KR101103246B1 - A method of microbe cultivation by mixed culture - Google Patents

Abstract

The present invention relates to a method for culturing microorganisms using a mixed culture, and more particularly, to grow microorganisms by adding and culturing other microorganisms with controlled cell activity when culturing microorganisms producing metabolites such as antibiotics and organic acids. It relates to a method for culturing microorganisms to improve the productivity of metabolites.

Microorganisms, mixed cultures, metabolites, antibiotics

Description

A method of culturing microorganisms using mixed culture {A METHOD OF MICROBE CULTIVATION BY MIXED CULTURE}

The present invention relates to a method for culturing microorganisms using a mixed culture, and more particularly, to grow and metabolize microorganisms by adding and culturing other microorganisms with controlled cell activity when culturing microorganisms producing metabolites such as antibiotics and organic acids. It relates to a method for culturing microorganisms to improve the productivity of the product.

Microorganisms are known to produce metabolites such as antibiotics, organic acids, amino acids, etc. during the culture (or fermentation) process. For example, mycelium microorganism Acremonium chrysogenum produces a substance called cephalosporin C ('CPC'), which is a widely used β-lactam antibiotic, cephalo, for the treatment of bacterial diseases. It is a raw material of sporin. Since the metabolites of microorganisms are usefully used in various fields such as medicine / pharmaceutical field, bioreaction process field, and the like, there is an increasing interest in a method of culturing microorganisms that can increase their productivity.

On the other hand, changes in morphology, inoculum status, types of microorganisms, and functional enzymes in cultivation of microorganisms have been reported to have a great influence on the growth and metabolites of microorganisms because they are closely related to metabolism of cells. . Among the important important substances produced from microorganisms, antibiotics in particular are closely related to these culture conditions.

In this regard, JP2004-275189 discloses a method for culturing a plurality of microorganisms, a preparation of a microorganism-derived preparation, and the like, for enhancing the probiotic functionality of lactic acid bacteria and Bifidobacterium microorganisms. This patent is characterized by culturing at least one of the microorganisms in the lactic acid bacteria and Bifidobacterium, and filamentous fungi such as fungi, respectively, followed by mixing and culturing each liquid culture in a liquid state or after drying.

However, this technique is intended to further improve the probiotics function of lactic acid bacteria or Bifidobacterium microorganisms, and does not disclose the purpose or effect for enhancing the productivity of metabolites such as antibiotics. Therefore, there is a continuing need in the art for the development of new culture techniques that can improve the productivity of metabolites.

The present invention has been devised under this technical background, and an object of the present invention is to provide a method for culturing new microorganisms for increasing productivity and efficiency of metabolites of microorganisms, particularly antibiotics, which are useful in various fields.

In order to achieve the above object, the present invention provides a method for culturing a microorganism, characterized in that by culturing a second microorganism with a controlled cell activity in the culture of the first microorganism producing a metabolite.

According to one embodiment, the second microorganism whose cell activity is controlled is a microorganism from which cell activity has been removed or a microorganism of which cell activity has been attenuated.

According to one embodiment, the metabolite is one or more selected from the group consisting of antibiotics, organic acids, amino acids, nucleic acids, alcohols and vitamins.

According to one embodiment, the metabolite is antibiotic, preferably cephalosporin C.

According to one embodiment, the first microorganism is Acremonium sp. Microorganism, the second microorganism is E. coli , Ankistrodesmus sp. And Streptomyces high At least one member selected from the group consisting of S. hygroscopicus subsp .

According to one embodiment, the first microorganism is Acremonium chrysogenum ( Acremonium chrysogenum ), the second microorganism is a microorganism of the genus Ankystrodes mousse cell activity is removed, E. coli or cell activity is weakened Attenuated Streptomyces hygroscopius subspecies ( S. hygroscopicus subsp. Hygroscopicus ).

According to one embodiment, the first microorganism may be incubated before mixing the second microorganism, the incubation time may be 10 hours to 300 hours.

