JPH0731468A - Method for continuous liquid culturing of mold - Google Patents

Abstract

PURPOSE:To simply and efficiently carry out the continuous liquid culturing of a mold excellent in productivity by inoculating the mold having the protease productivity into a specific liquid culture medium and culturing the mold while keeping the concentration, etc., of dissolved oxygen during the culturing constant. CONSTITUTION:A mold such as a filamentous fungus, having the protease productivity and belonging to the genus Aspergillus is inoculated into two or more culture media having substantially the same ratio (C/N ratio) of carbon to nitrogen as a liquid culture medium and cultured while keeping the concentration of dissolved oxygen or pH during the culturing constant. Thereby, the continuous liquid culturing of the mold is carried out.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquid culture method for filamentous fungi for the purpose of efficiently and stably producing a protease over a long period of time.

[0002]

2. Description of the Related Art Conventionally, in liquid culture of filamentous fungi,
Various techniques for efficiently producing protease have been reported (for example, JP-A-60-141482, JP-A-62-126975, and JP-A-64-2484).
No. 81, No. 62-248485, No. 63-71169, No. 63-219376, No. 63-248390, and No. 1-2.
96978, Japanese Patent Laid-Open No. 3-277280, etc.).

Among the above-mentioned conventional methods, JP-A-62-248
The method described in Japanese Patent Publication No. 485 is considered to be most related to the method of the present invention, but the method described in this publication is a method for improving the productivity of protease in a short-term culture (for example, 7 days). In this publication, there is no description regarding efficient and stable production of protease over a long period of time.

[0004]

When a filamentous fungus having a protease-producing ability is cultured in a liquid medium, the dissolved oxygen concentration and pH of the culture solution fluctuate (decrease or increase) from the initial values of the culture, and There has been a problem that the production amount is reduced and the protease cannot be stably produced over a long period of time. In order to solve this problem, means for adjusting the dissolved oxygen concentration while adjusting the agitation rotation speed in the culture tank or adjusting the pH of the medium by adding an alkali or an acid has been conventionally adopted. However, by rotating the stirring blade at a high speed, the mycelium of the filamentous fungus is damaged, and the production amount of protease is reduced. It has been pointed out that there are problems such as changes and adverse effects on the protease-producing ability of filamentous fungi.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies in order to overcome the problems of the above-mentioned conventional methods, and as a result, in the process of main culture for producing protease, the ratio of carbon to nitrogen ( C / N ratio) has substantially the same ratio, two or more kinds of media having different concentrations, and if necessary, one or more kinds of nutrient sources selected from a carbon source or a nitrogen source are used in combination,
The inventors have found that by culturing while maintaining the dissolved oxygen concentration and / or pH in the culture constant, it is possible to efficiently and stably produce a protease over a long period of time, and have completed the present invention.

That is, according to the present invention, in a method of inoculating a filamentous fungus capable of producing a protease into a liquid medium and continuously culturing the filamentous fungus, the liquid medium has a carbon-to-nitrogen ratio (C / N ratio). Continuous liquid culture of filamentous fungus, characterized by using two or more kinds having substantially the same ratio and different concentrations and culturing while maintaining the dissolved oxygen concentration or pH during the culture constant Method (invention method 1). Further, the present invention is a method for inoculating a filamentous fungus having a protease-producing ability into a liquid medium and continuously culturing the filamentous fungus, wherein the liquid medium has a substantially carbon-to-nitrogen ratio (C / N ratio). Culturing while maintaining the dissolved oxygen concentration and pH in the culture constant by using two or more types of media having the same ratio and different concentrations and one or more nutrient sources selected from carbon sources or nitrogen sources in combination. The present invention relates to a continuous liquid culture method for filamentous fungi (method 2 of the present invention).

The present invention will be described in detail below. The filamentous fungus used in the present invention is not particularly limited as long as it has protease-producing ability. Specifically, filamentous fungi having a protease-producing ability belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, the genus Mucor, the genus Rhizopus, for example, Aspergillus oryzae IFO434.
8, Aspergillus soya IAM2703, Aspergillus tamari IAM2156, Penicillium luteum AHU8022, Mucor Lasemmus AHU600
2. Resorpes Japanica IFO5441 etc. can be used. Among these filamentous fungi, those resistant to salt (for example, capable of growing even at a concentration of 5% or more of salt) are preferable from the viewpoint of preventing contamination of various bacteria.

