RU1839183C - Method of solid-phase cultivation of mycelial fungi - Google Patents

Description

The invention relates to biotechnology and may find application in the preparation of target enzymes in the process of growing on a solid nutrient medium. 5

Known methods for surface cultivation of micromycetes on a solid nutrient medium, including wheat bran, with the addition of biosynthesis inducers and ripper additives to increase the yield of target products 1.

 The disadvantage of this method is the inability to significantly increase the yield of enzymes due to the limited height of the nutrient layer, which makes it difficult to 15 airlift crops.

Closest to the technical nature of the invention is a cuvette method for solid-phase cultivation of fungal mushrooms, including sterilization of moistened wheat bran to 58-68% (it is possible to add 5-20% additives of cultivators), inoculation with seed material containing no spores less than O, 7bn; c1 g, thoroughly mixing the components, folding them into cuvettes 2–2.5 cm high and growing

cultures for 32-48 hours (depending on the individual characteristics of the microorganism) at a temperature of 30-35 ° C (depending on the thermophilicity of the culture), periodically purging with sterile air for aeration, cleaning the chamber, processing it with formalin , ammonia, steam .35

The main disadvantage of this method is the insufficiently high yield of the product.

The aim of the invention is to increase the yield of biomass.

This is achieved by the fact that in the method involving sterilization of moistened wheat bran, inoculation with seed, stirring, folding the resulting mixture into cuvettes 45 and growing, fungi of the genus Rhlzopus are used as seed, and periodically every 20-22 h. Fresh moistened wheat bran with a thickness of 1.5-50.0 cm is layered until a final layer thickness of 7-8 cm is reached and the growing process is carried out for 120-124 hours.

The method is as follows.

Cells for surface cultivation of 55 microorganisms measuring 40x75 cm with a side height of 12 cm and holes for aeration of the culture are washed, treated with formalin, ammonia and steam. Wheat bran is moistened to 58-65%, sterilized for 1-1.5 hours at 0.15 MPa. they are cooled and seed at a temperature of 40-45 ° С is introduced (for example, micromycete cultures Rhlzopus pygmaues pi-e and Rhizopus oryzae 1384-262 on wheat cuts with a spore content of at least 0.7 billion per 1 g) in an amount of 1 -2% to the volume of the nutrient medium. The mixture is thoroughly and quickly mixed, while the final temperature of the mixture should be 30-32 ° C. The resulting mixture is laid out in cuvettes with a layer of 2-2.5 cm. When using a thinner layer, the solid-phase nutrient medium dries quickly, and with a layer thickness of more than 2.5 cm, the spores are not uniformly growing during growth, and the aeration of the culture is reduced.

Germination of the culture is carried out at a temperature of 30-32RS (the most typical conditions for micromycetes) for 20-22 hours. Germination time 20-22 hours is characterized by a special physiological state of the culture. By this time, it is in an exponential phase of development, which is characterized by a high rate of reproduction and the beginning of the formation of a complex of exogenous enzymes, which are the target products. A time of less than 20 hours is not enough to ripen, achieve the highest rate of reproduction and development of producers.

A growing time of more than 22 hours leads to a sharp decrease in the rate of cell proliferation against the background of an increase in their biochemical activity.

After 20-22 hours of germination, a new nutrient layer is layered on the solid surface of the medium - sterilized moistened wheat bran 1.5-2.0 cm high, keeping the cultivation temperature constant (30-32 ° C). The operation is similarly repeated several times until a total height of the nutrient layer of 7-8 cm is reached. Using a height of the nutrient layer of less than 743 cm does not quite meet the intended goal due to economic considerations, and a layer height of more than cm is undesirable due to the prevalence of crop aging, reduction its aeration, a significant accumulation of products of biochemical activity of producers.

The recommended total growing time of 120-124 hours has been experimentally determined and is also associated with a change in the physiological activity of the culture and a decrease in the possibility of aeration. With a shorter duration of production when using nlol ..- inli, it is impossible to achieve the desired layer height, and therefore, significantly increase the yield of a product from a unit of the working surface. Moreover, the culture does not have the desired level of enzyme activity. A growing time of more than 124 hours is impractical due to the aging of the crop and a decrease in the yield of active biomass.

Example 1. 1 kg of wheat bran medium grinding with a starchiness of 18% is moistened with tap water to a final moisture content of 63%, sterilized in an autoclave at 0.15 MPa for 1 h. Prepared bran is cooled to 40-45 ° C, inoculated with inoculum mycelial Rhlzopus mushrooms (2% by weight of moistened bran with a spore content of 0.7 billion per g) are thoroughly mixed and placed in cuvettes (20x40x12 cm in size) 2 cm high. Cover and grow for 20 hours at 30 ° C. Then, a new batch of similarly prepared bran 1.5 cm high is layered onto a germinated nutrient medium, keeping the temperature constant (30 ° С). The operation is repeated after 40, 60 and 80 hours from the start of cultivation. Cultivation is continued until a total duration of 120 hours is reached, then the surface culture on the wheat bran is removed from the cuvette and the quality of the biomass is determined by the level of glucoamylase (HLA) and proteolytic (PA) activity, and the cuvette is treated.

