CS227392B1 - Method of preparing large amounts of highly physiologically active conidia of microorganism claviceps purpures(fr.)tul. - Google Patents

Abstract

The invention protects the preparation process of the large a number of highly physiologically active conidia microorganism Claviceps purpurea (Fr.) Tul. two-stage stationary cultivation on the surface of the liquid nutrient medium. In the first stage, which is inoculated with vegetative submerged inoculum or resting conidia in an aqueous suspension, the medium contains only a limited amount of assimilated carbon, which leads. to a complete stop extension vegetative undifferentiated growth of the spherical mycelium. In the second the medium is replaced by medium fresh, which contains an assimilable source carbon, preferably sucrose, in concentration 25 to 35 wt. and the culture is incubated until the end of conidial throttling.

Description

The invention relates to a process for the preparation of a plurality of highly physiologically active conidia of the microorganism Claviceps purpurea (Fr.) Tul.

Industrial cultivation of filamentous fungi is generally adversely affected by the need to prepare large volumes of inoculum to inoculate the production soil in large-volume fermenters. The preparation of large volumes of inoculum requires the propagation of vegetative fibers by submerged cultivation in a series of seed tanks of gradually increasing size. Increasing amounts of biomass are increasing in the course of propagation, but the quality of the inoculum decreases by such a process.

The youngest cells of the growing fiber ends and their branches are the active components of the inoculum, while the older, non-growing cells, further from the apex, in the conditions of submerged cultivation usually rapidly vacuolize, die and lyse. Tzv. A 'well-grown' inoculum, consisting of a 'homogeneous' dense fiber suspension, is therefore always a mixture of physiologically more or less active cells with inactive cells.

Moreover, these unfavorable results of the gradual aging of a portion of the population of propagated cells are usually accompanied by degenerative changes that always occur during repeated passage of the vegetative fibers of the production strains, be it degeneration at the level of physiological changes or at the level of genetic information changes.

The main reason why it is necessary to prepare large volumes of inoculum by the gradual propagation of biomass of growing vegetative hyphae is the difficulties associated with the preparation of large amounts of spore material. Spore filamentous fungi are usually prepared on sloped agar soils in test tubes or on loose soils in small Erlenmeyer flasks in quantities sufficient to prepare only a small amount of inoculum in flasks on shaking machines or in small pre-seed tanks.

Vegetative promycelium, prepared by synchronous germination of spores in submerged culture, represents a truly homogeneous population of physiologically maximally active cells, unlike the fibers obtained by its successive multiplication. Therefore, it is also an ideal form of inoculum.

In addition to the preparation of spore material by stationary cultivation on the surface of agar-hardened or loose sporulation soils, it is in many cases possible to prepare large amounts of spores by submerged cultivation. However, the quality of these spores never reaches the quality of spores throttled in surface stationary culture. This is usually manifested not only by considerable inhomogeneity in morphology (shape, size), but also in physiology. Submerged spores are always more or less vacuolized and have more or less reduced germination compared to stationary spores. Insufficiency of submerged spores also has a very unfavorable effect in that, even when stored at a low temperature, they die relatively quickly and are difficult to tolerate any kind of preservation.

Claviceps purpurea (Fr.) Tul is a typical industrial micro-organism to which everything has been said so far. The production strains of this fungus are used both in the field production of ergot and in the fermentation preparation of ergot alkaloids. The preparation of large quantities of highly virulent conidia is a prerequisite for the successful provision of both of these products.

Through continuous experimental research on the control of conidation of C. purpurea, we have been able to gradually identify and optimize cultivation conditions by allowing virtually synchronous conidation to produce a surprisingly large number of morphologically uniform and physiologically maximally active spores, even in so-called oligosporogenic strains.

