JP3498925B2 - Gliotoxin production by Gliocladium virens - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a cryogradium ( Gli
ocladium ), and a method for efficiently producing the antibiotic gliotoxin by solid-state culture using a new strain .

[0002]

BACKGROUND OF THE INVENTION Gliotoxin is an antibiotic showing activity against fungi and Gram-staining positive bacteria, including Gliocladium virens, Gliocladium fimbriatum, Trichodema viride, Aspergillus
It is known to be produced by Fumigatus, Penicillium sp. However, when the productivity of the strains to which these gliotoxin-producing strains belong is compared, there are many strains in which no production is observed or strains with a small production amount. Further, the production method conventionally used for the production of gliotoxin by these bacteria is tank culture using a liquid medium, which is not optimal for the culture of absolutely aerobic fungi. When culturing a fungus by the liquid culture method in a tank,
Since the oxygen demand is large, it needs to be agitated violently, but there are problems that the hyphae are damaged by the agitation and the productivity of the substance is lowered, or the target substance is not produced due to abnormal metabolism. In continuous culture and fed-batch culture for avoiding depletion of nutrients and self-suppression by products, there are problems such as difficulty in controlling various environments. As described above, many gliotoxin-producing strains are known, but the productivity differs for each strain, and the most suitable culture conditions also differ. For the above reasons, it is the current situation that the technology level for efficiently producing gliotoxin at low cost and high concentration has not been reached.

[0003]

[Problems to be Solved by the Invention] In order to efficiently produce gliotoxin at a low cost and a high concentration, among the known culturing techniques, a method having a low operating cost, a simple environmental control and a simple medium composition is selected. The first condition is to adopt it, and it is necessary to select strains with a truly high gliotoxin production capacity and to develop an optimal production environment for the selected strains.
The present invention finds a strain having a much higher production capacity than conventionally known gliotoxin-producing strains, and provides a low-cost and simple method for producing gliotoxin that can address the problem. is there.

[0004]

[Means for Solving the Problems] The present inventors have proposed a solid culture in which the apparatus cost and the operating cost are lower, oxygen transfer does not pose a problem, and the product recovery cost is low, as compared with the conventionally used liquid culture. Focusing on the above, we conducted a diligent study of the medium composition and culture conditions for efficient production of gliotoxin in solid culture, and found that the carrier and the food industry by-product substrate that keep the three-dimensional structure of the medium porous and the gas diffusion efficiency in the medium high By culturing at 25 to 27 ° C. for 4 to 5 days using a solid medium consisting of, it was possible to produce a high concentration of gliotoxin with an extremely inexpensive raw material and a simple culturing means. The carrier used here is preferably a substance which does not contain a component soluble in the solvent used in the gliotoxin extraction step and which keeps the growth of the culture bacterium suitable, and examples thereof include peat moss and vermiculite. Examples of the substrate include rice bran, rice bran, and rapeseed meal, which are by-products of the food industry, and those containing mainly carbohydrates and a small amount of protein are preferable. The mixing ratio of the carrier and the substrate is not strictly limited, but when the ratio of the substrate is too large, oxygen is deficient and the growth of the bacteria is adversely affected, and reduced gliotoxin is produced. Therefore, 30 to 10 parts by volume of the substrate is economically suitable for 70 to 90 parts by volume of the carrier.

The method for extracting gliotoxin from the culture is not particularly limited, and any conventionally known method may be used, such as reflux extraction of ethyl acetate or chloroform. Further, if it is necessary to isolate gliotoxin, various conventionally used isolation methods such as chromatography and fractional precipitation method utilizing the difference in solubility can be used.

[0006] Using such a technique, the antibiotic gliotoxin can be produced at low cost and efficiently, but the gliotoxin-producing ability of the conventionally known strain is not sufficient, and in order to make the present invention more effective, Selection of strains with high productivity of glyotoxin was necessary. Therefore, using soil collected from all over Japan as a sample, diligent examination on selection of strains capable of efficiently producing gliotoxin in solid culture,
Researched. That is, it was considered that the organ in which gliotoxin is produced by the fungus is a vegetative mycelium, and a strain having an extremely rich vegetative mycelium has high productivity. In addition, many strains form asexual spores when hyphae are elongated to a certain degree, their metabolic activity in vegetative hyphae is reduced, and they shift to a dormant state to start autolysis. Therefore, it was considered that the slower the asexual spore formation time and the less the strain, the higher the gliotoxin productivity.