According to the microbial culture method of the present invention, there is an excellent effect of improving the productivity of metabolites such as CPC by about 30% compared to the conventional culture method. Therefore, the present invention can be very useful in fields such as various pharmaceutical and pharmaceutical industries that require metabolites, and bioreaction process research.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention relates to a method for culturing a microorganism, characterized in that the culture of the first microorganism producing a metabolite is mixed by culturing a second microorganism with controlled cell activity.

At this time, the second microorganism whose cell activity is controlled is a microorganism whose cell activity has been removed or weakened. How to regulate the cell activity may vary depending on the type of microorganism, but microorganisms that attenuate cell activity are generally more desirable in terms of improving the productivity of metabolites than microorganisms (inactive microorganisms) from which cell activity has been removed.

When using microorganisms from which cell activity has been removed as a second microorganism, it is thought that enzymes or DNA particles in the second microorganisms affect the productivity of metabolites. In addition, in the case of using a microorganism with reduced cellular activity as the second microorganism, the second microorganism induces an immune response of the first microorganism and the like, leading to excessive antibiotic secretion and the like, thereby increasing the productivity of the metabolite.

In the present invention, the first microorganism that produces the metabolite may be used without particular limitation as long as it produces the metabolite. At this time, examples of the metabolite include antibiotics, organic acids, amino acids, nucleic acids, alcohols, vitamins, and the like, preferably antibiotics, and more preferably cephalosporin C.

In the present invention, the combination of the first microorganism and the second microorganism may be appropriately determined by those skilled in the art according to the type of metabolite to be obtained, the type of the microorganism, the culture characteristics or the active characteristics of the microorganism, but preferably antibiotics as the first microorganism. Acremonium sp. , Which produces the substance, is the second microorganism to be mixed and mixed with E. coli , Ankistrodesmus sp. Or Streptomyces hygroscopius subspecies. ( S. hygroscopicus subsp. ) May be used.

More preferably, Acremonium chrysogenum , which produces cephalosporin C, is used as a first microorganism, and the second microorganism includes microorganisms of the genus Ankystrodes mousse, cell activity of which cell activity is removed. S. ygroscopicus subsp. Hygroscopicus , etc., which have weakened E. coli, and attenuated cell activity, may be used.

On the other hand, as a method for removing or attenuating the cellular activity of the second microorganism, methods known in the art can be used without limitation.

For example, the following method may be used to remove the cellular activity of the second microorganism: The selected second microorganism may be cultured in each liquid medium except for agar, followed by vortex mixer with glass beads. The cells can be made inactive by mixing with a vortex mixer or the like to disrupt the cells. At this time, when the second microorganism is genus Anchistrodesmus, it is preferable to block the light during the stationary culture.

As a method of weakening the cellular activity of the second microorganism, a method of applying a vaccine manufacturing process, or a microorganism in a poor environment (using a medium other than the optimal medium, using a medium without essential components, culturing under conditions that are not optimal) Etc.) and the method of subculture.

For example, when E. coli is used as the second microorganism, it is passaged in sequence in the PDA (potato dextrose agar), agar, PDA, and agar medium, and then cultured for a predetermined time in PDB (potato dextrose broth) medium, respectively. It is possible to attenuate its cellular activity by the method.

On the other hand, the culture conditions at the time of the mixed culture of the first microorganism and the second microorganism can be appropriately determined by those skilled in the art according to the kind of the microorganisms used (particularly the kind of the first microorganism), the kind of the medium, and the like. For example, in one embodiment of the present invention, the first microorganism uses acremonium chrysogenum, and the second microorganism uses Escherichia coli, and the like, and the mixed culture conditions are 150-500 rpm, 20-40 ° C., 12 It is preferable that it is -240 hours. More preferably, it is preferable to incubate at 200-400 rpm, 23-35 ° C. for 48-200 hours, even more preferably 70-180 hours.

