JP2008054580A - Deferriferrichrysin highly productive variant, liquid medium for producing siderophore and method for producing siderophore - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an Aspergillus oryzae variant highly producing deferriferrichrysin, to provide a method for efficiently producing siderophore by using genus Aspergillus microorganism, and to provide a medium suitable for the method. <P>SOLUTION: Deferriferrichrysin highly productive variant 3129-7 strain (FERM P-20961) is provided, which belongs to Aspergillus oryzae and has mutation on a chromosome so that deferriferrichrysin production is ≥3 times compared with that of mother stock thereof. A liquid medium containing glycerol of e.g. ≤3% (v/v) and a liquid medium containing a thickening polysaccharide and having e.g. 3-30 mPa s viscosity are suitably usable for producing siderophore by culturing genus Aspergillus microorganism. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a high-producing mutant of Aspergillus oryzae, a liquid culture medium suitable for producing siderophores using Aspergillus microorganisms, and a method for producing siderophores using Aspergillus microorganisms.

  Iron ions are essential atoms for almost all living organisms except some lactic acid bacteria. Iron is used in the form of Fe (II) or Fe (III) in vivo, and functions as a prosthetic factor for a group of enzymes mainly involved in redox. However, in general, iron exists as iron ore in nature and hardly exists as a solubilized ionic state. Furthermore, since iron ions solubilized from iron ore by the action of microorganisms immediately become insoluble oxides and hydroxides, the amount of iron ions that can be used by organisms is extremely small.

  Bacteria, actinomycetes, and eukaryotic microorganisms produce a low-molecular iron ion chelate substance having a molecular weight of 1500 or less called a siderophore in order to efficiently acquire such a small amount of iron ions. By chelating iron ions with this siderophore, insolubilization of valuable iron ions is prevented, and iron ions are used preferentially. Iron ions are essential for living organisms, but if they are present in excess, they promote the generation of free radicals, which in turn harm the living organisms. Siderophores contribute greatly to the detoxification of iron ions as well as the acquisition of iron ions. It is known that siderophores are colorless in an unchelated state, but when chelated with iron ions, they show a red color and absorb visible light.

  Various siderophores have been identified to date, but the siderophores produced by filamentous fungi are called the hydroxamates family and generally contain N-hydroxyornithine as a constituent amino acid derivative. Aspergillus fungi, which are widely studied as research model filamentous fungi, industrial microorganisms, and pathogenic fungi, produce siderophores called ferrichromes and fusarinins in the hydroxamates family. In the former, glycine, serine and alanine form a cyclic peptide on a tripeptide of N-hydroxyornithine. On the other hand, the latter is characterized in that the N-position of some N-hydroxyornithine is acylated with mevalonic anhydride. By producing such a wide variety of siderophores, filamentous fungi preferentially acquire iron ions and use them for survival competition in nature.

  Here, in sake brewing, Aspergillus oryzae is grown on steamed rice to make “mochi” and used as a raw material for sake brewing. It is known that Aspergillus oryzae produces a large amount of siderophore (mainly deferriferriclysin) in this koji making, and this forms ferriclicin by chelating iron ions in brewing water, and sake is known to be colored. . Therefore, in sake brewing, ferrichromes, which are siderophores, cause quality degradation, and breeding of strains that do not produce ferrichromes as much as possible has been promoted.

  On the other hand, siderophores may be used as pharmaceuticals by the action of chelating iron, and development of a method for producing siderophores with high production is required.

  It is a first object of the present invention to provide an Aspergillus oryzae mutant strain that highly produces deferifericricin.

  Moreover, this invention makes it a 2nd subject to provide the method which can produce a siderophore efficiently using an Aspergillus microorganism, and the culture medium suitable for this method.

In order to solve the above-mentioned problems, the present inventors have conducted research and obtained the following knowledge.
(i) Mutant strain 3129-7 obtained by subjecting Aspergillus oryzae O-1013 strain (FERM P-16528) to mutation treatment with N-methyl-N′-nitro-N-nitrosoguanidine is Chrysin production was more than three times the parent strain.
(ii) In general, a medium containing glucose as a carbon source is used for culturing fungi, but in producing a siderophore by culturing an Aspergillus microorganism, the production amount is obtained by using a medium containing glycerol as a carbon source. Will improve.
(iii) In producing a siderophore by culturing Aspergillus microorganisms, the production amount is improved by using a liquid medium containing a thickening polysaccharide.

