JPH0363350B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing filamentous fungi. Up until now, many studies have been conducted on the use of plant-derived powders for the artificial cultivation of edible mushrooms (for example, Japanese Patent Application Laid-Open No. 1983-1999).
56614). However, there have been no reports yet of a case in which plant-derived powder is used for a purpose other than a nutrient source necessary for growth in liquid culture for the purpose of producing useful substances by filamentous fungi. 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 aimed at producing enzymes such as anticancer drugs, organic acids, antibiotics, proteases, glucoamylase, and glucose oxidase, L-amino acids, pigments, and enzyme inhibitors.
As a result of culturing each of the producing bacteria belonging to the genus Aspergillus, Cephalosporium, or Rhizopus in a culture solution containing plant-derived powder, the mycelial masses were miniaturized and at the same time the production of the desired useful substance was significantly increased. The present inventors have discovered that this can be promoted and have completed the present invention. That is, the present invention enables the cultivation of Penicillium spp., Aspergillus spp.,
The present invention relates to a method for culturing filamentous fungi, which comprises culturing filamentous fungi belonging to the genus Cephalosporium or Rhizopus aerobically. Among the plant-derived powders used in the present invention, there are various wood-derived wood powders. For example, sawdust of cedar, pine, toga, cypress, paulownia, hiba, lauan, etc. can be cited. Examples of the grass stem powder include rice, barley, wheat straw, haystack, bagasse, and the like. In addition, the seed coat may be of soybean, corn, rice hull, buckwheat hull, etc. The sawdust used may be in the form that is generally available, and in some cases it may be separated using a suitable mesh sieve. In addition, the seed coat can be used in its generally available form as it is or crushed into an appropriate size. When the plant-derived powder was added to the medium, spores and mycelia were observed to attach to the plant-derived powder, germinate using the spores as nuclei, and the hyphae were observed to grow. It can also be observed that they are growing without wearing clothes and without forming clumps. Thus, by adding the plant-derived powder, 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, and are anticancer agents such as mycophenolic acid and cyclosporin produced by filamentous fungi belonging to the genus Penicillium, Aspergillus, Cephalosporium, or Rhizopus. , organic acids such as gluconic acid, citric acid, malic acid, pyruvate, itaconic acid, araboascorbic acid, kojic acid, and alloisocitric acid, antibiotics such as penicillin and cephalosporin C, and enzymes such as protease, glucoamylase, and glucose oxidase. Class, L-
Examples include amino acids, pigments, and enzyme inhibitors such as compactin and mevinolin. The filamentous fungi used in the present invention are filamentous fungi that produce the above-mentioned useful substances, such as Penicillium brevicompactum that produces mycophenolic acid and compactin, Penicillium chrysogenum that produces penicillin G, and Penicillium that produces glucose oxidase.・Perprogenum,
Cephalosporium polyalerum, which is a cephalosporin C-producing bacterium, Aspergillus oryzae, Rhizopus derema, which is an amylase-producing bacterium,
Aspergillus cytoi, which is a protease-producing bacterium, Aspergillus pheinisis, and Aspergillus niger, which is a citric acid- and gluconic acid-producing bacterium.
Examples include Aspergillus terreus, which is an itaconic acid-producing bacterium. Specifically, mycophenolic acid-producing bacteria, Penicillium brevi-compactum,
AJ117096, (FERM P-5693, FERM BP-
55), Penicillium chrysogenum (P. chrysogenum), a penicillin-producing bacterium
ATCC10002AJ7343, Penicillium perprogenum No.778, a glucose oxidase producing bacterium
(P. purprogenum) AJ17045 FERM-P1846, Cephalosporium polyallerum (Cephalosporium polyallerum) as 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 plant-derived powder. For example, known media for mycophenolic acid production,
WLMuth et al , Autimicrob, Agents
The culture medium described in Chemtherap., 8 , 321-327 (1975) is used. As a medium for penicillin fermentation
Synthetic medium of Johnson et al. (Yusuke Sumiki “Antibiotics”)
above, p. 177, 1961) is used. As a medium for cephalosporin C fermentation, for example, AL.
DEMAIN, JF NEWKIRK, 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 plant-derived powder added may be 0.05 to 5%, preferably 0.1 to 1%. 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 to 250 rpm.The method of adding the plant-derived powder may be carried out singly or in combination of two or more. 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 testing 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 power measurement method (Jikken Kagaku Course, 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 accumulates and collects useful substances produced by filamentous fungi by adding plant-derived powder to a culture medium. The method of the present invention significantly increases the production amount of useful substances by adding plant-derived powder, and the residue containing the plant-derived powder from the culture solution after harvesting the product can be easily removed. Its practical industrial value is extremely large in that it can be incinerated.Example 1 Penicillium brevicompactum AJ117096
(FERM P-5693, FERM BP-55) were inoculated into the natural slant medium shown in Table 1, and after culturing at 27°C for 14 days, the spores were collected and added to 0.1M phosphate buffer (PH
6.8) and inoculated a certain volume of it into the mycophenolic acid production medium shown in Table 2 (50 ml of medium in a 300 ml Erlenmeyer flask) (approximately 10 ⁶ spores/
ml), and rotational culture was performed at 226 rpm for 12 days at 27°C. When adding sawdust, first sieve the sawdust (mainly composed of lauan wood) through a 30-mesh sieve, steam sterilize each, and then add 0.5%
It was added to the medium so that After culturing, mycophenolic acid in the culture solution was determined using high performance liquid chromatography, and L-aspartic acid was determined using a bioassay method. For bacterial growth, the entire culture medium (50 ml) was filtered naturally using Toyo Paper No. 2, and then washed with approximately 10 ml of distilled water.

【table】

[Table] The bacterial cells were washed and dried with paper at 105°C for 18 hours, and the weight of the paper and added sawdust was subtracted from the total dry weight. The results are shown in Table 3.

[Table] More than Tsushiyu
grain)

Claims (1)

[Claims] 1. Filamentous fungi belonging to the genus Penicillium, Aspergillus, Cephalosporium, or Rhizopus are grown aerobically in a liquid medium containing a plant-derived powder selected from at least one of wood powder, grass stalk powder, or seed coat. A method for culturing filamentous fungi, which is characterized by culturing the fungi.