KR102169686B1 - Method for preparing citric acid using Aspergillus sp. mutation by methanol - Google Patents

Abstract

The present invention relates to a method for producing citric acid using microorganisms of the genus Aspergillus, a method for producing mutated microorganisms for the production of citric acid, and to microorganisms in Aspergillus mutated through this. Microorganisms in the spore can significantly improve the production capacity of citric acid in a high concentration of methanol in the environment.

Description

Method for preparing citric acid using microorganisms of the genus Aspergillus mutated by methanol {Method for preparing citric acid using Aspergillus sp. mutation by methanol}

The present invention relates to a microorganism of the genus Aspergillus mutated for the production of citric acid, a method for producing the same, and a method for producing citric acid using the same.

Citric acid is an organic acid contained in the fruits and seeds of many plants such as lemons and tangerines, and is also called citric acid. Citric acid plays an important role in the body because it is an intermediate substance in the TCA cycle (Tricarboxylic Acid Cycle), which is a process of obtaining energy by breaking down carbohydrates, proteins, and fats in the body. In addition, citric acid is widely used in various industrial fields, and in particular, in the food industry, it is widely used as an acidulant and natural preservative to give the taste or sour taste of food.

Accordingly, as the demand for citric acid increases around the world, many studies are being conducted to increase the production of citric acid. The most representative method is the method of obtaining citric acid by fermenting carbohydrates using fungi, especially Aspergillus Niger ( Aspergillus) , which can discharge citric acid into the medium. niger ) fungi are used in many studies.

As another method for increasing the production of citric acid, it has been reported that when methanol is added to the liquid medium in which Aspergillus is grown and cultured, citric acid is produced more than that in the culture medium not containing methanol. (Wieczorek and Braver, 1998, I.-U. Haq et al. Bioresource Technology, 2003). This is because methanol increases the osmotic pressure of the cell membrane of Aspergillus and changes the shape and size of the mycelium and the shape and size of the mycelium to facilitate the excretion of citric acid from the mycelium. synthase) to promote citric acid production (I.-U. Haq et al. Bioresource Technology, 2003, Srivasta and Kamal, 1979).

However, it is essential to maintain the pH value in the fermentation of microorganisms for citric acid production, and since methanol is toxic, when methanol is used as an accelerator for citric acid production, the increase in the concentration of methanol causes the problem of inhibiting the growth of microorganisms. have. I.-U. Haq et al. (2003) found that when 1.0% (v/v) methanol was treated with Aspergillus, the production of citric acid anhydride was about two times higher than that of the control without methanol, but the concentration of methanol was 1.0% (v/v). When exceeding /v), it has been reported that the production of citric acid decreased sharply. In addition, U.S. Patent No. 4,791,058 describes that the yield of citric acid decreases rapidly when 3% (v/v) or more of methanol is treated with Aspergillus Niger.

Therefore, in conventional studies, it was found that the yield of citric acid was highest when methanol was treated with a concentration of about 1.0% (v/v), but the growth of microorganisms was rather suppressed by increasing the treatment concentration. Furthermore, even with the same paper, there is a limit that the production of citric acid does not increase consistently under methanol treatment.

Under this background, the present inventors have made intensive research efforts to develop a method for increasing the production of citric acid using microorganisms of the genus Aspergillus. As a result, when a microorganism of the genus Aspergillus is adapted and mutated to a high concentration of methanol (beneficial mutation) It was confirmed that the production rate of citric acid was remarkably improved and this tendency was maintained, and the present invention was completed.

One object of the present invention is (a) culturing the spores of microorganisms of the genus Aspergillus in a medium containing 1% (v/v) to 8% (v/v) of methanol to prevent microorganisms mutated by methanol. A first step of manufacturing; (b) a second step of separating the spores of the microorganisms mutated in the first step; (c) a third step of culturing the spores isolated in the second step in a medium containing 0.01% (v/v) to 8% (v/v) methanol; And (d) a fourth step of obtaining citric acid from the microorganism cultured in the third step or a medium thereof, to provide a method for producing citric acid.