Two consecutive signals received from the environment were found to be conditional. The first one is addressed to the growing spacelet that responds to it by terminating polar growth. Ending the growth of the sphellia is a prerequisite for initiating the conidation phase. The second signal, conditioned by further environmental change, is received by the already growing sphacell and decides on the conidiation experiment. The expression of conidiation is irreversible. Therefore, the sphellia cells, which, in response to the first signal, interrupted peak growth, have only two alternatives: when exposed to the second signal they start to constrict conidia, otherwise they gradually die.

The experimental demonstration of the significance of both signals was carried out by two-stage surface cultivation of sphingia by the method of culture medium exchange. The first stage was inoculated with a young vegetative inoculum represented by a sphellia at the beginning of the logarithmic growth phase, cultivated submerged from conidia, in a medium containing 3% by weight. sucrose. In the first step, the ending of the apical growth of sphellia was achieved by completely draining the sugar component from the culture medium. Growth was made possible by the residual sucrose introduced with the inoculum. Alternatively, the first stage was inoculated with resting conidia suspended in distilled water. In this case, traces of the sugar component were added to the first stage culture medium to allow a minimal increase in the continuous diaphragm diaphragm. Thus, during the first-day seven-day incubation, only a thin mycelium diaper grew at the medium level, the growth of which stopped completely at the end of the incubation. In the second step, the medium was replaced with the same medium but containing an additional 30 wt. sucrose. The addition of sucrose led to the differentiation of sphinga into the conidation phase and was manifested by intense spore formation. If the medium of the first stage was not replaced, conidia were created only sporadically, just as when unchanged medium was used for replacement. Very weak conidation was observed even after 14 days of cultivation in one stage on a medium with 30% by weight. sucrose.

The foregoing analysis of filamentous fungal sporulation and the specific clarification of conidia in C. purpurea allow us to understand the well-known fact that filamentous fungal conidia are much easier to produce in stationary cultures on the top of mycelial colony than in submerged mixed cultures. This is probably due to the polarized nature of the stationary colony and its substantially slower aging process, as opposed to the rapidly aging non-polar submerged mycelium.

These findings have been used in the development of a large number of highly physiologically active conidia of Claviceps purpurea (Fr.) Tul. by stationary cultivation of the microorganism on the surface of a liquid nutrient medium containing sources of assimilable carbon and nitrogen and a mineral nutrient salt at a temperature of 20 to 30 ° C according to the invention, which consists in that the cultivation is carried out in two stages, the liquid nutrient medium comprises assimilable nitrogen sources, mineral nutrient salts, and an assimilable carbon source, preferably sucrose, at a concentration of 0.02 to 0.2, preferably 0.05 to 0.1% by weight. and in a second step, the liquid nutrient medium comprises an assimilable carbon source, preferably sucrose, at a concentration of 25 to 35 wt%, preferably 30 wt%, and incubating the culture until the conidia constriction phase is complete.

Suitably, the liquid nutrient medium in the first step is inoculated with the submerged vegetative inoculum of the producing microorganism in the logarithmic growth phase or by resting spores in an aqueous suspension.

Depending on the operating conditions, the two cultivation steps usually take the same duration, usually 6 to 8 days.

The method according to the invention makes it possible, by conventional simple means, to obtain a material for a vaccine used in the infection of rye under field conditions, with a multiple increase in ergot production. Likewise, the conidia obtained by the process of the invention can be used to prepare large volumes of inoculum to inoculate the production soil in the fermentative production of ergot alkaloids.