In addition, the first microorganism is preferably used in terms of productivity of the metabolite using a culture in advance in the seed medium (seed medium) before the second microorganism is mixed and cultured.

At this time, the incubation time may vary depending on the type of microorganisms, for example 10 to 300 hours. Preferably, a sufficient incubation for 60 hours or more and mixed culture with the second microorganism are effective for increasing the productivity of the metabolite. Other specific culture conditions may also vary depending on the type of microorganism, the type of medium, etc., but may be cultured at 23 to 35 ° C. at 200 to 400 rpm, for example.

The seed medium for pre-culturing the first microorganism may be determined according to the type of the microorganism. For example, in the case of acremonium chrysogenium, basal seed medium may be composed of cellulose, glucose, corn steep liquor (CSL), and (NH ₄ ) ₂ SO ₄ . As the main medium, glucose, corn steep liquor, (NH ₄ ) SO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ , 0DL-methionine and a microelement solution may be used. In order to improve the morphological differentiation of microorganisms, soybean meal, cottonseed flour, CaCO ₃ may be further added to the basic seed medium.

Meanwhile, the mixing ratio of the first microorganism and the second microorganism may be appropriately determined and used by those skilled in the art according to the type of each microorganism, the amount of the medium, and the like. For example, the mixing ratio may be the first microorganism: the second microorganism = 100: 0.001 to 10 (volume ratio), preferably the first microorganism: the second microorganism = 100: 0.005 to 5 (volume ratio), more Preferably, the first microorganism: second microorganism = 100: 0.01-3 (volume ratio). When the mixing ratio of the microorganisms satisfies the above range, the degree of the immune response by the enzyme, the DNA particles, and the second microorganism in the second microorganism is maintained in an appropriate range to positively affect the productivity of the metabolite by the first microorganism. Can be.

As described above, the improvement of the yield of the metabolite obtained by the microbial culture method of the present invention includes the measurement of the concentration of the metabolite (eg, CPC) by high performance liquid chromatography (HPLC), the measurement of the dissolved oxygen amount (DO) of the biological culture solution, It can be confirmed by measuring the dry cell mass.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by the following examples.

Preparation Example: Cultivation of Microorganisms

1. Microorganism Types

As the first microorganism, Acremonium chrysogenum M35, a mutant species generated in Acremonium chrysogenum ATCC 20339, which produces CPC, was used. In addition, as a second microorganism for mixed culture, Streptomyces hygroscopicus subspecies hygroscopicus KCTC 9846, Escherichia coli JM109 ATCC 53323 or Ankystrodes mousse KMCC FC 107, respectively.

2. Medium and culture conditions of the first microorganism Acremonium chrysogenum M35

Stock cultures were maintained by transferring microorganisms every two weeks on a potato dextrose agar (PDA) sealant. The base seed medium consisted of 2.5% sucrose, 1.0% glucose, 2.5% corn steep liquor and 0.4% (NH ₄ ) ₂ SO ₄ . To improve the morphological differentiation of the microorganisms, 3.0% soy bean meal, 1.0% cottonseed flour and 0.5% CaCO ₃ were added to the base seed medium.

Main medium consists of 1.95% glucose, 5% corn steep liquor, 0.8% (NH ₄ ) SO ₄ , 0.3% KH ₂ PO ₄ , 0.5% K ₂ HPO ₄ , 0.5% DL-methionine and 0.4% trace element solution Was used.

In preparation of the basal seed medium, sugar, (NH ₄ ) ₂ SO ₄ and corn steep liquor were sterilized separately from the other components, and even when preparing the main medium, sugar, (NH ₄ ) ₂ SO ₄ and DL-methionine Sterilized separately from the other ingredients. CaCO ₃ and the like were added to the basic seed medium, and before sterilization, 1N NaOH was added to adjust the pH to 7.0. Linoleic acid 4% (v / v) was added to the main medium.