Another object of the present invention is a first step of culturing the spores of microorganisms of the genus Aspergillus in a medium containing 1% (v/v) to 8% (v/v) of methanol; And it is to provide a method for producing a mutated microorganism for citric acid production comprising a second step of separating the spores of the mycelium showing yellow in the front and white in the rear of the cultured microorganisms.

Another object of the present invention is to provide a microorganism of the genus Aspergillus that has been mutated, prepared by a manufacturing method.

In order to achieve the above object, the present invention is a first step of culturing the spores of microorganisms of the genus Aspergillus in a medium containing 1% (v/v) to 8% (v/v) methanol as an embodiment. ; And a second step of separating the spores of the mycelium showing yellow on the front side and white on the rear side of the cultured microorganisms, and a method for producing mutated microorganisms for citric acid production, and a mutated, produced by the production method It provides microorganisms of the genus Aspergillus.

As shown in FIG. 1, when cultured in a methanol environment, the growth of microorganisms in Aspergillus is somewhat inhibited, and accordingly, the production amount of citric acid is lowered or it is difficult to predict a uniform production amount. Accordingly, the present inventors found that when the spores of microorganisms of the genus Aspergillus were cultured in a medium containing a high concentration of methanol, a mycelium having a color and shape different from that of the microorganisms cultured in a medium not containing methanol appeared, and microorganisms having such a mycelium As a result of separating the spores and culturing them in a medium containing methanol, it was confirmed that the result of increasing the production amount of citric acid was continuously shown (Experimental Example 3, FIGS. 4 and 5). That is, it was confirmed that the microorganisms in Aspergillus, which were mutated primarily by adapting to methanol at a high concentration, can increase the production amount of citric acid consistently even if they are later cultured in a medium containing methanol at a low concentration.

Microorganisms belonging to the genus Aspergillus of the present invention are a kind of fungi, and may be used in the same terms as molds in the present invention.

For example Aspergillus ash Ranges (A. acidus), Aspergillus awamori (A. awamori), Aspergillus Cloud tooth bar (A. clavatus), Aspergillus nidul pullulans (A. nidulans), Aspergillus Niger ( A. niger ), Aspergillus versicolor Vuill . may include, but is not limited thereto as long as it can produce citric acid, the object of the present invention. As another example, the Aspergillus microorganism is Aspergillus assidus kozak ( A. acidus Kozak 41731), Aspergillus Awamori Nakazu 41844 ( A. awamori Nakaz 41844), A. clavatus Desmazieres 40071 ( A. 40071), Aspergillus nidulans 44342 ( A. nidulans (Ediam) G. Winter 44342), Aspergillus Niger Van Tayum 41018 ( Aspergillus niger van Tieghem 41018), Aspergillus Niger van Tay gum 44333 ( Aspergillus niger van Tieghem 44333), Aspergillus versicholcor 42602 ( A. versicolor (Vuill.) Tirab. 42602) may be included.

Hereinafter, a method for producing the mutated Aspergillus microorganism will be described in each step.

First, in the first step, spores of microorganisms of the genus Aspergillus are inoculated into a medium containing methanol and cultured.

Methanol is known as an accelerator that improves citric acid production by microorganisms of the genus Aspergillus, but it can be cultured in a methanol environment with a concentration of 1% (v/v) or 3% (v/v) or more depending on the species of microorganisms in the genus Aspergillus. In this case, the proliferation rate decreases due to low pH and toxicity, and thus there is a problem that the production amount of citric acid decreases. Furthermore, unlike the method of adding methanol to the medium once, there is no known method of mutating and mutating microorganisms by continuously exposing microorganisms to methanol. Accordingly, the present inventors first produced a mutant with increased production of citric acid as a microorganism mutated by methanol by exposing microorganisms in Aspergillus to a high concentration of methanol.