Example 2. 1 kg of wheat bran of medium grinding, 18% starch, is moistened with tap water to a final moisture content of 63%, sterilized in an autoclave at 0, 15 MPa for 1 hour. The sterilized bran is cooled and seed is introduced. The mixture is thoroughly mixed and laid out in cuvettes with a layer height of 2.5 cm. Cover and grow for 20 hours. Then a new nutrient layer of 1.5 cm is layered on the sprouted culture. The operation is repeated after 40, 60.80 hours (with a total cultivation time of not 120 hours. Then the culture is removed, the quality of the biomass is determined and used as intended. The cuvettes are processed.

Example 3. Wheat bran is preliminarily prepared in the same way as in examples 1, 2, it is laid out in cuvettes 3.0 cm high. They are covered with a lid and grown for 20 hours. The operation is repeated after 40, 60, 80 hours from the start of growing, layering on top wheat bran 1.5 cm high. After a total cultivation time of 120 hours, the culture is removed, the quality of the biomass is determined and used, and the cuvettes are processed.

EXAMPLE 4 Wheat bran (according to Examples 1, 2) is laid out in cuvettes of a 5th hundredth layer of 2.2 cm. Germination is carried out for 21 hours, and then the operation is repeated after 42, 63, 84 hours from the start of cultivation. each time the layer was kept at a height of 1.3 cm. After 124 hours from the beginning of the process, the culture was removed, the quality of biomass was determined and used. 4,

Example 5. Inoculated wheat bran (according to examples 1,2) are laid out in cuvettes with a layer height of 2 cm and germinated

5 for 22 hours. The operation is repeated after 44.66 and 88 hours at a height of each layer of 1.5 cm and after the last layering, growth is continued to a total duration of 128 hours, then the surface culture is removed from the cuvette, the biomass activity is determined and use.

Example 6. Inoculated wheat bran (according to examples 1,2) are laid out in cuvettes with a layer height of 2 cm and germinated

5 for 22 hours. The operation is repeated after 44, 66 and 88 hours at a height of each layer of 1 cm. Then, the culture is grown to a total process time of 124 hours. Then proceed as in the above examples.

0 Experimental data for examples 1-6 are shown in table. 1. Experimental data are presented for Rhlzopus pygmaues pi-e, a producer of glucoamylase and proteolytic enzymes. A similar dependence was noted for Rhizopus oryzae 1362-284.

The proposed solution has the following advantages compared to the prototype:

0 decrease in labor intensity due to removal of operations on processing the ditches, preparation of seed material and manual labor on laying out the inoculated nutrient medium. To get the same

5 the same amount of culture according to the prototype at a recommended layer height of 2-2.5 cm, the production cycle must be repeated 3-4 times with all operations for the preparation of cuvettes, rooms, seed,

0 basic cultivation;

save seed by 4-5 times, since for the implementation of the prototype method, each production cycle will require seed consumption of 1-2

5% by weight of moistened bran, according to the proposed technical solution, the seed is used only once in the first stage;

increase in the yield of the finished product (surface culture) 2.5-3.0 times

units of the growing area with almost the same level of biochemical activity of mycelial fungi.

(56) Stallions N.A. Shcheblykina L.V. Cultivation of the mold fungus Rhlzopus pygmaues pi-e on solid nutrient media. Microbiological Industry - General comparative characterization 5, -1972, vol. 2, p. 31.

Gracheva I. M. Technologists of enzyme preparations. M .: VO Agropromizdat, 1987.

The statistics of both methods are presented in Table 2.

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Table

Indicators

1. The list of technological operations of the process to obtain a similar amount of surface culture: preparation of ditches, sterilization and humidification of bran

2. Inoculation with seed

3. Growing, h

4. Culture removal 5. Processing, washing the cuvette 6. Sterilization and hydration of bran

7. Inoculation with seed

8. Growing, h 9. Next, the processes are repeated 3 times

 10. The height of the layer, cm 11. The yield of the product (surface culture from a unit to grow ln area), kg / cm2 12. Biochemical activity, units / g: HLA PS

Claims (1)

The claims METHOD OF SOLID-PHASE CULTIVATION OF MYCELIUM MUSHROOMS, which involves sterilization of moistened wheat bran, inoculation of jix seed, mixing, laying down the resulting mixture in cuvettes and growing, characterized in that, T a 6l egg 2 The value of indicators prototype on the proposed technical solution processing with formalin, ammonia, pa-, T5MPa for 1 h, W-58-68% -2% to the mass of bran 36-42 Manually Manually , 15 MPa for 1 4, W-58-68% 2% to the mass of bran 36-42 2-2.5 1.8-1.5  . 17-17.6 20-19 Treatment with formalin, ammonia, steam 0.15 MPa for 1 h, W-58-68% 1-2% to bran mass 20-22 0.15 MPa for 1 4, W-58-68% 2Q-22t 7-8 6.0-6.3 17.6-18.2 20-19 in order to increase the biomass yield, fungi of the genus Rnlzopus are used as seed, in the process of growing, periodically every 20 to 22 hours, fresh moistened wheat bran 2 cm thick is layered until the final layer thickness is reached. 7-8 cm and the growing process is carried out for 120 -124 hours