  The present invention has been completed based on the above findings, and provides the following mutant strains.

  Item 1. A highly deferifericin-producing mutant strain obtained by mutation treatment of Aspergillus oryzae, and the production amount of deferlifericin is more than three times that of the parent strain.

Item 2. Obtained by mutagenesis of Aspergillus oryzae, 2% glucose free of iron when (w / v) with shaking Czapek-Dox medium spores 10 ⁶ cells / ml inoculated 7 days at 30 ° C. cultures containing culture A deferifericrin high-producing mutant strain that produces 100 ppm or more of deferifericin in the supernatant.

  Item 3. Aspergillus oryzae deferlifericin high production mutant strain 3129-7 (FERM P-20961).

  Item 4. A liquid medium for producing siderophores by culturing Aspergillus microorganisms, which contains glycerol.

  Item 5. Item 5. The medium according to Item 4, wherein the glycerol concentration is 3% (v / v) or less.

  Item 6. Item 6. The medium according to Item 4 or 5, comprising a thickening polysaccharide.

  Item 7. Item 7. The medium according to Item 6, wherein the medium has a viscosity of 3 to 30 mPa · s.

  Item 8. A liquid medium for producing a siderophore by culturing an Aspergillus microorganism comprising a thickening polysaccharide.

  Item 9. Item 9. The medium according to Item 8, wherein the viscosity is 3 to 30 mPa · s.

  Item 10. Item 10. The medium according to any one of Items 4 to 9, which comprises a protein degradation product.

  Item 11. Item 10. The medium according to any one of Items 4 to 9, which comprises a mash or koji protease degradation product obtained by brewing cereals.

  Item 12. Item 12. A method for producing a siderophore comprising a step of culturing an Aspergillus microorganism using the medium according to any one of items 4 to 11, and a step of recovering a siderophore from the culture.

  Item 13. Item 13. The method according to Item 12, wherein the Aspergillus microorganism is Aspergillus oryzae and the siderophore is deferifericlysin.

  Item 14. Item 14. The method according to Item 13, wherein the Aspergillus oryzae is a highly fertile deferifericin mutant strain 3129-7 (FERM P-20961) of Aspergillus oryzae.

  According to the present invention, mutant Aspergillus oryzae was treated with N-methyl-N′-nitro-N-nitrosoguanidine, resulting in a mutant 3129-7 strain in which the production amount of deferifericrin was more than three times that of the parent strain. was gotten.

  In addition, when cultivating Aspergillus microorganisms in an iron-deficient medium to produce siderophore, the production of siderophore is improved by using glycerol as a carbon source and further adding a thickening polysaccharide to the liquid medium. .

Hereinafter, the present invention will be described in detail.
(I) Deferifericrin high production mutant The first deferifericricin high production mutant of the present invention is obtained by a mutation treatment of Aspergillus oryzae, and the production of deferlifericin is three times that of the parent strain. It is a mutant strain as described above.

  The mutation treatment method is not particularly limited. Typically, treatment with mutagens such as N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfonic acid (EMS), methylmethanesulfonic acid, 4-nitrosoquinone, nitrous acid, bromouracil. And physical mutation treatment such as ultraviolet irradiation and radiation (X-ray irradiation). In addition, mutant strains that have mutations on the chromosome due to cell fusion or genome shuffling are also included in the first mutant strain of the present invention.

  Aspergillus oryzae normally secretes and produces deferifericrin outside the cells. Therefore, as for the production amount of deferifericlysin, the parent strain and the mutant strain are cultured under the same conditions using a liquid medium, and the concentration of deferlifericin is compared in the culture supernatant. For example, the deferriferricin concentration may be calculated by chelating iron ions and then measuring the absorbance at 430 nm, which is a specific absorption wavelength of ferricricin, with a spectrophotometer. The composition of the medium and the culture conditions at the time of measuring the deferifericin concentration are not particularly limited. For example, at 30 ° C. using a medium in which 2% (w / v) glucose is added to a zapek dox medium without iron. The conditions for culturing for 7 days can be mentioned.

The second deferlifericin high-producing mutant of the present invention is obtained by a mutation treatment of Aspergillus oryzae, and 10 ⁶ spores are present in a Czapedoc medium containing iron and glucose 2% (w / v). Is a mutant that produces 100 ppm or more of deferlifericin in the culture supernatant when inoculated / ml and cultured with shaking at 30 ° C. for 7 days.

