JPH07112428B2 - Filamentous fungus culture method - Google Patents

Description

The present invention relates to a method for culturing filamentous fungi. Conventionally, substances similar to clay minerals, carbonaceous materials, synthetic resins and talc, alumina and zeolite, or carbon-silica composite materials or animal-derived materials (hereinafter referred to as "A powder") are added to the liquid medium. An example of culturing the filamentous fungus has not been reported yet.

Generally, when filamentous fungi are cultivated aerobically in a liquid medium, mycelia grow in pellet (spherical) or pulp-like lumps.
At times, one of the drawbacks in the production of useful substances using filamentous fungi was that the production of the useful substances intended was significantly reduced due to the large growth of these lumps.

In order to produce useful substances such as carcinostatic agents, organic acids, antibiotics, enzymes such as protease, glucoamylase and glucose oxidase, L-amino acids, polysaccharides, pigments and enzyme inhibitors, the present inventors As a result of culturing each of the producing bacteria belonging thereto in a culture solution containing A powder, it was found that the mycelial mass is miniaturized and at the same time the production of the intended useful substance is significantly promoted, and the present invention is completed. Came to do.

That is, the present invention relates to a method for cultivating a filamentous fungus, which comprises aerobically culturing the filamentous fungus in a liquid medium containing a powder belonging to powder A.

Among the A powders used in the present invention, the clay minerals include bentonite, kaolin, acid clay, vermiculite, wax stone, sericite, K-clay, pesticide clay, etc.
The carbonaceous material includes porous activated carbon, and microcrystalline charcoal powder, carbon black, thermax, mesocarbon, graphitized carbon black, graphite or carbon fiber. Examples of the composite material include a polypropylene-talc composite, a carbon-silica composite (FCC equilibrium catalyst), and an alumina-silica composite (smokysil). Animal-derived substances include shellfish, bone and silk. Use A
The powder may be the raw form that is generally available (powder),
It may be sterilized by drying at 180 to 200 ° C separately from the medium prior to addition to the medium, or steam sterilization under normal conditions, or steam sterilization together with the medium.

By adding the A powder to the medium, spores and hyphae adhered to the particles of the A powder, germination was performed by using these as nuclei, and the mycelia were observed to grow, and further adhered to the A powder. It is also observed that it grows without forming a lump. Thus, by adding A powder, mycelia grow without growing into large lumps,
The result is that the stirring efficiency of the medium is also improved, and the growth rate and the production of the desired useful substance are promoted.

The useful substance produced in the present invention is a filamentous substance produced by filamentous fungi, and examples thereof include anticancer agents such as mycophenolic acid, immunosuppressive agents such as cyclosporine, gluconic acid, citric acid, malic acid, pyruvic acid, and itacone. Acids, organic acids such as alaboascorbic acid, kojic acid, alloisocitrate, antibiotics such as penicillin, cephalosporin C, enzymes such as protease, glucoamylase, glucose oxidase, polysaccharides such as nigeran, pullulan, mannocarolose, etc. , L-amino acids, pigments and enzyme inhibitors such as compactin and mevinolin.

The filamentous fungus used in the present invention is not particularly limited as long as it is a filamentous fungus.
Glucose oxidase-producing bacterium Penicillium perprogenum, cephalosporin C-producing bacterium cephalosporium, polyaleulam, amylase-producing bacterium Aspergillus oryzae, Rhizopus terema, and protease-producing bacterium Aspergillus cytoii, Aspergillus phenylis, Examples include pullulan-producing bacterium pullularia, mannocarolose-producing bacterium Penicillium charlesii, citric acid, gluconic acid-producing bacterium Aspergillus niger, and itaconic acid-producing bacterium Aspergillus terreus.
Specifically, Penicillium brevi-compactu which is a mycophenolic acid-producing bacterium.
m), AJ117096, (FERM P-5693, FERM BP-55) Penicillium chrysogenum (P. chryso
genum) ATCC10002, AJ7343, glucose oxidase-producing bacterium Penicillium perprogenum No. 778 (P.
rprogenum) AJ17045 FERM-P1846, cephalosporin C
Cephalosporium polyalerum (Ceph)
alosporium polyaleurum) ATCC20359, AJ6993, and Aspergillus phenylis (As
pergillus phenicis) ATCC14332, AJ17063, glucoamylase and citric acid producing bacteria Aspergillus niger (A. niger) ATCC9642, AJ7172, mannocarolose producing bacteria
i) ATCC8730 etc. are used.

As the liquid medium used in the present invention, any medium can be used as long as it is a liquid medium in which filamentous fungi grow, except that A powder is added. For example, a known medium for producing mycophenolic acid, WLMuth et al , Antimicrob, Agent
s Chemtherap., 8, 321-327 (1975). As a medium for penicillin fermentation, a synthetic medium by Johnson et al. (Shusuke Sumiki, “Antibiotics”, p.177, 1961) is used. Examples of the cephalosporin C fermentation medium include ALDEMAIN, JFNEWKIRK, and D. HENDLIN, J. Bacterio
l. 85, 339 (1963) modified medium medium described (Table 4) is used. Czapek as a medium for mannocarolose production
As the Dox medium (Table 5) and the glucose oxidase producing medium, for example, the medium described in JP-B-52-109 is used (Table 7). As the medium for producing protease, glucoamylase and citric acid, the medium described in Tables 6 and 7 is used.

