Novel citric acid-producing bacterium and method for producing citric acid

Abstract

To provide microorganisms allowing for high yield production of citric acid in various culture conditions, and methods for producing citric acid by using the microorganisms.SOLUTION: The inventors searched microorganisms capable of producing citric acid at a high yield with various culture methods and achieved high yield citric acid production with various culture methods by using Aspergillus lacticoffeatus WU-2020. In particular, the strain WU-2020 produces citric acid at a high yield both in solid culture and semi-solid culture. Specifically, the invention provides a method for producing citric acid comprising a step of culturing Aspergillus lacticoffeatus WU-2020 which is deposited under accession number NITE P-03551 to the National Institute of Technology and Evaluation.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing citric acid using a novel citric acid-producing bacterium, and to the citric acid-producing bacterium.

Background Art

[0002] Citric acid is an organic compound contained in citrus fruits and the like, and is one of the hydroxy acids. Since it has a refreshing sour taste, it is widely used as a food additive. In addition, it is said that when citric acid is ingested, anaerobic respiration does not occur during exercise, so lactic acid is not produced and it is not easy to get tired, so it is also widely used as a supplement.

[0003] Citric acid is supplied by industrial fermentation production. For citric acid fermentation production, filamentous fungi (molds) of the genus Aspergillus, mainly strains of Aspergillus section Nigri, are mainly used. On the other hand, along with the citric acid production ability, the optimal culture method varies depending on the strain used, and various culture methods such as solid culture, semi-solid culture, and submerged shaking culture are also carried out at actual production sites.

[0004] As citric acid-producing bacteria, (1) Aspergillus niger that produces citric acid with a yield of 54% by solid culture using sweet potato starch hydrolyzate as a raw material (Non-Patent Document 1), (2) Aspergillus niger NRRL2001 that produces citric acid with a yield of 22.3% by submerged culture using cassava bagasse as a raw material (Non-Patent Document 2), (3) Aspergillus niger GCMC-7 that produces citric acid with a yield of 46.3% by semi-solid culture using a synthetic medium containing soluble starch (Non-Patent Document 3), (4) Aspergillus niger DS 1 that produces citric acid with a yield of 38.1% by solid culture using sugarcane bagasse as a raw material (Non-Patent Document 4), (5) Aspergillus niger GCMC-7 that produces citric acid with a yield of 64.1% by submerged culture using molasses as a raw material (Non-Patent Document 5), and (6) Aspergillus niger W5 that produces citric acid with a yield of 70.9% by submerged culture using turnip molasses as a raw material (Non-Patent Document 6) and other filamentous fungal strains have been reported.

[0005] On the one hand, in recent years, for cost reduction and sustainable production, the production of citric acid from unused biomass resources has become important. However, the shapes of these resources are diverse, and the culture methods vary depending on the resources to be used. With the conventional strains, changes were required for each culture method. In addition, since solid culture (including semi-solid culture) is less likely to be affected by stress such as impurities compared to liquid culture, a strain suitable for the new solid culture is particularly important.

[0006] Therefore, there is a demand for providing microorganisms capable of producing citric acid with high yields by various culture methods.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a microorganism capable of producing citric acid in a high yield by various culture methods, and a method for producing citric acid using the microorganism.

Means for Solving the Problems

[0009] In the present invention, a search was made for a microorganism capable of producing citric acid in a high yield by various culture methods, and high citric acid production by various culture methods using Aspergillus lacticophytus WU-2020 strain was achieved. In particular, the WU-2020 strain is capable of producing citric acid in a high yield by solid culture or semi-solid culture.

[0010] Specifically, the present invention provides a method for producing citric acid, including a step of culturing Aspergillus lacticophytus WU-2020 strain deposited with the National Institute of Technology and Evaluation. Deposit Number NITE P-03551 In the production method of the present invention, the culture may be solid culture, and the culture substrate of the medium in the solid culture may be a biomass raw material.

[0011] In the production method of the present invention, the biomass raw material may be selected from a mixture of cassava meal and rice bran, corn, sugarcane, wheat, and barley.

[0012]

[0013] In the production method of the present invention, the culture may be semi-solid culture, and the carbon source of the culture substrate of the medium in the semi-solid culture may be selected from glucose, fructose, mannose, xylose, cellobiose, xylobiose, starch, soluble starch or xylan, or a combination thereof.

[0014] In the production method of the present invention, when the culture is a semi-solid culture, the carrier of the medium in the semi-solid culture may be selected from bagasse, straw, rice straw, wheat straw, cotton fiber, filter paper, pulp, cut papers, dried tangerine peel (Chen Pi), dried product of apple peel and / or core part, glass wool, or spun wool yarn, or a combination thereof.