  The cause of the mutation and the method for measuring the concentration of deferlifericin are as described for the first mutant. The composition of the Czapek Dox medium without iron and 2% glucose (w / v) is described in the examples.

As a specific example of the first and second mutants described above, the Aspergillus oryzae deferifericrisin high-producing mutant 3129-7 (independently named FERM P-20961 on July 14, 2006) National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (deposited at Tsukuba Center, Central 1-1-1 Tsukuba, Ibaraki, Japan). The method for obtaining this strain and the characteristics of the strain will be described later.
(II) Liquid medium for producing siderophore by culturing Aspergillus microorganism The first liquid medium for producing siderophore by culturing Aspergillus microorganism of the present invention is a liquid medium containing glycerol. By including glycerol as a carbon source, the production amount of siderophore is higher than in a medium containing glucose, which is another general-purpose low-molecular carbon source.

  The glycerol concentration in the liquid medium is preferably 3% (v / v) or less, and more preferably about 1 to 2.5% (v / v). With the above glycerol concentration, the production of siderophore can be sufficiently improved. In addition, within the above glycerol concentration range, siderophores can be produced at a high rate without affecting the bacterial metabolic system.

  As a carbon source in the medium, in addition to glycerol, polymer carbon such as various starches and dextrins derived from corn, wheat, rice, beans, potatoes, sweet potatoes, cassava, etc., as long as the effects of the present invention are not affected. Sources: Low molecular carbon sources such as monosaccharides and oligosaccharides (especially disaccharides) such as glucose, sucrose, fructose, mannitol, sorbitol, galactose, maltose, erythritol, lactose, xylose, inosit, trehalose May be. However, if a low molecular carbon source other than glycerol is included, glycerol is the only low molecular carbon source because it may be first assimilated to inhibit the effect of increasing siderophore production by adding glycerol. It is preferable.

  The liquid medium preferably further contains a thickening polysaccharide. In the liquid medium of the present invention, siderophores are produced in a larger amount by containing both glycerol and thickening polysaccharide. The kind of thickening polysaccharide is not specifically limited, A well-known thickening polysaccharide can be used. Such known thickening polysaccharides include sodium carboxymethylcellulose (CMC), alginic acid, xanthan gum, gum arabic, carrageenan, guar gum, pectin, tamarind seed gum, agar and the like. Since these are substances that are approved for use as food additives, they are suitable for the production of siderophores for food and pharmaceutical applications. A thickening polysaccharide can be used individually by 1 type or in combination of 2 or more types.

  The content of the thickening polysaccharide is such that the viscosity of the liquid medium is preferably 3 mPa · s or more, more preferably 5 mPa · s or more, even more preferably 6 mPa · s or more, and even more preferably 9 mPa · s or more. I just need it. Further, the amount is preferably 30 mPa · s or less, more preferably 15 mPa · s or less. If it is the said thickening polysaccharide density | concentration, the production of siderophore can fully be improved. Moreover, if it is the range of the said thickening polysaccharide density | concentration, it can stir easily and can fully ensure the dissolved oxygen required for siderophore production. In the present invention, the viscosity of the medium is a value measured using a TVB-10 viscometer manufactured by Toki Sangyo Co., Ltd. For example, when using CMC, it is preferable that about 0.15-0.45 (w / v)% is contained in a culture medium.

  The second liquid medium for producing siderophores by culturing the Aspergillus microorganism of the present invention is a liquid medium containing a thickening polysaccharide. The kind and content of the thickening polysaccharide are as described for the first liquid medium. When the second liquid medium of the present invention contains the thickening polysaccharide in the above range, the production amount of siderophore is increased.

  The first and second liquid media may contain other components necessary for the growth of Aspergillus microorganisms. The nitrogen source only needs to contain inorganic nitrogen compounds such as nitrates and ammonium salts, and organic nitrogen compounds such as amino acids and peptides. A nitrogen source can be used individually by 1 type or in combination of 2 or more types.

  As the nitrogen source, it is preferable to add an organic nitrogen compound because microorganisms grow better and, as a result, the production amount of siderophore increases. However, since iron suppresses the production of siderophore, it is desirable to use an organic nitrogen compound with a low iron content.

  The first and second liquid media of the present invention preferably contain about 0.05 to 5% (w / v) of a protein hydrolyzate (particularly casamino acid), preferably 0.5 to 2.5. % (W / v) is more preferable. Further, it is preferable to add a protein hydrolyzate (particularly casamino acid) so that the iron content of the medium derived therefrom is 1 ppm or less. If it is the range of the said addition amount, while microorganisms will fully grow, production of a siderophore is hard to be suppressed with the iron which may be contained in a protein hydrolyzate, As a result, a siderophore can be produced efficiently.

