JP2003299494A - Method for biologically producing polyenic polycarboxylic acid or salts thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To heighten production efficiency of a polyenic polycarboxylic acid or salts thereof. <P>SOLUTION: The polyenic polycarboxylic acid or the salts thereof are produced by culturing microorganisms belonging to the genus Talaromyces by using a medium using a carbon source containing a maltose, a saccharified starch or a liquefied starch in an amount of 20-60 wt.% based on the whole carbon source. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for biologically producing a polyene polycarboxylic acid or a salt thereof.

[0002]

PRIOR ART AND PROBLEM TO BE SOLVED BY THE INVENTION It is disclosed in WO99 that salts of polyene polycarboxylic acids represented by the following formulas (1) and (2) are produced by culturing Thallomyces fungi. / 46231.

[0003]

[Chemical 1]

[0004]

[Chemical 2]

In the above equations (1) and (2),
A represents an alkali metal such as H or Na.

However, the production method disclosed in the above publication has a problem that the concentration of the finished culture is low and the production cost is increased.

Therefore, an object of the present invention is to enhance the production efficiency of the above polyene polycarboxylic acid or salts thereof.

[0008]

The present invention provides 20-60.
The above problem is solved by culturing a fungus of the genus Talalomyces in a medium containing a carbon source in a weight percentage to produce a polyene polycarboxylic acid or a salt thereof.

By using a predetermined amount of carbon source, it is possible to suppress the growth of bacteria and improve the productivity of polyene polycarboxylic acid or salts thereof.

The polyene polycarboxylic acid or its salt is (3Z, 8Z, 10E) -6-ethyl-3,
8,10-Tridecatriene-1,3,4,8,9-pentacarboxylic acid (hereinafter referred to as "S1 compound") or a salt thereof, (3Z, 8Z, 13Z, 15E) -6,1
1-Diethyl-3,8,13,15-octadecatetraene-1,3,4,8,9,13,14-heptacarboxylic acid (hereinafter referred to as "S2 compound") or a mixture thereof. The carbon source concentration is 15% by weight
When the above is completed, although the finished concentration of culture is high, S
The composition ratio of one compound is considerably higher than that of the S2 compound, resulting in a mixture in which the composition ratio of the S1 compound: S2 compound is far from the desired 1: 1. In order to avoid this, if the carbon source and the nitrogen source used in the medium are separately sterilized, more S2 compound can be produced. Further, when maltose, saccharified starch or liquefied starch is used as the carbon source, the productivity of polyene polycarboxylic acid or its salt can be increased.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The method for biologically producing a polyene polycarboxylic acid or a salt thereof according to the present invention is a method of culturing a microorganism belonging to the genus Talaromyces using a predetermined medium to produce a polyene polycarboxylic acid or a salt thereof. is there.

The polyene polycarboxylic acid or its salt is used as an industrial dispersant, and specifically, (3Z, 8Z, 10E) -6-ethyl-3,8,
10-tridecatriene-1,3,4,8,9-pentacarboxylic acid (S1 compound), (3Z, 8Z, 13Z, 1
5E) -6,11-Diethyl-3,8,13,15-octadecatetraene-1,3,4,8,9,13,14
-Heptacarboxylic acid (S2 compound) or a salt thereof. The biological production method according to the present invention is obtained as a mixture of the S1 compound (or a salt thereof) and the S2 compound (or a salt thereof).

The salts of the above S1 compound and S2 compound are
Examples thereof include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, organic base salts such as ammonium salt and trimethylamine salt, and amino acid salts such as alginate salt. Further, the carboxyl group forming a salt may be the whole or a part thereof. The pentasodium salt of the S1 compound has the following formula (3)
In addition, the 7-sodium salt of the S2 compound is a compound represented by the following formula (4).

[0014]

[Chemical 3]

[0015]

[Chemical 4]

The above S1 compound or a salt thereof and the S2 compound or a salt thereof are used as an industrial dispersant as described above. Among them, the S2 compound or a salt thereof is the S1 compound or a salt thereof. In many cases, the dispersive power is stronger than that in Example 1, and the performance as an industrial dispersant is often improved. Therefore, a production method in which the production ratio of the S2 compound or its salt is high is more preferable.

The composition ratio of the S1 compound or salt thereof and the S2 compound or salt thereof produced according to the present invention is not particularly limited, but the S2 compound or salt thereof is preferably higher for the above reasons. In the present invention, if the concentration of the carbon source is increased, as will be described later, the concentration of the finished culture is increased, but the composition ratio of the S1 compound or its salts is considerably higher than that of the S2 compound or its salts. For this reason, the composition ratio of both is close to the composition ratio in which the S1 compound or its salt is present in a larger amount than the S2 compound or its salt, and the composition ratio in which the S1 compound or its salt is equivalent to the S2 compound or its salt. Is more preferable.