In a 2L Erlenmeyer flask containing 200 ml of the basic seed medium, acremonium chrysogenum M35 was pre-incubated for 72 hours at 27 ° C. and 280 rpm.

3. Medium and culture conditions of the second microorganism

(1) Streptomyces hygroscopius subspecies hygroscopicus

To culture Streptomyces hygroscopius subspecies hygroscopicus, a type of actinomycete, a medium consisting of 0.4% yeast extract, 1.0% malt extract, 0.4% glucose and 2.0% agar at pH 7.3 was used. . At this time, the culture temperature was 26 ℃.

(2) Escherichia coli

As a culture medium of Escherichia coli, one composed of 0.5% yeast extract, 1.0% tryptone, 1.0% sodium chloride and 1.5% agar at pH 7.0 was used. At this time, the culture temperature was 37 ℃.

(3) Ankistrodesmus

The algae genus Anchistrodesmus was cultivated in Jwor (Jaworski's medium) at a light intensity of 3,000 lux for 10 hours per day. At this time, the culture temperature was 20 ℃.

4. Removal of cellular activity of the second microorganism

In order to remove the cell activity in the ankystrodes mousse, the light was blocked first in JM medium except agar (agar), and then incubated for 32 hours at 52 ℃ and then incubated for 24 hours at 25 ℃. This process was repeated three times.

Then, 5 ml of the obtained culture was mixed with six glass beads (glass bead 1, Glastechnique Mfg., Germany) for 10 minutes with a vortex mixer to lyse the cells.

5. Weakening Cellular Activity of Second Microorganisms

The method of weakening the cell activity of the second microorganism is as follows.

(1) Streptomyces hygroscopius subspecies Hygroscopicus was passaged at 30 ° C. for 7 days in PDA, agar, PDA, and agar medium, respectively, followed by 7 days at 30 ° C. and 120 rpm in PDB medium. It was.

(2) Escherichia coli was subcultured at 41 ° C. for 7 days while continuously changing the medium to PDA, agar, PDA and agar medium, and finally at 41 ° C. and 120 rpm for 7 days in potato dextrose solution (PDB) medium. Incubated.

(3) Ankistrodesmus genus was 60 days at 28 ° C. in JM medium containing no Ca (NO ₃ ) _{2 ·} 4H ₂ O, MgSO ₄ .7H ₂ O, NaNO ₃ and Na ₂ HPO ₄ .12H ₂ O. During stationary culture. At this time, the intensity of illuminance was maintained at 1,000 lux for 10 hours per day.

[Examples 1 to 14 and Comparative Examples 1 and 2: Mixed Culture of Microorganisms]

Examples 1-12

Acremonium chrysogenum M35 (first microorganism) pre-incubated for 72 hours and the second microorganism from which cell activity has been removed or attenuated, were subjected to 27 ° C. in a 250 ml Erlenmeyer flask containing 20 ml of main medium. , 340 rpm mixed culture for 7 days. At this time, the type and amount of the second microorganisms mixed and cultured are shown in Table 1 below.

Comparative Example 1

The experiment was carried out under the same conditions as in Examples 1 to 12 except that the second microorganism was not added.

Examples 13-14

In addition, acremonium chrysogenum M35 (first microorganism) pre-cultured for 72 hours and a second microorganism from which cell activity was removed or attenuated were respectively stored in a 5 L tank stirred microbial incubator (Kobiotech Co., Ltd., Korea). The mixture was cultured for 7 days at 340 rpm. At this time, the operating volume was 3.0L and the air flow rate was 1.2vvm. The type and amount of the second microorganisms mixed and cultured with the first microorganism in this example are also shown in Table 1 below.

Comparative Example 2

The experiment was carried out under the same conditions as in Examples 13 to 14 except that the second microorganism was not added.