  Each of the first and second liquid media of the present invention may contain a mash or koji protease degradation product obtained by brewing cereals as a nitrogen source.

  The kind of cereal is not particularly limited, and cereals used for brewing such as rice, wheat, buckwheat, and wax can be used without limitation. For example, moromi and koji obtained from brewing using rice include sake moromi and koji (ordinary moromi and koji, liquefied moromi and koji), shochu mash and koji, miso mash and koji. High-quality sake, miso moromi and koji are preferred. In addition, shochu using wheat and beer brewing mash and koji can also be used.

  In the present invention, moromi refers to cereals, water, koji or yeast, or a mixture of both, fermented, fermented, and immediately after charging. Moreover, koji refers to a solid material remaining after squeezing a liquid part (eg, alcoholic beverage) from moromi.

  The mash or koji protease degradation product can be obtained, for example, as follows. Since sputum is a solid, protease should be allowed to act on fluidized water and buffer. Moreover, since the moromi is liquid or fluid, it is sufficient to act on the protease as it is. The action temperature and time of the protease are not particularly limited, and may be any conditions under which the protease exhibits activity.

  The kind of protease is not particularly limited, and a known protease can be used without limitation. In addition, proteinase (endopeptidase) may be used to decompose the protein into peptides, and peptidase (exopeptidase) may be used to decompose the protein into amino acids. It is sufficient that the protein is degraded to such an extent that the microorganism to be cultured can be used.

  A koji-containing solution or a product obtained by allowing protease to act on moromi may be added to the medium as it is. Among these protease degradation products, amino acids and peptides produced by the degradation of proteins by proteases, undegraded products of water-soluble proteins, water-soluble components such as small amounts of alcohol, water-insoluble proteins, cereal residues, koji molds, And water-insoluble components such as yeast. Since alcohol is contained only in a small amount, it does not substantially inhibit the growth of the organism.

  Alternatively, the water-insoluble component may be removed by filtering or centrifuging the product obtained by allowing protease to act on the koji-containing liquid or moromi, and the remaining water-soluble component may be added. In order to facilitate isolation of the target product, it is preferable to remove water-insoluble components.

  When adding the protease decomposition product of koji or moromi to the medium as it is, or when adding the water-insoluble component to the medium, it can be added after further drying. Thereby, the addition amount can be accurately controlled.

  The content of the mash or koji protease degradation product in the medium is preferably about 0.05 to 5 w / v in terms of the dry weight of the protease degradation product, 0.2 to 1.5 w / v % Is more preferable, and about 0.3 to 0.7 w / v% is even more preferable. Within the above range, the growth of the organism is sufficiently promoted, and the iron content derived from the protease degradation product in the obtained medium is not excessively increased. As a result, the siderophore can be obtained efficiently. . In addition, it is preferable that a protease degradation product is contained so that the iron content of the culture medium derived from it may be 1 ppm or less.

  The first and second liquid media of the present invention include minerals such as P, K, S, Mg, Zn, Cu, biotin, and thiamine that are necessary for or promote the growth of Aspergillus microorganisms. Such vitamins may be included.

  The pH of the medium may be in a range where Aspergillus microorganisms grow, and may be about 3 to 6.

If the 1st and 2nd liquid culture medium of this invention is used, the secretory production amount of the siderophore of Aspergillus microorganisms will improve. Therefore, the first and second liquid culture media of the present invention can be suitably used for the production of siderophores by Aspergillus microorganisms. The liquid medium of the present invention is particularly suitable for ferrichrome production, and particularly suitable for the production of deferlifericin by Aspergillus oryzae.
(III) Method for Producing Siderophore The method for producing siderophore of the present invention is a method comprising the steps of culturing Aspergillus microorganisms using the liquid medium of the present invention described above and the step of recovering siderophores from the culture. is there. The microorganism belonging to the genus Aspergillus is not particularly limited as long as it is a bacterial species that produces siderophore. -Niger, Aspergillus soya, Aspergillus usami and so on. Of these, Aspergillus oryzae is preferred because of its well-known safety and properties. The main siderophore produced by Aspergillus oryzae is deferlifericin. Among them, it is preferable to use the Aspergillus oryzae deferifericricin high production mutant strain 3129-7 (FERM P-20961).