The methanol may be, for example, a concentration of 1% (v/v) to 8% (v/v), and as another example, a concentration of 4% (v/v) to 8% (v/v). have. At a concentration of less than 1% (v/v), adaptation to methanol may not occur sufficiently, and at a concentration of more than 8% (v/v), mold growth may be reduced due to pH reduction and toxicity due to methanol.

The spores are germ cells of microorganisms of the genus Aspergillus, and may be used in the same meaning as conidia in the present invention. Inoculation of the spores may be used without limitation as long as it is a method used in the art to cultivate the spores of microorganisms of the genus Aspergillus in a medium.

In the present invention, the term "culture" means to grow microorganisms under artificially controlled environmental conditions. The medium for cultivation of the present invention can be used without limitation, as long as citric acid production, which is the object of the present invention, is possible in the art for microorganisms of the genus Aspergillus.

The medium may be, for example, a solid medium. Solid medium refers to the state of the medium containing the substrate used during fermentation in a solid state rather than a liquid. Fermentation in a solid state using a solid medium is advantageous for the production of large amounts of chemicals because the energy requirement is lower than that of water fermentation (SmF) and there is less wastewater.

Next, in the second step, the spores of the mycelium showing yellow in the front and white in the back of the microorganisms cultured in the first step are separated. The front side refers to the side in the direction in which the spores were inoculated, and the back side refers to the side opposite to the front side.

As a result of culturing the microorganisms of the genus Aspergillus in a medium containing a high concentration of methanol, the present inventors obtained mycelium showing yellow in the front and white in the back (FIG. 2). As a result of separating the spores of the microorganisms and culturing them in a medium containing methanol again, the amount of citric acid produced by the microorganisms was remarkably increased, and it was confirmed that this tendency was maintained during subculture. The medium not containing methanol It was confirmed that the microorganisms cultured in different colors and sizes from the microorganisms cultured in were mutated (benefial mutation) by methanol (Experimental Examples 2 and 3). Therefore, a microorganism having a mycelium indicating yellow in the front and white in the back refers to a microorganism that has been mutated by adapting to methanol.

The mycelium is a group of mycelium, which is a nutrient cell of a fungus, and in the fungus, the mycelium is branched into several branches and tangled like threads.

The method of separating the spores may be used without limitation as long as it is a method of separating spores of molds in the art.

On the other hand, in the mold cultured in a medium containing no methanol, since the back surface of the mycelium is white and the front surface is black, microorganisms mutated by methanol can be distinguished with the naked eye as shown in FIG. 2.

After the second step, a third step of inoculating and culturing the spores of the separated mutated microorganisms in a medium containing methanol to determine whether citric acid is produced; And it may further include a fourth step of determining a microorganism having increased production of citric acid as a mutated microorganism compared to the microorganism cultured in a medium containing no methanol.

The present inventors confirmed that when the microorganisms in Aspergillus are adapted at a high concentration, the growth rate does not decrease after at least the second passage, and the maximum citric acid production amount is 2.4 times higher even in a methanol environment (Experimental Example 3, Figs. 4 and Figs. 5). Accordingly, Aspergillus niger 44333 mutated by methanol was deposited under the deposit number KACC93201P (May 30, 2014).

The first step or the third step may be irradiating ultraviolet rays during microbial culture. UV irradiation is intended to increase long-term adaptability by stimulating microorganisms to be cultured.

The present inventors confirmed that when irradiation with ultraviolet rays during cultivation, the size of the mycelium of microorganisms in Aspergillus mutated by adaptation to methanol increased, and long-term adaptability was improved.

Ultraviolet irradiation can be used without limitation in the art of irradiating a microorganism with ultraviolet rays. For example, it is possible to use a method of irradiating for 5 to 20 minutes with a UV lamp of 20 to 60 watts. If excessive amount of ultraviolet rays are irradiated for a long time, it may rather inhibit the growth of microorganisms.