In the method of the present invention, before the method of the present invention, that is, the main culture for producing a protease, is started, pre-culture is carried out to proliferate the cells in advance to secure a required cell concentration. This pre-culturing is carried out by inoculating the filamentous fungal cells or spores into a liquid medium usually used for liquid culture of filamentous fungi, until the desired bacterial cell concentration is obtained, usually until the stationary phase. That is, glucose, soluble starch, saccharose, dextrin, glycerin, etc. as the carbon source of the medium used for preculture, and peptone, meat extract, yeast extract, soybean powder, soybean isolated protein, gluten, polypeptone, soyton etc. as the nitrogen source. Can be used respectively. Also, micronutrients necessary for the growth of microorganisms such as various vitamins, various inorganic salts, and nucleic acid compounds may be used if necessary.

The C / N ratio of the medium used for the preculture may be arbitrarily set according to the type of filamentous fungus to be used. For example, when a filamentous fungus belonging to the genus Aspergillus is used as the filamentous fungus, the filamentous fungus belonging to the genus Aspergillus is effective by setting the C / N ratio of the medium in the range of 5 to 50, preferably 10 to 30. Can be propagated. Cultivation conditions of filamentous fungi can be appropriately selected from the following ranges: culture temperature: 20-40 ° C., medium pH: 3-8, and aeration rate: 0.1-2 vvm. The culture method is a batch system,
Either continuous method may be adopted.

The culture obtained from the above pre-culture is used as it is, or only the bacterial cells are separated and used for main culture for producing a protease. In the method 1 of the present invention, the ratio of carbon to nitrogen (C /
One of the characteristics is that two or more kinds of media having substantially the same N ratio) but different concentrations are used.
In the present specification, “substantially the same” means that the target numerical value is included within ± 5% of the standard numerical value.

The various nutrient sources in the liquid medium used for the main culture may be any as long as filamentous fungi can be used. Illustrating such a nutritional source,
As the carbon source, glucose, soluble starch, sucrose, dextrin, glycerin, soy sauce, etc., as the nitrogen source, peptone, meat extract, yeast extract, soybean powder,
Bran, soybean protein isolate, casein, gluten, polypeptone, soyton and the like can be used respectively. Also, micronutrients necessary for the growth of microorganisms such as various vitamins, various inorganic salts, and nucleic acid compounds may be used if necessary.

The C / N ratio of the medium may be appropriately set depending on the filamentous fungus to be used as in the case of the preculture. For example,
When producing a protease using a filamentous fungus belonging to the genus Aspergillus, the C / N ratio of the medium is 6 to
It is set to 30, preferably 12 to 20. The concentration of carbon or nitrogen in the medium may be appropriately set depending on the filamentous fungus to be used. For example, when a protease is to be produced using a filamentous fungus belonging to the genus Aspergillus, the carbon concentration in the medium is 0.05 to 10% (W /
V), preferably 0.1 to 5.0% (W / V). If the carbon concentration is determined, the nitrogen concentration is
It is automatically set according to the C / N ratio. In this way, when the concentration of the medium and the C / N ratio are appropriately selected from the above range, the first medium is prepared by a conventional method.

Next, in the method 1 of the present invention, although the C / N ratio is substantially the same as that of the above-prepared medium,
It is necessary to prepare one or more media with different concentrations.
At that time, when the medium to be prepared next is continuously cultured using only the previously prepared medium, the dissolved oxygen concentration or pH in the culture solution changes (increases or decreases). It must have a concentration that can induce a change (decrease or increase). Therefore, it is important to carry out a preliminary test before the main culture to determine a concentration range satisfying the above conditions in advance. Generally, the concentration range satisfying such conditions varies depending on the microorganism used, but when filamentous fungus belonging to the genus Aspergillus is used, the concentration range is about 0.1 to 10 times the concentration of the initially prepared medium. is there. Such a medium is easily prepared, for example, by previously analyzing the respective contents of carbon and nitrogen for each nutrient source and appropriately combining various nutrient sources according to a predetermined C / N ratio and concentration. can do.