[0015] In the production method of the present invention, when the culture is a liquid culture, the medium in the liquid culture may be an SLZ medium.

[0016] In the production method of the present invention, when the culture is a liquid culture, the liquid culture may further include a step of adding a thickening agent.

[0017] In the production method of the present invention, the thickening agent may be selected from carboxymethyl cellulose, gelatin, carrageenan, agar, or polyethylene terephthalate.

[0018] In addition, the present invention provides Aspergillus lacticophytus WU-2020 strain deposited with Deposit Number NITE P-03551 the National Institute of Technology and Evaluation.

Advantages of the Invention

[0019] According to the present invention, it is possible to provide a microorganism capable of producing citric acid in a high yield by various culture methods, and further provide a method for producing citric acid using the microorganism.

Brief Description of the Drawings

[0020]

Figure 1

Mode for Carrying Out the Invention

[0021] 1. Method for producing citric acid One embodiment of the present invention includes a step of culturing Aspergillus lacticoffiatas WU-2020 strain deposited with the National Institute of Technology and Evaluation, Deposit Number NITE P-03551 and is a method for producing citric acid.

[0022] Therefore, in the present invention, microorganisms capable of producing citric acid in high yields by various culture methods were searched for, and high citric acid production by various culture methods such as solid culture method, semi-solid culture method and liquid culture method using Aspergillus lacticoffiatas WU-2020 strain was achieved. In particular, the WU-2020 strain is capable of producing citric acid in high yields by solid culture or semi-solid culture.

[0023] (1) Citric acid production by solid culture The "solid culture method" or "solid culture" is used in this specification in the generally used meaning. That is, as the name implies, solid culture is used as a culture method for growing microorganisms on the surface or inside of a solid substance containing a small amount of moisture.

[0024] When attempting to industrially utilize such solid culture, the culture itself containing the culture medium and the bacterial cells can be utilized as in traditional fermented food production, and oxygen for respiration can be directly utilized from the atmosphere, so no power for oxygen supply such as in liquid culture is required. There are advantages such as being able to utilize inexpensive biomass raw materials as the culture medium, having a small culture tank per product due to low cost and low moisture.

[0025] In this specification, the "biomass raw material" is used in this specification in the generally used meaning. That is, it refers to reusable organic resources (excluding fossil fuels such as petroleum) derived from animals and plants. Generally, it mainly refers to wood, seaweed, food waste, paper, animal carcasses and excrement, plankton, etc.

[0026] In the production method of the present invention, when the culture is a solid culture, the culture medium substrate in the solid culture may be a biomass raw material as an example of the culture medium substrate.

[0027] In the present invention, examples of the biomass raw material can be selected from a mixture of cassava meal and rice bran, corn, sugarcane, wheat, and barley.

[0028] (2) Citric acid production by semi-solid culture In this specification, the "semi-solid culture method" or "semi-solid culture" is used in the generally used meaning. That is, semi-solid culture means culturing using a semi-solid medium in which a carrier is impregnated with a liquid medium.

[0029] The medium used for the production of citric acid by semi-solid culture can produce citric acid at a sugar concentration of up to 230 g / L of glucose.

[0030] Also, as examples of the carbon source of the medium used for the production of citric acid by semi-solid culture, glucose, fructose, mannose, xylose, cellobiose, xylobiose, starch, soluble starch or xylan, or a combination thereof can be used.

[0031] Also, as an example of the composition of salts in the medium used for the production of citric acid by semi-solid culture, for inorganic nitrogen salts, ammonium salts such as ammonium sulfate can be used.

[0032] Also, the pH of the medium used for the production of citric acid by semi-solid culture can be used between 2.70 and 5.70.

[0033] Also, as examples of the carrier of the medium used for the production of citric acid by semi-solid culture, bagasse, straw (rice straw, wheat straw, etc.), cotton fiber, cut papers such as filter paper and pulp, newsprint, dried citrus peel (Chenpi), dried apple peel and core, glass wool or spun yarn, or a combination thereof can be used.

[0034] (3) Citric acid production by liquid culture In the present specification, the "liquid culture method" or "liquid culture" is used in the commonly used meaning. That is, liquid culture is a method of culturing mainly microorganisms in an aqueous solution (culture medium) containing substances necessary for growth such as a carbon source, a nitrogen source, and salts, and is used as a term for solid culture in which the culture medium is solidified with agar or the like.