[0007] As a result of continuing these studies, as a strain satisfying the above-mentioned condition, Glioc
The present invention has been completed by discovering the strain ladium virens ) NT-10 (FERM P-14123). That is, the present invention uses the technique of solid culture for the production of gliotoxin, and the production strain Gliocladium virens NT-10.
It is an important point that it becomes a more effective technology by using the strain (FERM P-14123).

The microorganism of the present invention is a strain isolated from a long potato continuous cropping soil in Rokunohe-cho, Kamikita-gun, Aomori Prefecture. The isolation of this fungus from soil was performed by the dilution plate method. That is, 1 g of soil is dispersed in 99 ml of sterile dilution water (Tris buffer) and diluted 10 times and 100 times with the same dilution water,
1 ml of each was dispensed into a sterile petri dish, 10 ml of rose bengal agar medium containing chloramphenicol was poured into this dish, mixed well, solidified, and cultured at 28 ° C. for 3 days to separate from colonies that appeared.

This strain has the following properties. Growth status in each medium Oatmeal agar medium Vigorous growth (d = 67 mm at 28.5 ° C for 3 days) Initially few aerial mycelia, white colonies, but later fluffy aerial mycelia. There is little formation of green asexual spores in culture for 5 days or more. Malt extract agar medium Vigorous growth (d = 79 mm at 28.5 ° C for 3 days) There are relatively many aerial mycelia, and colonies initially appear white and then amber. Does not form asexual spores. Potato-Glucose agar medium Vigorous growth (d = 79mm for 3 days at 28.5 ℃) Abundant aerial hyphae are fluffy, and lysis starts from the center of the colony, and the colony backside becomes amber to brown. Green asexual spores are formed in the culture for 5 days or more, but it is extremely small, and is formed only on the aerial hyphae at the end of the colony. Sabouraud agar medium Slightly vigorous (d = 55 mm for 3 days at 28.5 ° C) There are few aerial mycelia, and colonies are white to slightly amber. Does not form asexual spores

Physiological and ecological properties Suitable growth temperature: 25-32 ° C Growth limit: Not grow above 10 ℃ or above 40 ℃ Optimum pH: 5-7 pH range: Can grow in at least 3-10 range

Type of strain of the present invention KHDOMSH, W. GAMSAND THANDERSON: COMPENDIUM in which the species classification of the genus Trichoderma and the genus Gliocladium is described
OF SOIL FUNGI, ACADEMIC PRESS, LONDON, UKVOLUME 1
, 859pp. (1980), and MARIFI: A REVISION OF G
ENUS TRICHODERMA, mycological Paper, No.116, COMMONW
EALTH MYCOLOGICAL INSTITUTE, KEW, SURREY, UK 56pp.
The characteristics of Gliocladium virens in (1969) were compared with the strain of the present invention. Growth on oatmeal agar is extremely fast (colon diameter 5.8 cm at 20 ° C for 5 days), and phialides form halos at narrow angles to each other, forming a large green ball-like asexual spore mass. Asexual spores are oblong
It is 4.5-6 × 3.5-4 μm and the surface is smooth. Since these morphological characteristics are consistent with those described in the literature, the strain of the present invention is classified as Gliocladium virens , but in a rich medium, the property of producing high concentration of gliotoxin and autolyzing, and asexual spores In the solid medium having a natural product as a substrate, the amount of mycelia is extremely large and the asexual spore formation is slow. For this reason, the strain of the present invention has been entrusted to the Institute of Biotechnology, Institute of Biotechnology, with the accession number FERM P-14123. The mutant strain in the present invention, the strain is mutated by natural mutation or radiation or chemicals,
Strains producing high concentrations of gliotoxin are also included.

The effects of the present invention will be specifically described below with reference to examples.

Example 1 A method for separating the strain of the present invention from soil will be described. 1 g of raw soil was added to 99 ml of sterilized dilution water (Tris buffer), and the mixture was shaken reciprocally for 15 minutes to disperse, to obtain primary dilution. After stirring this well, take 10 ml with a sterilized pipette and
Add to ml of sterile dilution water. This was used as a secondary dilution, and at the same time, a tertiary dilution was made. Five pieces of each 1 ml were put into the sterilized petri dish from the second and third dilution solutions. To this, 10 ml of each rose bengal agar medium, which was kept warm at around 50 ° C, was dispensed. After the medium was solidified, it was cultured in a room temperature chamber at 28 ° C. for 3 days, and the strain of the present invention was isolated from the filamentous fungal colonies that appeared. Sterile dilution water was prepared with the following composition. Trisaminomethane 1.21 g Distilled water 1000 ml pH 7.2 (corrected with hydrochloric acid) The composition of the Rose Bengal agar medium was as follows. _{KH 2 PO 4 1.0 g MgSO 4} · 7H 2 O 0.5 g Peptone 5.0 g Glucose 10.0 g rose bengal 0.033g agar 20.0 g Chloramphenicol 50.0 mg Distilled water 1000 ml