The mutated microorganism prepared by the above manufacturing method may be one that produces citric acid in a medium containing 0.1 to 8% (v/v) methanol. In the production of citric acid, a lower concentration of methanol than the concentration of methanol required for mutation in microorganisms can be used.

In another aspect, the present invention provides a mutated microorganism prepared by the above manufacturing method. The microorganism may be Aspergillus niger 44333 of the accession number KACC93201P, which is the same as described above.

As another aspect, the present invention provides a method for producing citric acid using a microorganism of the genus Aspergillus, which has been prepared by the above production method.

The method for producing citric acid comprises: (a) culturing the spores of microorganisms of the genus Aspergillus in a medium containing methanol to prepare a microorganism mutated by methanol; (b) a second step of separating the spores of the microorganisms mutated in the first step; (c) a third step of culturing the spores isolated in the second step in a medium containing 0.01% (v/v) to 8% (v/v) methanol; And (d) a fourth step of obtaining citric acid from the microorganism cultured in the third step or a medium thereof.

The microorganism mutated by the first step may be cultured in a color and size different from that of the microorganism cultured in a medium containing no methanol, as described above. For example, the front of the mycelium may be yellow and the back may be white (FIG. 2).

The medium used in the first step may contain 1% (v/v) to 8% (v/v), for example, 4% (v/v) to 8% (v/v) of methanol. . As for methanol, as described above.

The method of separating spores in the second step may be used without limitation as long as it is a method of separating spores of mold in the art, and is the same as described above.

The medium used in the third step may be one containing 0.1% (v/v) to 4% (v/v) of methanol. Microorganisms in Aspergillus genus, which have been mutated by adapting to methanol, greatly increased the production of citric acid in a methanol environment of 0.1% (v/v) or higher.

The medium may be, for example, a solid medium, as described above.

The third step may be repeated two or more times, for example. The present inventors found that when the microorganisms in Aspergillus genus in which random mutations occurred by methanol were cultured once (w2 in FIG. 3), when passaged two or more times (w3 in FIG. 3), the proliferation rate did not decrease and methanol It was confirmed that citric acid production capacity was increased by showing a high citric acid production amount of up to 2.4 times even in the environment.

The medium of the first step may contain, for example, a higher concentration of methanol than the medium of the third step. The methanol concentration used for the production of citric acid in the third step is preferably a lower concentration than the methanol concentration required for the mutation of microorganisms.

In the first step or the third step, ultraviolet rays may be irradiated during microbial culture. UV irradiation is intended to increase long-term adaptability by stimulating microorganisms to be cultured. When UV rays are irradiated during cultivation, long-term adaptability of microorganisms in Aspergillus to methanol may increase.

The method for obtaining citric acid in the fourth step may be any method used in the art to obtain citric acid produced in molds without limitation.

The microorganisms in Aspergillus genus mutated by adapting to methanol according to the present invention can consistently improve the production capacity of citric acid even in a high concentration of methanol environment.

1 shows the mycelium diameter (MGR) of microorganisms in Aspergillus according to the treatment concentration of methanol.
Figure 2 shows the front (right) and rear (left) of a medium in which mycelium and spores of microorganisms of the genus Aspergillus mutated by adapting to methanol according to the present invention are cultured.
Figure 3 shows the relative diameter (relative MGR) of the mycelium according to the number of subcultured weeks (w) of the microorganism of the genus Aspergillus mutated according to the present invention.
Figure 4 shows the degree of citric acid production according to the presence or absence of mutation of microorganisms in Aspergillus genus by bromocresol green.
Figure 5 shows the amount of citric acid produced in the mutated Aspergillus genus microorganisms prepared according to the present invention.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1. Preparation of solid medium containing methanol

A PDA medium was prepared by mixing 1L of distilled water and 39g of PDA (Potato Dextrose Agar, Difco) in an Erlenmeyer flask container, and then sterilized with an autoclave at 121°C for 15 minutes. After lowering the temperature of the medium to 45° C., 10% tartaric acid was added and the pH was adjusted to 3.5 to prevent contamination by other microorganisms. Then, the temperature of the medium was adjusted to 30° C., and 26.32 mL of methanol was added to 500 mL of the PDA medium to prepare a medium having a concentration of 5% (v/v). In the same way, a medium containing 1, 2, 3, 4% (v/v) methanol was prepared and poured into a petri dish to prepare a solid medium.