First microorganism 2nd microorganism Culture space Kinds Kinds Content (ml) Example 1 a-1 b-1 0.05 250ml Erlenmeyer flask Example 2 a-1 b-1 0.10 250ml Erlenmeyer flask Example 3 a-1 b-1 0.15 250ml Erlenmeyer flask Example 4 a-1 b-1 0.20 250ml Erlenmeyer flask Example 5 a-1 b-2 0.05 250ml Erlenmeyer flask Example 6 a-1 b-2 0.10 250ml Erlenmeyer flask Example 7 a-1 b-2 0.15 250ml Erlenmeyer flask Example 8 a-1 b-2 0.20 250ml Erlenmeyer flask Example 9 a-1 b-3 0.05 250ml Erlenmeyer flask Example 10 a-1 b-3 0.10 250ml Erlenmeyer flask Example 11 a-1 b-3 0.15 250ml Erlenmeyer flask Example 12 a-1 b-3 0.20 250ml Erlenmeyer flask Example 13 a-1 b-1 30 5 ml incubator Example 14 a-1 b-3 30 5 ml incubator Comparative Example 1 a-1 - - 250ml Erlenmeyer flask Comparative Example 2 a-1 - - 5 ml incubator

a-1: Acremonium Chrysogenum M35

b-1: genus of ankystrodesmus with depleted cellular activity

b-2: Streptomyces hygroscopius subtype Hygroscopicus with reduced cellular activity

b-3: Escherichia coli with weakened cellular activity

[Test Example: Evaluation of Characteristics of Mixed Culture Microorganisms]

1. Method of Analysis

(1) Every 24 hours, CPC concentration was measured by high performance liquid chromatography (HPLC, Young Lin M930, Young Lin Instrument Co., Korea) using a UV detector set at 254 nm with μ Bondapak C-18 of a reversed phase column. Acetonitrile-phosphate buffer was used as the mobile phase. As elution mixture, 98% (v / v) phosphate buffer and 2% (v / v) acetonitrile having a flow rate of 0.9 mL / min were used, and CPC zinc salt (Sigma, USA) was used as a standard sample.

(2) Cell morphology was studied with micrographs taken using Image Pro 3.0 software (Media cybernetics, Inc. USA) and optical microscope (Nicon eclipse 80i, Nicon Co., Japan).

2. Results

1 is a graph showing CPC concentrations according to time zones in Examples 1 to 4 and Comparative Example 1. FIG. Referring to Figure 1, it can be seen that the CPC concentration of Examples 1 to 4 is higher than the CPC concentration according to Comparative Example 1. In particular, the CPC concentration of Example 4 rapidly increased from 3.7 g / L at 84 hours of incubation to 7.9 g / L at 132 hours of incubation. Therefore, it can be seen from the results of FIG. 1 that the CPC production of Acremonium chrysogenum M35 was positively affected by the mixed culture with Ankystrodesmus genus from which cell activity was removed. At this time, it is thought that the cause of the influence is due to the enzyme of the microorganism of the genus Ankystrodes mousse.

2 is a graph showing CPC concentrations according to time zones of Examples 5 to 8 and Comparative Example 1. FIG. 2, it can be seen that the CPC concentration of Examples 5 to 8 is higher than the CPC concentration of Comparative Example 1. In addition, in Examples 5 to 8 and Comparative Example 1, the CPC concentration reached its peak at 120 hours of incubation. Among them, the CPC concentration of Example 6 obtained by adding 0.10 mL of streptomyces with attenuated cell activity to the mixed culture showed the maximum value, and the CPC concentration was 8.7 g / L after 120 hours of incubation. However, after 132 hours had elapsed, the concentrations of CPC of Examples 5 to 8 and Comparative Example 1 dropped drastically.

3 is a graph showing CPC concentrations according to time zones of Examples 9 to 12 and Comparative Example 1. FIG. Referring to FIG. 3, Examples 9 to 12 reached the highest CPC concentrations for 108 hours of incubation. In particular, the CPC concentration of Example 12, which was mixed and cultured with 0.20 mL of E. coli attenuated cell activity, showed a maximum value. Increased sharply. On the other hand, in Comparative Example 1, the CPC concentration reached the highest and about 5.3 g / L at 120 hours of incubation, and the CPC concentration decreased from 120 hours.