[0013]

[Example 2] Gliotoxin productivity in solid culture Comparison of Gliocladium virens NT-10 strain (FERM P-14123) (strain of the present invention) obtained by the above method with known strains (see Table 1) Did. The medium was prepared by mixing 76 parts by volume of soil improving material mainly composed of peat moss with 12 parts by volume of malt and 12 parts by volume of water, filling an Erlenmeyer flask, and sterilized by high pressure steam at 120 ° C. The test strain was inoculated into the obtained sterilized medium in the state of asexual spore suspension, cultured at 25 ° C. for 5 days and extracted by the chloroform reflux method to measure the amount of gliotoxin produced. still,
The quantification of gliotoxin was carried out using the TLC area method, and the Rf value was confirmed and quantified from the spot area by a calibration curve prepared with standard gliotoxin. The results are shown in Table 1, and it was proved that the strain of the present invention had much higher productivity.

[0014]

[Table 1] Gliotoxin production comparison ──────────────────────────── strain mg / medium fresh weight 100g ────── ────────────────────── The present strain 35.7 Gliocladium virens (IFO 06355) 0.0 Gliocladium virens (IFO 09166) 15.7 Gliocladium virens (IFO 09169) 0.0 Trichoderma viride (IFO 05720) 0.0 Aspergillus fumigatus (soil) 7.0 ─────────────────────────────

[0015]

Example 3 The production period of gliotoxin by the strain of the present invention was investigated. The medium was prepared by mixing 76 parts by volume of soil improving material mainly composed of peat moss with 12 parts by volume of water and 12 parts by volume of water, filling a polypropylene bag with a ventilation filter, and sterilizing with high pressure steam at 120 ° C. The bactericidal medium was inoculated with the strain of the present invention in the state of an asexual spore suspension and cultured at 26.5 ° C.
The quantification of gliotoxin was the same as in Example 1. The results are shown in FIG. 1, and gliotoxin production was confirmed from the second day of culture, and the maximum concentration was reached on the fourth day.

[0016]

[Example 4] A solid medium was prepared using the materials commonly distributed as food industry by-products and agricultural materials as substrates, and the same culture as in Example 1 was performed to compare the gliotoxin productivity on the 5th day. However, the medium was prepared by mixing 25 parts by volume of the substrate with 75 parts by volume of the soil-improving material mainly composed of grass charcoal as a carrier so that the water content is most suitable for the growth of the bacterium of the present invention.

[0017]

[Table 2]             ───────────────────────                 Substrate gliotoxin mg / d.m. 100g             ───────────────────────                   143               Rice bran 186               Coffee meal <9               Sawdust <9               Chicken droppings 91               Rapeseed meal 118               Skim milk 146               Cow dung compost <9               Burke compost <9             ────────────────────────

The results are shown in Table 2, and it was proved that 100 mg or more of gliotoxin was produced per 100 g of the dry matter of the medium in the culture using rice bran, rice, skim milk, and rapeseed meal as a substrate.

[Brief description of drawings]

FIG. 1 shows the relationship between the number of culture days and the amount of gliotoxin production according to Example 2.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12P 17/18 C12P 17/18 C12R 1: 645) (56) Reference US Pat. No. 5268173 (US, A) Phytopathology, Vol. 82, No. 2 (1992), p. 230-235 Phytopathology, Vol. 81, No. 7 (1991), p. 738-741 Mycol. Res. , Vol. 95, No. 10 (1991) p. 1242-1248 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12P 17/00-17/18 JSTPlus (JOIS) BIOSIS / WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A Gliocladium virens NT-10 strain capable of producing gliotoxin.
A method for producing gliotoxin, which comprises culturing (FERM P-14123) in a solid medium and extracting gliotoxin from the culture. 2. The method for producing gliotoxin according to claim 1, wherein the solid medium uses as a substrate at least one by-product of the food industry selected from the group consisting of dough, rice bran and rapeseed meal. 3. Gliocladium virens NT-10 strain (FERM P-14123) capable of producing gliotoxin.