The culture method may be any method such as batch, fed-batch or continuous. The culture temperature and time vary depending on the bacterial species and strain, but may be cultured at about 25 to 35 ° C. for about 3 to 7 days.
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

As an acquisition parent strain of a deferifericlysin (Dfcy) high production mutant strain , Aspergillus oryzae O-1013 strain (FERM P-16528) was used. Spore suspension of Aspergillus oryzae O-1013 strain (10 ⁸ cells / ml) and saturated N-methyl-N′-nitro-N-nitrosoguanidine (NTG) solution are mixed in equal amounts and reacted at 30 ° C. for 30 minutes. I let you. The spore after centrifugation was washed twice with water to remove NTG, and then the spore group subjected to the mutation treatment was recovered. The spore after mutation treatment was applied to Cz-dox, 2% (w / v) Glucose, 0.25% (v / v) Triton X-100, 2% (w / v) Agar plate, and then cultured at 30 ° C. for 3 days. did.

The Dfcy production amount of the grown strain was measured by the method described later, and 3129-7 strain was selected as the strain having the largest Dfcy production amount. This strain has been deposited as FERM P-20961 at the National Institute of Advanced Industrial Science and Technology on July 14, 2006.
<Culture method>
The parent strain and each mutant-treated strain were applied from a preserved slant to a potato dextrose agar plate (manufactured by Nissui) to form spores. Spores of each strain were collected, and iron-restricted zapek dox medium (0.2% (w / v) NaNO ₃ , 0.1% (w / v) K ₂ HPO ₄ , 0.05% (w / v) 10 ⁶ cells / ml were inoculated into 40 ml of a liquid medium in which 2% (w / v) glucose was added to KCl, 0.05% (w / v) MgSO ₄ .7H ₂ O, pH 6.0), 30 Cultured with shaking at 7 ° C. for 7 days.
<Dfcy production measurement method>
10 μl of 0.2 M citrate buffer (pH 4.0) and 10 μl of 3000 ppm FeCl ₃ solution were added to 100 μl of the culture supernatant, and iron was chelated to deferriferricin (Dfcy) to obtain ferriclysin (Fcy).

  This was subjected to reverse phase chromatographic analysis using HPLC (manufactured by SHIMADZU; Prominence), and the Fcy peak was identified using the wavelength 430 nm, which is an absorption wavelength specific for Fcy, as an index. Fcy was quantified from the peak area. The Dfcy amount was calculated by multiplying the quantified Fcy amount by 0.93 (744/800) from the molecular weight ratio.

  Table 1 below shows the amount of Dfcy produced in the culture supernatant of the parent strain and the mutant strain.

  In addition, this mutant strain 3129-7 has a reduced spore-forming ability on the glucose plate and a reduced succinic acid-producing ability as compared to the parent strain O-1013.

Production of deferlifericin Aspergillus oryzae was cultured under the same conditions as in Example 1 to produce deferifericlysin. However, the following four types of liquid media were used as the media.
Medium 1:
A medium in which 2.5% (w / v) glucose (Nacalai Tesque) is added to an iron-restricted zapek dox medium 2
Iron-restricted zapek dox medium supplemented with 2.5% (v / v) glycerol (Nacalai Tesque) Medium 3:
A medium in which 2.5% (v / v) glycerol (Nacalai Tesque) and 0.3% (w / v) CMC (Nacalai Tesque) are added to an iron-restricted zapek dox medium.
2.5% (v / v) glycerol (Nacalai Tesque), 0.3% (w / v) CMC (Nacalai Tesque), 1.5% (w / v) casamino acid Medium 5 supplemented with (Difco):
2.5% (v / v) glycerol (Nacalai Tesque), 0.3% (w / v) CMC (Nacalai Tesque), 0.5% (w / v) liquefied koji What added protease decomposition product When the viscosity of the culture media 3-5 was measured using the Toki Sangyo Co., Ltd. TVB-10 viscometer, it was the range of 9.0-9.5 mPa * s.
<Liquefied soot decomposition method>
Distilled water (50 ml) was added to 10 g of the lyophilized product of the liquefied koji, homogenized, and 0.2 g of protease S Amano (manufactured by Amano Enzyme), a commercially available protease, was added and reacted at 50 ° C. with stirring for 3 hours. After inactivating the enzyme by heating at 100 ° C. for 10 minutes, the insoluble residue was removed by centrifugation, and the supernatant was lyophilized to obtain a powder of a liquefied soot decomposition product.