Figure 4 shows a photograph of the morphological changes of the Acremonium chrysogenum M35 observed in a phase contrast microscope when mixed culture in the conditions of Example 13, Example 14 and Comparative Example 2 in a 5L incubator. In Figure 4 (a) is a photograph of the three-day culture of Comparative Example 2, (b) is a photograph of the five-day culture of Comparative Example 2, (c) is a photograph of the five-day culture of Example 13 , (d) is a photograph at the time of culture for 5 days in Example 14.

Referring to (a) to (d) of FIG. 4, it can be seen that when three days of Comparative Example 2 were cultured, many filamentous fungi and expanded mycelium fragments were present. However, when culturing Comparative Example 2, Example 13, and Example 14 for 5 days, respectively, it can be seen that the morphological changes of Acremonium chrysogenum M35 are similar.

5 is a graph showing CPC concentrations according to time zones of Example 13, Example 14, and Comparative Example 2. FIG. Referring to FIG. 5, in Example 13, the maximum value of the CPC concentration appeared on day 6, and the value was 2.0 g / L. Example 14 appeared on day 4 the maximum value of CPC concentration was 2.2 g / L. On the other hand, in Comparative Example 2, the maximum value of CPC concentration appeared on day 5, and the value was 1.7 g / L.

From the results of FIGS. 1 to 5, when Acremonium chrysogenum M35 is mixed and cultured with a second microorganism with or without cellular activity, CPC production is improved compared to when Acremonium chrysogenum M35 is cultured alone. I could confirm that. Among them, mixed culture with microorganisms with reduced cellular activity was more effective for CPC production by Acremonium chrysogenum M35 than mixed culture with microorganisms with reduced cellular activity. This was thought to be due to the fact that living microorganisms are closely related to the immune response caused by acremonium chrysogenum, and that immune response (defense response) induces excessive secretion of antibiotics in immersion culture. Particularly, it was found that CPC production was increased more rapidly and rapidly when the mixed culture of Acremonium chrysogenum M35 and E. coli with weakened cell activity was combined.

1 is a graph showing the CPC concentration by time zone of Examples 1 to 4 and Comparative Example 1.

2 is a graph showing CPC concentrations according to time zones of Examples 5 to 8 and Comparative Example 1. FIG.

3 is a graph showing CPC concentrations according to time zones of Examples 9 to 12 and Comparative Example 1. FIG.

FIG. 4 is a photograph of morphological changes of acremonium chrysogenum M35 when mixed and cultured under the conditions of Example 13, Example 14, and Comparative Example 2 in a 5L incubator using a phase contrast microscope.

5 is a graph showing CPC concentrations according to time zones of Example 13, Example 14, and Comparative Example 2. FIG.

Claims (9)

When culturing Acremonium chrysogenum to produce cephalosporin C, Cell activity is removed or weakened as not keystrokes in death mousse microorganism (Ankistrodesmus sp.), Escherichia coli and the one member selected from the group consisting of Streptomyces high-gloss high-gloss nose kusu subspecies nose kusu (S.hygroscopicus subsp. Hygroscopicus) A method for culturing acremonium chrysogenium, characterized by culturing by mixing. delete delete delete delete delete delete The method according to claim 1, The acremonium chrysogenium is Acromonium, characterized in that it is pre-cultured prior to mixing with one species selected from the group consisting of microorganisms of the genus Ankystrodes mousse, Escherichia coli and Streptomyces hygroscopicus subspecies S. hygroscopicus subsp. Method for culturing chrysogenium. The method according to claim 8, The method of culturing acremonium chrysogenium is characterized in that the incubation for 10 hours to 300 hours in advance.

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