Another feature of the method 1 of the present invention resides in that two or more kinds of the above-mentioned media are continuously used while being switched, and the culture is carried out while keeping the dissolved oxygen concentration or pH during the culture constant. That is, while monitoring the dissolved oxygen concentration or pH in the culture broth, when the dissolved oxygen concentration or pH in the culture broth becomes higher than a predetermined value, switch to a medium with a higher concentration than the medium used previously and reverse. When it becomes lower than a predetermined value, the medium before the switching is supplied to carry out the culturing, whereby the dissolved oxygen concentration or pH during the culturing can be kept constant. On the contrary to the above case, when the dissolved oxygen concentration or pH in the culture solution becomes lower than the predetermined value, the medium is switched to a medium having a lower concentration than the previously used medium and, conversely, becomes higher than the predetermined value. In that case, the medium before switching may be supplied and cultured.

As other conditions during the culture, the usual conditions adopted when producing a protease using a filamentous fungus may be appropriately selected and used. For example, the dissolved oxygen concentration is adjusted to 0 ppm or more, preferably 4 to 6 ppm. The pH is adjusted to 3-8, preferably 5-7.

Next, the method 2 of the present invention is a further improvement of the method 1 of the present invention described above. In addition to the medium in the method 1 of the present invention, at least one nutrient selected from a carbon source or a nitrogen source is further added. It is characterized by culturing while using a source together and maintaining the dissolved oxygen concentration and pH during culturing constant. As the nutrient source used for the culture, the same one as that used when preparing the above-mentioned medium can be exemplified. When the dissolved oxygen concentration or the pH fluctuates from a predetermined value while carrying out the method 1 of the present invention, a nutrient source is added to maintain the dissolved oxygen concentration and the pH at the predetermined values. Good. The other method conditions in the method 2 of the present invention are the same as those in the method 1 of the present invention.

As a means for recovering the protease from the culture broth obtained as described above, for example, the culture broth is filtered by a conventional method to separate the cells, and if necessary, dialysis, salting out, and ion exchange. Examples of the purification method include resin and gel filtration.

[0018]

INDUSTRIAL APPLICABILITY The present invention is a method for culturing a filamentous fungus while maintaining the dissolved oxygen concentration and / or pH at a constant value by a simple method, and efficiently proteases for a long time (for example, 60 days or more). ), It can be stably produced, and is extremely significant in industry.

[0019]

EXAMPLES The present invention will be described in detail below with reference to examples. Example 1 (1) Preculture 5% (W / V) soluble starch, 1% (W / V) soyton, 0.03% (W / V) yeast extract, 0.5% (W / V)
V) 3 liters (L) of a liquid medium (pH 6.3) containing 1 potassium phosphate and 5.0% (W / V) sodium chloride was added to 3 types of jar fermenters, and heat-sterilized under normal pressure. The medium was inoculated with a spore suspension of Aspergillus oryzae IFO4348 (the number of spores was 1 × 10 ⁶ cells / ml), and the cells were cultured at an aeration rate of 1 vvm, a stirring rate of 400 rpm, and a culture temperature of 30 ° C. After 48 hours from the start of the culture, three kinds of continuous culture were carried out as follows.

(2) Main Culture Test-1 (Invention Method 1) The following two types of liquid medium-1 or-
2 was continuously supplied from the supply port of the jar fermenter at a dilution rate of 0.015 hr ⁻¹ , and the stirring speed was 400 rpm and the aeration rate was 1 vvm while maintaining the culture solution volume to 3 L.
The continuous culture was carried out. In addition, when the dissolved oxygen concentration is 5 ppm or less, the liquid medium-1 is supplied, and the dissolved oxygen concentration is 5 pp.
When it was more than m, liquid medium-2 was supplied. By this means, the dissolved oxygen concentration is 5 ± 0.1ppm
It was possible to control in the range of.