Example 1

(a) Vegetative inoculum

Submerged vegetative inoculum was prepared in 500 ml beakers filled with 80 ml inoculum broth of the following composition (in% by weight): sucrose 3, corn steep liquor 0.4, crystals. magnesium sulfate 0.1, potassium dihydrogen phosphate 0.04, crystal. calcium nitrate 0.144, target. Ammonium citrate 1, pH 5.8 (adjusted with ammonia) After filling, the soil was sterilized in an autoclave for 20 minutes at 0.1 MPa, and the cooled soil was inoculated with a conidial suspension prepared from spore-generation cultures grown on agar-hardened sporulation soil of the following composition: wort 5 ° Bé, casein acid hydrolyzate 1% w / w, crystalline magnesium sulfate 0.1% w / w, agar 3% w / w, pH 6.3 (adjusted with potassium hydroxide) Sterile inoculum broth was used to prepare the suspension. The conidia obtained by wiping off the surface of the spore generation was filtered by sucking into a sterile 10 ml pipette with a slightly cut tip overlaid with two layers of gauze to remove fibers and mycelium from the suspension and to prevent pellets from growing through the growth centers The density of the conidia suspension thus prepared was tentatively checked by subtraction to Bo. Each flask was inoculated with about 0.3 X 10 ⁹ conidia. The vegetative submerged inoculum was incubated on a shaker at 24 ° C for 30 hours.

bj First stage

In the first stage, vegetative airy white mycelium (sphcelia) was grown in stationary culture at the level of sucrose-free soil of this composition (in% by weight); corn steep liquor 0.4, crystal. magnesium sulfate 0.1, potassium dihydrogen phosphate 0.04, crystal. calcium nitrate 0.144, target. ammonium citrate 1, pH 5.8 (adjusted with ammonia). The soil was filled into Blacke flasks (5.5 cm) of 30 ml and sterilized in an autoclave at 20 psi for 20 minutes. The cold soil was inoculated with 1 ml of vegetative submerged inoculum. After mixing the inoculated soil, it was incubated at rest at 24 ° C for 7 days.

(c) Second stage

In the second stage, the spacellia mycelium, grown during the first stage, was differentiated into a sporulation phase and conidia constricted. This was accomplished by pouring the spent first-stage soil under sterile cautious sutures and again carefully layering the mycelium with 30 ml of sporulated soil of the following composition (in% by weight): sucrose 30, maize display 0.4, crystallized magnesium sulfate 0.1, potassium dihydrogen phosphate 0.04, crystalline calcium nitrate 0.144, target ammonium citrate 1, pH 6.2 (treated with ammonia) Sporulation broth was sterilized in 30 ml tubes After the mycelium was layered, the flasks were stoppered and incubated again 7 days at 24 [deg.] C. On average, 18 * 10 < ⁹ > ⁹ conidia were obtained from one flask (soil surface area was 25 cm &^lt; ^{2 >} ).

Example 2

The cultivation was performed as in Example 1, except that an aqueous conidia suspension containing 0.03 XX 10 ⁹ resting conidia in 1 ml was used to inoculate the first stage and 0.075% w / w was added to the first stage culture medium. sucrose. On average, 21 X 10 ⁹ conidia were obtained per flask.

The conidia obtained were used to infect rye in a field trial as compared to conidia prepared by the routine submerged culture method. The conidia concentration in both comparisons was set equal to 20 X 10 ⁹ live conidia (as determined by the vital erythrosine staining method) in 10 L of the vaccine. With this amount of vaccine of both types, the area of 1 ore was repeatedly infected at three different sites. An average of 1.84 kg of ergot was harvested after infection with the control vaccine, while an average of 4.12 kg of ergot was obtained using a vaccine prepared from conidia according to the invention.

Claims (1)

SUBJECT Process for the preparation of a large number of highly physiologically active conidia of the microorganism Claviceps purpurea (Fr.) Tul. by stationary cultivation of the microorganism on the surface of a liquid nutrient medium containing assimilable carbon and nitrogen sources and a mineral nutrient salt at a temperature of 20 to 30 ° C, characterized in that the cultivation is carried out in two stages, OF THE INVENTION of a lovable nitrogen, a mineral nutrient salt and an assimilable carbon source, preferably sucrose, at a concentration of 0.02 to 0.2, preferably 0.05 to 0.1 wt%, and in a second stage the liquid nutrient medium comprises an assimilable carbon source %, preferably sucrose, in a concentration of 25 to 35 wt.%, preferably 30 wt. and incubating the culture until the conidia constriction phase is complete.