The above-mentioned Talaromyc
The microorganism belonging to the es) genus is a kind of fungi. An example of this is Talalomyces. sp. No. 10092
(Talaromyces. Sp. No. 10092)
(Deposit No. FER, Institute of Biotechnology, Industrial Technology Institute)
MBP-6250).

The above-mentioned medium contains a high concentration of carbon source. The content of this carbon source is 2 with respect to the whole medium.
It is preferably 0% by weight or more, and more preferably 25% by weight or more. 20
If it is less than wt%, the concentration of the above-mentioned polyene polycarboxylic acid or salts thereof obtained becomes too low, resulting in inefficiency. In addition, the osmotic pressure of sugar is low, other microorganisms are likely to propagate, and various bacteria are easily contaminated. On the other hand, the upper limit is
60% by weight or less is preferable, and 50% by weight or less is preferable. 6
It may be higher than 0% by weight, but the production rate of the above S1 compound or its salts increases, the proportion of the S2 compound or its salts significantly decreases, and the viscosity of the culture solution becomes too high, making it difficult to handle. Moreover, the growth of microorganisms belonging to the genus Talalomyces is extremely slowed down. As a result, the number of days required for culturing becomes long and it becomes unsuitable for industrial production, so 50% by weight is preferable.

Examples of the carbon source include glucose, xylose, fructose, sucrose, maltose, raw starch, liquefied starch, saccharified starch, modified starch (oxidized starch, hydrolyzed starch, etc.). Among these, maltose, liquefied starch, saccharified starch and the like are preferable from the viewpoint of decomposition and absorption.

In addition to the carbon source, the medium contains nitrogen sources, inorganic salts and trace elements as essential components.
Examples of the nitrogen source include inorganic nitrogen such as sodium nitrate, ammonium nitrate and ammonium sulfate, organic nitrogen such as amino acids, natural products such as corn steep liquor and soybean powder. Among these, inorganic nitrogen such as sodium nitrate and ammonium sulfate, natural products such as corn steep liquor and the like are preferable from the viewpoints of contained components and price. These may be used alone or in combination. Examples of the inorganic salts and trace elements include calcium carbonate, magnesium sulfate, iron sulfate and the like. Regarding the use amount of the above-mentioned nitrogen source, inorganic salts and trace elements, it is only necessary to select the condition that the production amount of the above polyene polycarboxylic acid or its salt increases under the condition that the above-mentioned use amount of the carbon source is satisfied.

The above-mentioned carbon source and nitrogen source may be sterilized after they are mixed, but it is more preferable to sterilize them separately and then mix them to prepare a medium. This is because when a carbon source and a nitrogen source are mixed and sterilized, the sugar content and the nitrogen content may react with each other to form another compound due to a Maillard reaction or the like. This reaction product may interfere with the enzymatic reaction and growth of the above microorganisms, which may reduce the production amount of the above polyene polycarboxylic acid or its salt. In addition, when both are mixed and sterilized, the enzymatic reaction may be hindered as described above, but this hindrance is caused by the reaction of the S2 compound or its salts rather than the enzymatic reaction for the production of the S1 compound or its salts. The enzymatic reaction for production is more hindered. For this reason,
Among the obtained polyene polycarboxylic acids or salts thereof, the production amount of the S2 compound or salts thereof is decreased, and the production ratio of the S1 compound or salts thereof is relatively increased, so that the S2 compound or salts thereof is increased. This is because it is not preferable when it is desired to produce.

The sterilization conditions are not particularly limited as long as the purpose can be achieved, and may be 120 ° C. for about 20 minutes. The pH of the medium is preferably 3-8, more preferably 4-6. Moreover, about temperature, 20-35 degreeC is good and 25-30
C is preferred. These conditions are in a preferable range from the viewpoint of the production amount of the polyene polycarboxylic acid or its salt and the growth of the microorganism. The amount of dissolved oxygen is preferably 10% or more, and more preferably 20% or more with respect to the amount of saturated dissolved oxygen. Further, the air supply amount is preferably 0.3 to 1.5 VVM, and more preferably 0.5 to 1 VVM. This "VVM"
Means the amount of air fed / the amount of medium per minute. Air is sufficient as the gas to be fed at this time. Furthermore, the internal pressure in the culture tank is preferably 0.02 to 0.15 MPa, 0.0
5 to 0.1 MPa is preferable.

As the method for producing the above polyene polycarboxylic acid or salts thereof, the following method can be adopted. First, the above-mentioned microorganism is inoculated into a seed culture medium and seed-cultured for several days. Next, at a stage where the above-mentioned microorganism has grown to some extent, a part of the seed culture solution is inoculated into the preculture medium and precultured. Then, the preculture liquid in which the microorganisms have grown to some extent is inoculated into the main culture medium that satisfies the above conditions, and under certain conditions,
Perform main culture. This produces the desired polyene polycarboxylic acid or salt thereof, after a predetermined period of time, by separating the microorganism from the main culture, by collecting the residual liquid,
The desired polyene polycarboxylic acid or salt thereof is recovered.