Liquid medium-1: 1.25% soluble starch, 0.6% soytone, 0.03% yeast extract, 0.5
% Monopotassium phosphate, 5.0% sodium chloride, pH 6.3 Liquid medium-2: 3.5% soluble starch, 1.8% soytone, 0.03% yeast extract, 0.5% monopotassium phosphate, 5 Table 1 shows the elemental analysis values of various nutrient sources used in the preparation of 0.0% sodium chloride, pH 6.3 medium. The C / N ratios of liquid media-1 and -2 are both 14.8.

[0022]

[Table 1]

Test-2 (Method 1 of the present invention) The following two types of liquid medium-3 or-
4 was continuously supplied at a dilution rate of 0.015 hr ^-1 from the supply port of the jar fermenter, and while maintaining the amount of culture solution to 3 L, stirring rotation speed 400 rpm, aeration amount 1 vv
Continuous culture was performed at m. The liquid medium-3 was supplied when the pH was 6.3 or lower, and the liquid medium-4 was supplied when the pH was 6.3 or higher. By this means,
Was controllable within the range of 6.3 ± 0.1.

Liquid medium-3: 1.25% soluble starch, 0.6% soytone, 0.03% yeast extract, 0.5
% Monopotassium phosphate, 5.0% sodium chloride, pH 6.3 Liquid medium-4: 3.5% soluble starch, 1.8% soytone, 0.03% yeast extract, 0.5% monopotassium phosphate, 5 0.0% sodium chloride, pH 6.3 The C / N ratios of liquid culture media -3 and -4 were both 1
It is 4.8.

Test-3 (Control) Liquid culture medium-5 was held in the preculture liquid at a dilution rate of 0.015 hr ^-1 so that the culture liquid of the jar fermenter was kept at 3 L, and the dissolved oxygen concentration was 5 ppm. Change the stirring speed as shown below, and add p with sodium hydroxide or hydrochloric acid.
H was adjusted to 6.3, and continuous culture was performed at an aeration rate of 1 vvm. Liquid medium-5: 3.5% soluble starch, 1.8% soytone, 0.03% yeast extract, 0.5% monopotassium phosphate, 5.0% sodium chloride, pH 6.3.

Table 2 shows the amount of filamentous fungal cells and the protease activity of the above-mentioned three types of continuous culture solutions over time. The protease activity value was measured by the Anson Hagiwara modified method, and the amount of enzyme that liberates 1 μg of tyrosine-like substance in 1 minute was 1 unit (u). As is clear from Table 2, it was revealed that the method of the present invention can stably produce protease for a long period of time, as compared with the conventional method.

[0027]

[Table 2]

Example 2 (Invention Method 2) In the method of Test 1 of Example 1, soy sauce was further used as a carbon source in combination with a dissolved oxygen concentration of 5 ± 0.1 ppm and a pH of 6. The filamentous fungus was continuously cultured under the control of 3 ± 0.1. As a result, as also shown in Table 2 above, the protease production amount was increased by about 20 to 30% as compared with the case of the test 1 of Example 1, and moreover, it was stable for a long period of time as in the case of test 1. It became clear that a protease could be produced.

Claims (2)

[Claims] 1. A method of inoculating a filamentous fungus capable of producing a protease into a liquid medium and continuously culturing the filamentous fungus, wherein the liquid medium has substantially the same ratio of carbon and nitrogen (C / N ratio). A continuous liquid culture method for filamentous fungi, which comprises using two or more kinds of media having different ratios and different concentrations, and culturing while maintaining the dissolved oxygen concentration or pH during culturing constant. 2. A method for inoculating a liquid medium with a filamentous fungus capable of producing a protease and continuously culturing the filamentous fungus, wherein the liquid medium has substantially the same ratio of carbon and nitrogen (C / N ratio). Culturing while maintaining a constant dissolved oxygen concentration and pH in the culture by using two or more types of media having different ratios and different concentrations and one or more nutrient sources selected from carbon sources or nitrogen sources in combination. A continuous liquid culture method for filamentous fungi, which is characterized in that