[0035] In the production method of the present invention, when the culture is liquid culture, an SLZ medium can be used as an example of the medium in the liquid culture.

[0036] In the production method of the present invention, when the culture is liquid culture, the liquid culture may further include a step of adding a thickening agent.

[0037] Examples of the thickening agent in the production method of the present invention may include carboxymethyl cellulose, gelatin, carrageenan, agar, and polyethylene terephthalate.

[0038] The thickening agent concentration can be used at 1.0 to 10.0 g / L, and 2.0 to 6.0 g / L is preferable.

[0039] 2. Aspergillus lacticophytus WU-2020 strain Another embodiment of the present invention is Aspergillus lacticophytus WU-2020 strain deposited with Deposit Number NITE P-03551 the National Institute of Technology and Evaluation.

[0040] The microorganism according to the present invention is presumed to belong to Aspergillus lacticophytus, and the strain name is WU-2020 strain. Further, the microorganism according to the present invention has been deposited with the Patent Biological Depositary Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture 292-0818). Deposit and its Deposit Number isNITE P-03551 It is as follows.

[0041] In addition, regarding the microorganism according to the present invention, mutants obtained by mutating with known methods such as ultraviolet irradiation and radiation irradiation while having a high citric acid-producing ability, and mutants mutated in nature are also included.

[0042] By using the Aspergillus lacticoffiatas WU-2020 strain of the present invention, citric acid can be produced at a high yield under various culture conditions such as solid culture, semi-solid culture, or liquid culture.

[0043] Hereinafter, it will be described in detail with reference to examples. All documents mentioned in this specification are incorporated herein by reference in their entirety. The examples described herein are illustrative of embodiments of the present invention and should not be construed as limiting the scope of the present invention.

Examples

[0044] <Isolation of Citric Acid-Producing Bacteria> Soils collected from various parts of Japan were used as samples to search for citric acid-producing bacteria, especially filamentous fungi.

[0045] As the isolation medium, the following Selective Agar Medium A and Selective Agar Medium B were used. The modified CD medium is a medium obtained by replacing the carbon source of the Czapek-Dox minimal medium from 30 g / L of glucose to 90 g / L, and has the following composition. 90 g of glucose, 2 g of sodium nitrate, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.01 g of ferrous sulfate heptahydrate, and 0.5 g of potassium chloride were dissolved in 1 L of ion-exchanged water. The pH was adjusted to 6.0 with hydrochloric acid. The medium obtained by adding agar to this modified CD medium to a concentration of 20 g / L is referred to as Selective Agar Medium A.

[0046] As the selective agar medium B, a modified SLZ medium with 30 g / L of agar added and solidified was used. That is, each component of the SLZ medium was made into a 2-fold concentration (glucose concentration 240 g / L; final concentration 120 g / L), autoclaved for steam sterilization, and then 50 g / L of citric acid was added. This 500 mL was cooled to about 40 °C (Solution A). On the other hand, 60 g of agar was suspended in 1 L of ion-exchanged water, autoclaved for steam sterilization, and then cooled to about 40 °C (B agar suspension). 5 mL of Solution A and 5 mL of B agar suspension were mixed at about 40 °C, placed in a petri dish with an inner diameter of 15 cm, and solidified at room temperature. This is called the selective agar medium B. The composition of the SLZ medium consists of 120 g / L of glucose, 3 g / L of ammonium sulfate, 1 g / L of potassium dihydrogen phosphate, 1 g / L of dipotassium hydrogen phosphate, 0.50 g / L of magnesium sulfate heptahydrate, 0.22 g / L of manganese sulfate pentahydrate, 0.010 g / L of iron(III) chloride hexahydrate, 0.075 mg / L of zinc chloride, and the pH was adjusted to 3.0.

[0047] 5 mL of physiological saline was dispensed into a test tube with a capacity of 20 mL, and 0.01 g of soil was added. It was stirred for 3 minutes while rotating and shaking with a vortex mixer, left to stand for 5 minutes, and 0.1 mL of the upper suspension was inoculated into the above-mentioned selective agar medium A. It was statically cultured at 30 °C for 7 days, and the colonies that appeared were transplanted onto the selective agar medium A. It was further statically cultured on the transplanted selective agar medium A for 7 days, and filamentous fungi (molds) that produce black spores were selected. For pure separation, the spores of the obtained filamentous fungi were collected with a platinum loop, suspended in physiological saline, and 0.1 mL of this suspension was inoculated into a normal Czapek-Dox agar medium (Note: 30 g / L of glucose), and statically cultured for 7 days to separate single colonies. This operation was repeated 2 more times, and 100 strains were obtained as the filamentous fungi for which the pure separation operation was performed, and they were further subjected to the following selection test.