The polyene polycarboxylic acid or salt thereof produced according to the present invention is used as an industrial dispersant as described above, and its fields of application are pigments, paints, cosmetics, dyes, processed papers, and ceramics. , A wide range of fields such as construction and civil engineering. Furthermore, the polyene polycarboxylic acid or a salt thereof produced by the present invention is
Being a compound produced by a microorganism, it has biodegradability. Therefore, it is also environmentally friendly.

[0026]

EXAMPLES The present invention will be described more specifically with reference to Examples and Comparative Examples below.

(Examples 1 to 4, Comparative Examples 1 and 2) <Seed culture> 3% by weight of sucrose, 2% by weight of pharma media, 1% by weight of dry yeast powder, and 0.1% by weight of 1 potassium phosphate. 100 ml of the medium obtained by dissolving in water was added to
It was placed in a 00 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes to obtain a seed culture medium. The above seed culture medium was added under sterile conditions to Talalomyces. sp. No. 10
092 (Talaromyces. Sp. No. 100)
92, FERM BP-6250) 0.2% inoculation,
Culture was carried out with shaking at 25 ° C. and 200 rpm for 3 days.

<Pre-cultivation> 100 ml of the same medium as the seed culture medium in which the above-mentioned microorganism was cultivated was placed in a 500 ml Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes,
It was used as a pre-culture medium. The above preculture medium was aseptically inoculated with 0.05% of the culture solution obtained by the seed culture, and the culture was performed at 25 ° C. for 20 days.
The culture was performed with shaking at 0 rpm for 2 days.

<Main culture> In the case of Example 1, 27% by weight of Koso Syrup (manufactured by Japan Corn Starch Co., Ltd., solid content: 75% by weight) containing 50% by weight of maltose as a carbon source and corn steep liquor (as a nitrogen source) Manufactured by Nippon Shokuhin Kako Co., Ltd., hereinafter abbreviated as “CSL”) 6% by weight,
0.2% by weight of calcium carbonate was used as the inorganic salt. Similarly, the main culture media of Examples 2 and 4 and Comparative Examples 1 and 2 are as shown in Table 1. In Examples 1, 2, and 4, the above carbon source, nitrogen source, and inorganic salts were mixed to prepare a 3 liter medium, which was autoclaved at 121 ° C. for 20 minutes to prepare a main culture medium. In addition, in Example 3, as a carbon source, a final concentration of koso syrup of 47% by weight (solid content: 3
(5.3% by weight), the final concentration of calcium carbonate as an inorganic salt was 0.2% by weight of 2.5 liters, and the mixture was autoclaved at 121 ° C. for 20 minutes. Also, as a nitrogen source,
CSL final concentration 9 wt% for 0.5 liter, 12
It was autoclaved at 1 ° C. for 20 minutes. And
Both were mixed and used for the main culture as 3 liters.

The above main culture medium was placed in a 5 liter fermenter (manufactured by Mitsuwa Biosystems Co., Ltd.) and 5% of the preculture liquid in which the above microorganism was cultured was inoculated. And temperature 25
℃, DO 10% or more (with stirring), pH lower limit value 4.6
(Adjusted with sodium hydroxide solution), internal pressure 0.5k
g / cm ² , air flow rate 3 liters / min (ie 1
VVM) for 7 days. And the obtained culture solution 1
After adjusting the pH to around 6 using 0 ml of an aqueous sodium bicarbonate solution and centrifuging at 3000 rpm for 10 minutes,
A supernatant was obtained. The supernatant was subjected to liquid chromatography to obtain Abs. The amount of S1 compound and S2 compound produced was measured by detection by absorption at 255 nm. As a column to be used at this time, Mightysil RP-18φ4.6 ×
75 mm was used. Furthermore, acetonitrile: distilled water: phosphoric acid = 550: 450: 1 was used as a mobile phase. Further, heptyl 4-hydroxybenzoate was used as an external standard, and the concentrations of the S1 and S2 compounds were calculated from the peak area ratios. The results are shown in Table 1.

[0031]

[Table 1]

(Results) Examples 1, 2, 4 and Comparative Example 1,
From 2, it was revealed that the total amount of the S1 compound and the S2 compound was increased by increasing the concentration of the carbon source. Further, it was revealed from Example 3 that the ratio of the production amount of the S2 component can be increased by separately sterilizing the carbon source and the nitrogen source.

