JPH0322149B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing filamentous fungi. Conventionally, an example of culturing filamentous fungi by adding powder belonging to diatomaceous earth to a liquid medium for the purpose of producing useful substances is in penicillin G fermentation using filamentous fungi belonging to Penicillium chrysogenum. The only known example is that celite was added at a high concentration of 10% for the purpose of immobilization (DICWang et al., 1986 Japanese Society of Fermentation Engineering Abstracts, p. 112), and a low concentration of diatomaceous earth was added to a liquid medium. There have been no reports of cases in which filamentous fungi have been cultured with the addition of these substances. Generally, when filamentous fungi are cultured aerobically in a liquid medium, the hyphae grow into pellet-like (spherical) or pulp-like lumps, and sometimes these lumps grow so large that the production of the desired useful substance is significantly reduced. One of the drawbacks in the production of useful substances using filamentous fungi is the decrease in the production of useful substances. The present inventors aim to produce useful substances such as anti-inflammatory agents, organic acids, antibiotics, enzymes such as protease, glucoamylase, and glucose oxidase, L-amino acids, pigments, and enzyme inhibitors, which belong to filamentous fungi. As a result of culturing each of the producing bacteria in a culture solution containing diatomaceous earth, the inventors discovered that the mycelial masses were made finer and at the same time, the production of the target useful substance was significantly promoted, leading to the completion of the present invention. Ivy. That is, the present invention uses 8% of powder belonging to diatomaceous earth.
The present invention relates to a method for culturing filamentous fungi, which is characterized by culturing filamentous fungi aerobically in a liquid medium containing no more than . The diatomaceous earth used in the present invention has long been used as a supernatant, and there are various trade names depending on the manufacturer and particle size. for example
Johns-Manville products include various Celites "Celite 501-577,""Hyflo Super Cell," and "Filter Cell," while Showa Kagaku Kogyo Co., Ltd. products include various "Radiolite,""RapidFlow," and various "Torite." There is Hakusan Kogyo Co., Ltd.
There are various products such as ``Zemlite'' and ``Zemlite Super,'' Grefco products include various ``Dicalite,'' and Eagle-
Picher's products include various ``Celatom'' products.
There is. The diatomaceous earth used may be in its commonly available form (powder) and may be dry heat sterilized at 180-200°C separately from the medium before being added to the medium, or steam sterilized under normal conditions, or Steam sterilization may be performed together with the culture medium. By adding diatomaceous earth to the medium, spores and hyphae adhere to the diatomaceous earth particles, germinate using these as nuclei, and the hyphae are observed to grow.Furthermore, they do not adhere to the diatomaceous earth and do not form clumps. It can also be observed that they are growing without any problems. Thus, by adding diatomaceous earth, the mycelia grow without forming large clumps, the stirring efficiency of the medium is improved, and the growth rate and production of the desired useful substance are promoted. The useful substances produced in the present invention are useful substances produced by filamentous fungi, such as mycophenolic acid, anticancer agents such as cyclosporin, gluconic acid, citric acid, malic acid, pyruvic acid, itaconic acid, and araboascorbic acid. , organic acids such as kojic acid and aloisocitric acid, antibiotics such as penicillin and cephalosporin C, enzymes such as protease, glucoamylase, and glucose oxidase, L
-Amino acids, pigments and compactin, enzyme inhibitors. The filamentous fungus used in the present invention is not particularly limited as long as it is a filamentous fungus, and includes, for example, Penicillium brevicompactum that produces mycophenolic acid and compactin, Penicillium and chrysogenum that produce penicillin C, and glucose oxidase-producing bacteria. Penicillium perprogenum, Cephalosporium polyalerum which is a cephalosporin C producing bacterium, Aspergillus oryzae which is an amylase producing bacterium, Rhizopus derema, and Aspergillus derma which is a protease producing bacterium.
Cytoi, Aspergillus phenisis, Aspergillus niger, which produces citric acid and gluconic acid, and Alpergillus, which produces itaconic acid.
We can mention Tereus et al. Specifically, Penicillium brevi compactum, a mycophenolic acid-producing bacterium,
compactum), AJ117096, (FERM P-5693,
FERM BP-55), Penicillium chrysogenum (P. chrysogenum), a penicillin-producing bacterium
ATCC10002, AJ7343, Penicillium perprogenum No. which is a glucose oxidase producing bacterium.
778 (P. purprogenum) AJ17045 FERM−P1846,
Cephalosporium polyallerum (Cephalosporium polyallerum) is a cephalosporin C-producing bacterium.
polyaleurum) ATCC20359 AJ6993, protease producing bacteria include Aspergillus phenicis (Aspergillus phenicis) ATCC14332 AJ17063,
Aspergillus niger (A. niger) ATCC9642 as a glucoamylase and citric acid producing bacterium
AJ7172 etc. are used. The liquid medium used in the present invention may be any liquid medium in which filamentous fungi can grow, except for the addition of diatomaceous earth. For example, a well-known medium for producing mycophenolic acid, WL
Muth et al, Antimicrob, Agents
The culture medium described in Chemtherap., 8 , 321-327 (1975) is used. As a medium for penicillin fermentation
Synthetic medium of Lohnson et al. (Yusuke Sumiki “Antibiotics”)
above, p. 177, 1961) is used. As a medium for cephalosporin C fermentation, for example, AL
DEMAIN, JFNEWKIRK, and D.
HENDLIN, J. A modified medium (Table 4) of the medium described in Bacteriol. 85 , 339 (1963) is used. As the culture medium for producing glucose oxidase, for example, the culture medium described in Japanese Patent Publication No. 109/1982 is used (Table 6). As the culture medium for protease, glucoamylase and citric acid production, the media listed in Tables 5 and 6 are used. The amount of diatomaceous earth added does not exceed 8%,
Preferably it is 0.2 to 2%. The culture temperature may be any temperature as long as the filamentous fungus that produces the useful substance can grow, but it is preferable to use the optimal culture temperature that produces the useful substance. Culture is carried out with rotation or aeration under conditions suitable for the production of each useful substance. The rotation speed is 200-250 rpm. Diatomaceous earth can be added either singly or in combination. Various useful substances produced by the method of the present invention can be quantified and collected by each known method. Mycophenolic acid and citric acid in the culture solution were analyzed and quantified using high-performance liquid chromatography, aspartic acid was determined using the bioassay method, penicillin G was determined using the national assay method using Staphyloccus aureus, and cephalosporin C was determined using Comamonas.
ferrigena growth inhibition circle method was used. Glucoamylase activity was measured using the saccharification measurement method (Jikken Kagaku Koza, Vol. 24, p. 272, 1961), and protease activity was measured using the Casein-Folin color A method (Hagiwara, Enzyme Research Methods, Vol. 2, p. 237, 1956). The activity of glucose oxidase was measured by a pressure detection method using a Warburg manometer.As explained above, the present invention relates to a method for accumulating and collecting useful substances produced by filamentous fungi by adding diatomaceous earth to a culture medium. The method of the present invention significantly increases the production amount of useful substances by adding diatomaceous earth, and is industrially suitable in that diatomaceous earth can be easily recovered from the culture solution by incineration or other methods after harvesting the product. Its practical value is extremely large.Example 1 Penicillium brevicompactum AJ117096
(FERM P-5693, FERM BP-55) were inoculated onto the natural slant medium shown in Table 1, and after culturing at 27°C for 10 days, the spores were collected and added to 0.1M phosphate buffer (PH
6.8) and inoculated a certain volume of it into the medium shown in Table 2 (50 ml of medium in a 300 ml Erlenmeyer flask) (approximately 10 ⁶ spores/ml), and incubated at 27°C for 12 days.
Rotary culture was performed at 226 rpm. When diatomaceous earth shown in Table 3 was added, it was sterilized by heating at 180° C. and then added to the medium at a concentration of 1%. After culturing,
Adjust the culture solution to PH10-10.5 with sodium hydroxide solution.
After adjusting and rotating at 150 rpm for 20 minutes, the Toyo paper
Pass through No. 2 to obtain a clear liquid. Mycophenolic acid in this liquid was determined by high performance liquid chromatography, and L-aspartic acid was determined by bioassay method. Bacterial growth was determined by washing the entire culture medium (50 ml) of bacterial cells with approximately 10 ml of distilled water, drying the bacterial cells together with paper at 105℃ for 18 hours, and subtracting the weight of paper and diatomaceous earth from the total dry weight. Indicated. Table 3 shows the results.