  Table 2 below shows the amount of Dfcy produced in the culture supernatant obtained by culturing the mutant strain 3129-7 using the media 1 to 5.

  As can be seen from Table 2, the production amount of Dfcy was higher when glycerol was used than when glucose which is widely used as a carbon source was used. Moreover, the production amount of Dfcy was further increased by adding CMC as a thickener.

  Moreover, by adding casamino acid or liquefied koji protease degradation product in addition to sodium nitrate as a nitrogen source, the production amount of Dfcy was remarkably increased. In particular, when the liquefied koji enzyme degradation product was added, the production amount of Dfcy increased 1.5 times or more as compared with the case of adding casamino acid.

The iron concentration derived from casamino acid in the medium 4 supplemented with 1.5% (w / v) casamino acid was measured as follows and found to be 21 ppb. Further, the iron concentration derived from the liquefied koji in the medium 5 to which 0.5% (w / v) liquefied koji decomposition product was added was measured as follows and found to be 482 ppb. In all cases, the value was 1 ppm or less.
<Iron concentration measurement method>
0.1% (w / v) aqueous solutions of casamino acid and liquefied koji protease degradation products were prepared, respectively, and the Fe content was measured by an atomic absorption spectrometer (Akinalst 800, manufactured by Perkin Elmer). And 96.4 ppb.

  Therefore, the concentration of iron derived from casamino acid in the medium 4 supplemented with 1.5% (w / v) casamino acid was 21 ppb, and the liquefaction in the medium 5 supplemented with 0.5% (w / v) liquefied koji decomposition product. It was calculated that the iron concentration derived from soot decomposition product was 482ppb.

Examination of types and concentrations of thickening polysaccharides In a liquid medium consisting of 2.5% (v / v) glycerol, 1.5% (w / v) casamino acid, 50 ppm chloramphenicol, and thickening polysaccharides, Aspergillus 10 ⁶ cells / ml of the spores of Oryzae 3129-7 were inoculated and cultured at 30 ° C. with shaking for 7 days.

  The thickening polysaccharides are CMC 0.15% (w / v), 0.3% (w / v), 0.45% (w / v), and sodium alginate 0.3% (w / v), purified agar powder 0.15% (w / v). Moreover, the control medium which does not add thickening polysaccharide was also used.

  The Dfcy concentration in the culture solution and the viscosity of the medium are shown in Table 3 below.

  From Table 3, Dfcy productivity was improved by the increase in viscosity as compared with the control, regardless of the type of thickener added. From these facts, it can be seen that Dfcy productivity can be improved by increasing the viscosity of the medium by adding a thickener.

Claims (14)

A highly deferifericin-producing mutant strain obtained by mutation treatment of Aspergillus oryzae, and the production amount of deferlifericin is more than three times that of the parent strain. Obtained by mutagenesis of Aspergillus oryzae, 2% glucose free of iron when (w / v) with shaking Czapek-Dox medium spores 10 ⁶ cells / ml inoculated 7 days at 30 ° C. cultures containing culture A deferifericrin high-producing mutant strain that produces 100 ppm or more of deferifericin in the supernatant. Aspergillus oryzae deferlifericin high production mutant strain 3129-7 (FERM P-20961). A liquid medium for producing siderophores by culturing Aspergillus microorganisms, which contains glycerol. The medium according to claim 4, wherein the glycerol concentration is 3% (v / v) or less. The culture medium of Claim 4 or 5 containing a thickening polysaccharide. The medium according to claim 6, wherein the medium has a viscosity of 3 to 30 mPa · s. A liquid medium for producing a siderophore by culturing an Aspergillus microorganism comprising a thickening polysaccharide. The medium according to claim 8, which has a viscosity of 3 to 30 mPa · s. The culture medium according to any one of claims 4 to 9, comprising a protein degradation product. The culture medium according to any one of claims 4 to 9, which contains a mash or koji protease degradation product obtained by brewing cereals. A method for producing a siderophore comprising the steps of culturing an Aspergillus microorganism using the medium according to any one of claims 4 to 11, and a step of recovering the siderophore from the culture. The method according to claim 12, wherein the microorganism belonging to the genus Aspergillus is Aspergillus oryzae, and the siderophore is deferifericin. The method according to claim 13, wherein the Aspergillus oryzae is a highly fertile deferifericin mutant strain 3129-7 (FERM P-20961) of Aspergillus oryzae.