Example 5 Maltose was used as a carbon source.
3% of saccharified starch (solid content 60% by weight) containing 0% by weight
3.8% by weight, 6% by weight of CSL as a nitrogen source, 0.2% by weight of calcium carbonate as an inorganic salt, and the above carbon source, nitrogen source and inorganic salts were mixed to prepare a 3 liter medium. Then, it was autoclaved at 121 ° C. for 20 minutes to obtain a main culture medium. The content rate of the carbon source in this main culture medium was 20.3% by weight (solid content). Main culture was carried out in the same manner as in Example 1 except that this main culture medium was used. The results are shown in Table 1.

Example 6 Maltose was used as a carbon source in an amount of 5
A saccharified starch containing 0% by weight (solid content: 60% by weight) was used at a final concentration of 58.8% by weight (1058 g), and calcium carbonate was used as an inorganic salt at a final concentration of 0.2% by weight (6 g). 2.5 liter of carbon source-containing liquid was prepared and autoclaved at 121 ° C. for 20 minutes.
In addition, CSL as a nitrogen source has a final concentration of 9% by weight (270
g) prepare 0.5 liter of a nitrogen source-containing solution,
It was autoclaved at 121 ° C. for 20 minutes. Then, the carbon source-containing liquid and the nitrogen source-containing liquid were mixed to prepare 3 liters of the main culture medium. The carbon source content in this main culture medium was 35.3% by weight (solid content).
Main culture was carried out in the same manner as in Example 1 except that 3 liters of this main culture medium was used. The results are shown in Table 1.

(Comparative Example 3) Glucose was used as a carbon source in an amount of 2
0.3% by weight (609 g), 6% by weight (180 g) of CSL as a nitrogen source, 0.2% by weight (6 g) of calcium carbonate as an inorganic salt, and the above-mentioned carbon source, nitrogen source and inorganic salts were used. Prepare 3 liters of medium by mixing
It was autoclaved at 1 ° C. for 20 minutes to obtain a main culture medium. The content ratio of the carbon source in this main culture medium is 2
It was 0.3% by weight (solid content). Main culture was carried out in the same manner as in Example 1 except that this main culture medium was used. The results are shown in Table 1.

Comparative Example 4 Glucose was used as a carbon source in a final concentration of 35.3% by weight (1059 g), and inorganic salts were
Using 0.2% by weight (6 g) of calcium carbonate, these were mixed to prepare 2.5 liters of carbon source-containing liquid.
It was autoclaved at 21 ° C. for 20 minutes. Also,
Prepare 0.5 liter of nitrogen source-containing liquid containing CSL as a nitrogen source at a final concentration of 9% by weight (270 g),
Autoclaved for 20 minutes. Then, the carbon source-containing liquid and the nitrogen source-containing liquid were mixed to prepare 3 liters of the main culture medium. The carbon source content in this main culture medium was 35.3% by weight (solid content). Main culture was carried out in the same manner as in Example 1 except that 3 liters of this main culture medium was used. The results are shown in Table 1.

[0037]

EFFECTS OF THE INVENTION According to the method of the present invention, by using a predetermined amount of carbon source, it is possible to suppress the growth of bacteria and improve the productivity of polyene polycarboxylic acid or its salts. .

When maltose, saccharified starch or liquefied starch is used as the carbon source, the productivity of polyene polycarboxylic acid or its salt can be increased.

If the carbon source and the nitrogen source are separately sterilized, more S2 compound can be produced.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hidehiro Kubo             1-8-10 Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui Prefecture             No. Rengo Co., Ltd. Fukui Research Center (72) Inventor Yoshio Kimoto             1-8-10 Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui Prefecture             No. Rengo Co., Ltd. Fukui Research Center F term (reference) 4B064 AD08 CA05 CD09 CD19 CE10                       DA16

Claims (3)

[Claims] 1. A polyene polycarboxylic acid or a salt thereof is cultured by culturing a microorganism belonging to the genus Talaromyces using a medium containing 20 to 60% by weight of the total carbon source containing maltose, saccharified starch or liquefied starch. A method for biologically producing a polyene polycarboxylic acid or a salt thereof, which produces: 2. The method for biologically producing a polyene polycarboxylic acid or a salt thereof according to claim 1, wherein the carbon source and the nitrogen source used in the medium are separately sterilized. 3. The polyene polycarboxylic acid or salt thereof is (3Z, 8Z, 10E) -6-ethyl-3,8,
10-tridecatriene-1,3,4,8,9-pentacarboxylic acid, (3Z, 8Z, 13Z, 15E) -6,1
The poly (ethyl ether) according to claim 1 or 2, which is 1-diethyl-3,8,13,15-octadecatetraene-1,3,4,8,9,13,14-heptacarboxylic acid, or salts thereof. A method for biologically producing enepolycarboxylic acid or a salt thereof.