Experimental Example 1. Mold culture in methanol environment

Aspergillus Aspergillus Kozak, Aspergillus Awamori Nakazu 41844, Aspergillus clavatus demagier 40071, Aspergillus nidulans sold by the Rural Development Administration Microbial Bank (KACC) with 8 kinds of Aspergillus fungi (Ediam) G. Winter 44342, Aspergillus Niger Van Tayum 41018, Aspergillus Niger Van Tayum 44333, Aspergillus Versikolkor (Bouil) Tirab. 42602, A.sp was prepared.

In the center of the solid medium containing methanol of 0, 1, 2, 3, 4% (v/v) concentration prepared in Example 1, each 100 µl of spore solution (about 2.5 x 10 ⁴ spores) of each microorganism species Inoculated. Thereafter, the cells were cultured in an incubator at 34° C. for 5 days, and the diameter (MGR) of the mycelium was measured and shown in FIG. 1.

As a result, the mycelium size of Aspergillus clavatus demagier 40071 ( A. clavatus ) was constant despite methanol treatment. However, in Aspergillus Aspergillus Kozak ( A. acidus ) and Aspergillus Awamori ( A. awamori ), when methanol was treated with a concentration of 1% (v/v) or more, the proliferation of mycelium decreased, and Aspergillus versi Colkor (buil) Tirab. 42602 ( A. versi ) increased the diameter of mycelium when treated with a low concentration of methanol, but the growth of mycelium decreased again at a methanol concentration of 2% (v/v) or higher. In addition, Aspergillus Niger Banteay gum 44333 (A. niger 44333), Aspergillus Niger Banteay gum 41018 (A. niger 41018), in the case of Aspergillus nidulans ( A. gidulans ), the proliferation of mycelium was inhibited by methanol treatment.

Therefore, when methanol is treated, the growth of microorganisms in Aspergillus is somewhat inhibited, and thus there is a problem in that it is difficult to uniformly obtain citric acid production.

Experimental Example 2. Preparation of mutated Aspergillus microorganisms

In Experimental Example 1, 100 µl of a spore solution of Aspergillus Awamori Nakazu 41844, Aspergillus Niger Van Tayum 41018, Aspergillus Van Tay gum 44333, which are microorganisms of the genus Aspergillus with the largest diameter of mycelium (about 2.5 x 104 spores) each was inoculated in the center of a solid medium containing methanol at a concentration of 5% (v/v) prepared in Example 1, and incubated in an incubator at 34° C. for 1 week.

FIG. 2 shows the shape of the first strain (w1) in which Aspergillus van Tay gum 44333 (A. niger 44333) was cultured in methanol at a high concentration (5%v/v), and the back of mycelium (left ) Is white, and the front (right) is yellow. This means that the fungus cultured in a medium containing no methanol showed a different color and shape from that of the mycelium in white and black in the front. On the other hand, black in FIG. 2 represents spores (conidia).

As shown in FIG. 2, spores of mycelium (black part in FIG. 2) showing different colors and shapes from the fungi cultured in a medium containing no methanol were separated and cultured weekly in the same manner as above, and the diameter of the cultured mycelium ( MGR) was measured.

As a result, in the first culture (w1) and the second culture (w2), the microorganisms of the genus Aspergillus cultured in a medium containing a high concentration of methanol had a smaller mycelial diameter compared to microorganisms cultured in a medium containing no methanol. However, from the third culture (w3), it was confirmed that the size of the mycelium was greatly increased despite the treatment with high concentration of methanol (FIG. 3). In addition, after the fourth culture (w4, 5, 6), it was confirmed that the mycelium of a similar size as in the third culture appeared.