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[Table] When Celite 535, Celite 545, and Filter Cell were added, the accumulation of mycophenolic acid and L-aspartic acid significantly increased in all cases. On the other hand, although there was no significant difference in the degree of bacterial growth compared to the case without the addition, the size of the pellets became finer due to these additions. Example 2 Penicillium brevi compactum AJ117096 (FERM P-5693,
Table 4 shows the results of rotary culture of FERM BP-55) at 27°C for 12 days at 226 rpm in the medium shown in Table 2 containing various concentrations of Celite 545. Celite
Mycophenolic acid accumulation was significantly promoted by addition of 0.4 to 8% of 545.

[Table] Example 3 According to the method shown in Example 1, penicillin G producing bacteria Penicillium clingenum ATCC10002 and AJ7343 were grown in the medium shown in Table 1, and cephalosporin C producing bacteria Cephalosporium
Polyaleurum ATCC20359, AJ6993, Aspergillus feinisis, a protease producing bacterium
ATCC14332, AJ17063, glucoamylase and citric acid producing bacterium Aspergillus niger
ATCC9642, AJ7172, and Penicillium perprogenum, a glucose oxidase-producing bacterium
Suspensions of FERM-P1846 and AJ17045 mycelia and spores were inoculated into the respective production media shown in Tables 5, 6, and 7 (50 ml medium filled in a 300 ml Erlenmeyer flask), and cultured by rotation at 27°C (226 rpm). I went there. The culture time was 5 days for penicillin G, cephalosporin C, and glucoamylase production, 4 days for protease production, 6 days for glucose oxidase production, and 10 days for citric acid production. The amount of each product accumulated in the culture solution was measured by the method described above. Table 8 shows the results of measuring the amounts of these producing bacteria in the presence and absence of diatomaceous earth.

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【table】

【table】

【table】

[Table] In all cases of diatomaceous earth used, Celite 545 was added to each production medium at a concentration of 1%. In both cases, an increase in accumulation was observed with the addition of celite. On the other hand, the mycelial pellets were generally refined by the addition of Celite.

Claims (1)

[Claims] 1. A method for culturing filamentous fungi, which comprises culturing filamentous fungi aerobically in a liquid medium containing not more than 8% of powder belonging to diatomaceous earth.