[0048] The spores of the filamentous fungus that had undergone a pure separation operation were collected with an inoculation loop, suspended in physiological saline, and 0.1 mL of this suspension was inoculated onto selective agar medium B. It was statically cultured at 30 °C for 3 days, and 20 strains were selected as those forming colonies. Furthermore, for the selected 20 strains, they were inoculated onto normal Czapek-Dox agar medium (Note: glucose 30 g / L) and statically cultured at 30 °C for 7 days. After culturing, the formed spores were collected with an inoculation loop, suspended in physiological saline, and 0.1 mL of this suspension was inoculated onto selective agar medium B. It was statically cultured at 30 °C for 3 days, and 10 strains were selected as those forming colonies.

[0049] For the selected 10 strains, a citric acid production test using bagasse as a carrier was conducted, and the WU-2020 strain was selected as a strain showing a citric acid production amount with a yield of 50% or more using glucose or sucrose.

[0050] The WU-2020 strain showed the morphology of a filamentous fungus of the genus Aspergillus and formed black spores (conidia), so it was considered to belong to Aspergillus Section Nigri (black mold).

[0051] Furthermore, for the WU-2020 strain, genomic DNA was extracted according to a conventional method, the regions of the rDNA-ITS, β-tubulin gene, and calmodulin gene were amplified by PCR, and their nucleotide sequences were determined. By comparing the partial nucleotide sequences of these three genes with the sequences of the same genes of standard strains belonging to other Aspergillus Section Nigri, it was identified as Aspergillus lacticoffeatus (see Figure 1).

Example

[0052] <Citric Acid Production by Solid Culture> Citric acid production by solid culture of Aspergillus lacticoffeatus WU-2020 was carried out as follows.

[0053] To the dried cassava meal, add water at 5 times its weight and let it stand for 1 day. Using filter paper (Whatman No. 4), perform suction filtration until no filtrate comes out, and obtain the water-added cassava meal. Mix 300 g of the water-added cassava meal and 30 g of rice bran in a plastic Tupperware, and sterilize it in an autoclave at 110 °C for 15 minutes to obtain a solid medium. Collect the conidia of Aspergillus lacticoffieatus WU-2020 formed on a synthetic slant medium with a platinum loop (about 1×106 pieces), suspend it in 1 mL of pure water to prepare a conidia suspension. Inoculate 1 ml of the conidia suspension per 50 g of the solid medium and culture it at 30 °C for 3 days. After culturing, use pure water at 5 times the volume of the initial weight of the solid medium and stir for 15 minutes to prepare an extract. Analyze the citric acid concentration in the extract using a citric acid quantification kit (F kit, Roche Diagnostics).

[0054] The medium composition is as follows. The synthetic slant medium consists of 90 g / L of glucose, 2 g / L of ammonium nitrate, 10 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate heptahydrate, 0.022 g / L of manganese sulfate pentahydrate, 0.01 g / L of copper sulfate pentahydrate, and 30 g / L of agar, and the pH is adjusted to 6.0.

[0055] As a result of determining the citric acid concentration in the extract on the 3rd day of culture, the citric acid production was 360 g per 1 kg of the dried cassava meal weight. There has been no previous report on citric acid production by solid culture from unused biomass such as cassava meal.

Example

[0056] <Citric acid production by semi-solid culture> Citric acid production by semi-solid culture of Aspergillus lacticoffieatus WU-2020 and Aspergillus lacticoffieatus CBS101833 (Note: The type culture of Aspergillus lacticoffieatus in the strain preservation institution = reference strain, obtained by transfer from the strain preservation institution IFM) was carried out as follows.

[0057] Collect the conidia of Aspergillus lacticoffiatas WU-2020 or Aspergillus lacticoffiatas CBS101833 formed on a synthetic slant medium using a platinum loop (about 1×10 6 cells), and suspend them in 1 mL of SS medium to prepare a conidia suspension. Next, inoculate the conidia suspension into a bagasse (carrier) containing 15 mL of SS medium, and let it stand and culture at 30 °C for 3 days. After culturing for a predetermined number of days, suspend the bagasse in 200 mL of warm water (60 °C) and stir for 15 minutes. Filter the above suspension through filter paper (Whatman No. 4), and use the filtrate as the citric acid fermentation broth.