Therefore, the fungus in Aspergillus has caused a mutation by adapting to methanol, and whether such mutation can be distinguished with the naked eye as shown in FIG. 2.

Experimental Example 3. Confirmation of production of citric acid using mutated microorganisms

(1) Confirmation using bromocresol green

In order to confirm the citric acid production effect of the microorganisms in Aspergillus genus mutated according to Experimental Example 2, bromo cresol green, an acid base indicator, was used. The indicator is yellow when it becomes acidic, and bluish when it is slightly acidic, neutral, or basic. Therefore, as the amount of citric acid produced from microorganisms and discharged to the medium increases, the yellow color around the mycelium becomes stronger.

As a control group, a group in which Aspergillus Niger 44333 (non-adapt.), which was initially cultured in methanol, was cultured in a medium without methanol or a medium containing 5% v/v of methanol was used.

As a result, as shown in FIG. 4, when the non-mutated microorganisms (non-adapt.) are cultured in a medium containing methanol, growth is very inhibited and citric acid is hardly produced, whereas benefial is mutated by methanol. mutation) In the case of microorganisms (Adapt.), it was confirmed that the inhibition of proliferation was low even with treatment with a high concentration of methanol, and the yellow color was very strong in the medium, indicating that the production of citric acid by the microorganism was very high. On the other hand, when the non-mutated microorganisms were cultured in a medium containing no methanol, the proliferation rate was high, but the production of citric acid was very low compared to the mutated microorganisms.

(2) Quantification of citric acid

The spores of the microorganisms mutated in Experimental Example 2 were inoculated in CZA (Czapek Dox Broth Agar, Sigma-Aldrich) medium to be 104 (spores)/ml. After incubation at 34° C. for 4 days, the medium with the grown mold was cut into small pieces, and the pieces were placed in a 50 ml tube and heated to 100° C. in a hot water bath, so that the solid pieces became completely liquid. The liquid-formed solution was filtered through a paper filter to filter out mycelium and spores.

The filtered materials were dried at 65° C. in a dry oven, and then dry cell mass (g/kg) was measured.

The filtered and remaining liquid was cooled to 50° C., and then 10% v/v tartaric acid was added and heated at 100° C. for 2 hours to hydrolyze agar to obtain a sample. Thereafter, the amount of citric acid was measured according to the method of JR Marier and M. Boulet (J. Dairy Sci. 41, 1683-92, 1958). Specifically, after cooling the sample to 32°C in a water bath, 12.5% by weight of the sample, 16.25% by weight of pyridine, and 71.25% by weight of acetic anhydride were added to a cuvette of a spectrophotometer and reacted for 30 minutes. Then, the amount of citric acid (mg/kg) was measured at a wavelength of 420 nm in a spectrophotometer.

As a result, as shown in FIG. 5, the mold (adap) that has been mutated by adapting to methanol produces about 400 mg/kg of citric acid (ca) under 1% v/v methanol, whereas it is first cultured in methanol, that is, Mold (n-adap) not mutated by methanol produced about 170 mg/kg of citric acid under the same concentration (1% v/v) of methanol (about 2.4 fold difference). Therefore, it was confirmed that molds adapted to high concentrations of methanol could produce significantly larger amounts of citric acid than molds that did not adapt to them.

In addition, it was confirmed that the fungi that were not adapted to methanol significantly reduced not only the production capacity of citric acid but also the growth of microorganisms due to the toxicity and pH change of methanol.

Accordingly, microorganisms in Aspergillus that have been mutated by adapting to methanol according to the present invention can significantly improve the production capacity of citric acid even in a high concentration of methanol environment.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof, are included in the scope of the present invention.

National Institute of Agricultural Sciences Agricultural Genetic Resource Center (KACC) KACC93201P 20140530

Claims (15)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete Aspergillus niger 44333, deposited under the accession number KACC93201P.

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