The addition amount of A powder remarkably differs depending on the type of powder, but it is 0.
It is from 01 to 10%, preferably from 0.01 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 optimum culture temperature for producing the useful substance. Rotation or aeration-agitation culture is performed under conditions suitable for the production of each useful substance. The rotation speed is 200-250 rpm. As a method of adding the A powder, there are single or a combination of two or more kinds.

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 by high performance liquid chromatogram, and aspartic acid was quantified by Staphyloccus aureus by bioassay method.
Cephalosporin C is a national test method using Comamonas
The ferrigena growth inhibition method was used. Glucoamylase activity is measured by the saccharification assay (“Experimental Chemistry Course” 2
4, p2721961), protease activity is measured by Casein-Fol
in Coloring Method A (Hagiwara "Enzyme Research Method Volume 2, p237 1956),
The glucose oxidase activity was measured by a pressure measurement method using a Warburg manometer. For the determination of mannocarolose, acetone was added to the culture solution to precipitate the polysaccharide, and the precipitate was dehydrated with acetone, dried in vacuum and weighed.

As described above, the present invention relates to a method for accumulating and collecting a useful substance produced by a filamentous fungus by adding A powder to a medium, and the method of the present invention produces a useful substance by adding A powder. The amount is remarkably increased and the industrial practical value is extremely large in that the A powder can be easily recovered or removed by a method such as incineration from the culture solution after collecting the product.

Example 1 Penicillium brevicompactum AJ117096, (FERM P-
5693, FERM BP-55) was inoculated into the natural slant medium shown in Table 1, and after culturing at 27 ° C for 10 days, spores were collected and suspended in 0.1M phosphate buffer (pH6.8). A certain amount of the medium shown in Table 2 (300 ml Erlenmeyer flask was filled with 50 ml medium) was inoculated (about 10 ⁶ spores / ml), and rotary culture was carried out at 27 ° C. for 12 days at 226 rpm. When powder A shown in Table 3 was added, it was sterilized by heating at 180 ° C. or steam sterilization at 120 ° C. for 10 minutes in advance and then added to the medium so that the concentration shown in Table 3 was obtained. After culturing,
The culture broth was adjusted to pH 10 to 10.5 with sodium hydroxide solution and rotated at 150 rpm for 20 minutes, and then filtered using Toyo Paper No. 2 to obtain a transparent liquor. Mycophenolic acid in this solution was quantified by a high performance liquid chromatogram, and L-aspartic acid was quantified by a bioassay method. For the growth of the bacteria, the cells in the entire medium (50 ml) were washed with about 10 ml of distilled water, and the cells were dried with paper for 18 hours at 105 ° C. The value obtained by subtracting the weight of the paper and the powder A added from the total dry weight. Indicated by.
The results are shown in Table 3.

When the clays, carbonaceous substances, complex substances, and shellfish shown in Table 3 were added to the medium, they were all mycophenolic acid and
The accumulation of L-aspartic acid was markedly increased. On the other hand, the degree of growth of the bacterial cells was not significantly different from that without addition, but the pellet size was refined by these additions.

Example 3 According to the method shown in Example 1, the penicillin G-producing bacterium Penicillium clingenum AT grown in the medium shown in Table 1 was used.
CC10002, AJ7343, Cephalosporin C-producing fungus Cephalosporium polyaleulam ATCC20359, AJ6993, Mannocarolose-producing fungus Penicillium charlesii AT
CC8730, Aspergillus phenylis ATCC14332, AJ17063 which is a protease producing bacterium, Aspergillus niger ATCC9642, A which is a glucoamylase and citric acid producing bacterium
J7172, and glucose oxidase-producing bacteria Penicillium perprogenum FERM-P1846, AJ17045 Mycelium and spore suspensions were added to the respective production media shown in Tables 4, 5, 6 and 7 (50 ml culture medium in a 300 ml Erlenmeyer flask). , And rotary culture (226 rpm) was performed at 27 ° C. Culture time is 5 days for penicillin G, cephalosporin C, mannocarolose, and glucoamylase production, 4 days for protease production, and 6 days for glucose oxidase production.
Culture was carried out for 10 days, and for citric acid production, 10 days. The accumulated amount of each product in the culture solution was measured by the method described above. Table 8 shows the results of measuring the accumulated amounts of these producing bacteria in the presence and absence of activated carbon F-17.

The addition of activated carbon F-17 increased the accumulation in all production media. On the other hand, mycelium pellets were generally made finer by the addition of activated carbon.

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Claims (2)

[Claims] 1. A clay material selected from (a) bentonite, K-clay or pesticide clay, (b) a carbonaceous material selected from porous microcrystalline carbon, non-porous microcrystalline carbon or graphite,
A liquid medium containing at least one of (c) carbon-silica synthetic resin composite material and (d) powder of animal-derived material selected from shellfish, bone or silk at a concentration of 0.01% -2%. A method for culturing a filamentous fungus, which comprises aerobically culturing the filamentous fungus. 2. The method according to claim 1, wherein the carbon-silica synthetic resin composite material is a composite material of synthetic resin and talc, alumina and zeolite, or carbon and silica.