilic ketones such as acetone. By this mixing operation, pullulan is deposited and recovered, and then, pullulan is recovered by drying or by directly drying.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction content]

[0017]

According to the method for producing polymeric pullulan of claim 1, the culture is carried out by performing a heat treatment on condition that the culture solution after the culture is kept at a temperature of 50 to 75 ° C. for 0.3 to 3 hours. The cells in the liquid die, and the pullulan-degrading enzyme, mainly amylase, is deactivated. As a result, it is possible to prevent a decrease in the molecular weight in the step of purifying the culture medium after the culture is completed and recovering pullulan, and it is possible to produce a high-molecular weight pullulan.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

According to the method for suppressing blackening of the medium for producing polymeric pullulan according to claim 2, the aureobasidium pullulans, which is a pullulan-producing bacterium, is cultured, and then the heat treatment according to claim 1 is performed. The cells in the culture broth die,
The pullulan-degrading enzyme, mainly amylase, is deactivated.
This makes it possible to suppress the production of black pigment in the step of purifying the culture solution and collecting pullulan after the completion of the culture, and thus suppressing the blackening of the medium.

Claims (2)

[Claims] 1. A method for producing pullulan by culturing a pullulan-producing bacterium, Aureobasidium pullulans, wherein the culture solution after culturing is at a temperature of 50 to 75 ° C. for 0.3 to 3 hours. A method for producing polymer pullulan, which comprises performing a heat treatment on the condition that the polymer pullulan is held. 2. A method for suppressing blackening of a production medium for polymer pullulan, which comprises culturing a pullulan-producing bacterium Aureobasidium pullulans and then performing the heat treatment according to claim 1.