[0058] The medium composition is as follows. The synthetic slant medium is the same as the medium composition described in Example 2. The SS medium for the culture test consists of 140 g / L of glucose, 2 g / L of ammonium nitrate, 10 g / L of potassium dihydrogen phosphate, 0.25 g / L of magnesium sulfate heptahydrate, 0.22 g / L of manganese sulfate pentahydrate, 0.020 g / L of iron(III) chloride hexahydrate, and the pH is adjusted to 4.25. Also, the bagasse used for the culture was a mixture of 2.6 g of the fraction classified at 600 μm - 250 μm (short branched ones) and 1.3 g of the fraction classified at 250 μm or less (powdered ones) using a sieve. Similarly, instead of glucose, sucrose and maltose were used to produce citric acid by semi-solid culture.

[0059] The citric acid production amounts in the citric acid fermentation broths of Aspergillus lacticoffiatas WU-2020 and Aspergillus lacticoffiatas CBS101833 (reference strain) were determined by HPLC analysis. The results are shown in Table 1. As shown in Table 1, Aspergillus lacticoffiatas WU-2020 showed a higher citric acid production ability compared to Aspergillus lacticoffiatas CBS101833 (reference strain).

Table 1

Example

[0060] <Citric Acid Production by Liquid Culture> Citric acid production by liquid culture of Aspergillus lacticofiratus WU-2020 was carried out as follows.

[0061] The test fungus conidia formed on the koji juice agar medium were suspended in 0.8% (v / v) Tween 80, and a conidia suspension was prepared by adjusting the conidia concentration to 3×108 cells / mL. Next, 1 mL of the above conidia suspension was inoculated into 60 mL of SLZ medium (the composition is described in Example 1) dispensed into a 500 mL Erlenmeyer flask with a shoulder (Sakaguchi flask), and cultured at 30°C and 120 rpm with reciprocal shaking for 12 days. 1 mL of the culture solution was taken, centrifuged at 15000 rpm for 15 minutes, and the supernatant obtained was used as the citric acid fermentation broth.

[0062] The citric acid production amount in the citric acid fermentation broth of Aspergillus lacticofiratus WU-2020 was determined by HPLC analysis. Aspergillus lacticofiratus WU-2020 produced 15.4 g / L of citric acid in liquid culture. Although the citric acid production ability was lower than that in semi-solid culture, citric acid production was also possible in liquid culture.

Example

[0063] In the liquid culture of Example 4, when soluble starch was used as the carbon source, 52.0 g / L of citric acid was produced. This value was a higher production amount than when glucose was used. When the viscosity of the medium was high, such as in the medium using soluble starch, a tendency for the citric acid production ability to increase was observed. Therefore, in order to achieve a citric acid production amount as high as that in the case of semi-solid culture, carboxymethyl cellulose (CMC, final concentration 2.5 mg / mL) was added as a thickener to the liquid medium, and citric acid production by Aspergillus lacticofiratus WU-2020 was carried out.

[0064] The cultivation method was the same as in Example 4 except that a thickener was added. The results are shown in Table 2. As shown in Table 2, by adding a thickener, Aspergillus lacticofiratus WU-2020 can produce a high amount of citric acid even in liquid culture.

Table 2

Claims

1. A method for producing citric acid, comprising a step of culturing Aspergillus lacticoffiatas WU-2020 strain deposited with the National Institute of Technology and Evaluation, Independent Administrative Institution under the deposit number NITE P-03551.

2. The production method according to claim 1, wherein the culturing is solid-state culturing, and the culture substrate of the medium in the solid-state culturing is a biomass raw material.

3. The production method according to claim 2, wherein the biomass raw material is selected from a mixture of cassava meal and rice bran, corn, sugarcane, wheat, and barley.

4. The production method according to claim 1, wherein the culturing is semi-solid culturing, and the carbon source of the culture substrate of the medium in the semi-solid culturing is selected from glucose, fructose, mannose, xylose, cellobiose, xylobiose, starch, soluble starch or xylan, or a combination thereof.

5. The production method according to claim 1 or 4, wherein the culturing is semi-solid culturing, and the carrier of the medium in the semi-solid culturing is selected from bagasse, straw, rice straw, wheat straw, cotton fiber, filter paper, pulp, cut papers, dried tangerine peel (Chenpi), dried peel and / or core of apple, glass wool, or spun wool, or a combination thereof.

6. Aspergillus lacticoffiatas WU-2020 strain deposited with the National Institute of Technology and Evaluation, Independent Administrative Institution under the deposit number NITE P-03551.

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