The method of producing drug for combating plant diseases

 

(57) Abstract:

The invention relates to the microbiological production of the drug to combat bacterial and fungal plant diseases. The method of producing drug for combating plant diseases containing as an active start complex streptothricin antibiotics involves preparing a seed, growing producer - Streptomyces griseus strain 420 in a fermentation medium containing sources of nitrogen, carbon, mineral salts and 0.01 -0,2% lysine, followed by concentration and drying of the culture fluid and standardization of the drug. In the preparation of inoculum selected clones with preferential formation of streptothricin With c and D by means of thin layer chromatography. Suitable for cultivation of a producer to use the medium of the following composition (%): corn flour 3-5, molasses 1,5-2,0, lysine 0,01-0,2, (NH4)2SO4or NH4NO30,6-0,7 KN2RHO40,01-0,09, NaCl, 0,05-0,25, MgSO40,005-0,05, caso30.5 to 1.5, propanol of 0.1-0.5, water - the rest. The method allows to reach exit antibiotic 8-9 kg/m3the culture fluid. 2 C.p. f-crystals.

The invention relates to microbiological PR is

One of microbiological drugs used to combat plant diseases, is phytobacterial that as the beginning of the current contains a complex streptothricin antibiotics with the prevalence of streptothricin C and D (Melnikova E. A., Makarova, J., Wood E. B. Antibacterial activity and toxicity of antibiotic phytobacteriology and its streptothricin // Physiologically active substance.- Kiev, 1980.- N 12, PP 93-96).

A method of obtaining the drug ficolin, containing in its composition phytobacterial, Ficolin used to deal with bacterial and fungal plant diseases: root rot of cereals, bacterial diseases of vegetable crops and other (Gavrilin, Century, Roboduck C. D., Pelikanova A. G., Popkov, P. , Sayenko I. A., Sepetuka I. M., Astartes E. C. the results of the study of vitravene // Sat. scientific papers. Preparations of microbiological synthesis agriculture.- M 1981, pages 25-30).

To obtain phetolamine use the culture of Streptomyces lavendulae strain 12-10 Unibankpremyer. Growing culture are deep way on liquid nutrient medium containing sources of carbon, nitrogen and mineral salts; after Fe is already installed, containing 100000 units of antibiotic,

Antibiotic complex contains streptothricin With 17-27%; streptothricin D-45-55%.

The disadvantage of this method of obtaining vitravene is low levels of antibiotic in the finished product and the low output at the final stage of production.

Recently, for the production of vitravene encouraged to use another type of microorganism, namely Streptomyces griseus, in particular a strain of S. griseus-420, due to its high productivity, providing a greater output per unit of production volume.

When using a culture of S. griseus strain 420 receive the drug ficolin-300 containing 300000 unit/year Antibiotic complex product contains streptothricin With 22% and streptothricin D - 54%. Production testing of vitravene-300 showed its high effectiveness in the fight against plant diseases (Bykov, A., White, E. B., Stroeva I. A., Novikov I. I. Activity of vitravene derived from two strains producers // plant Protection against pests and diseases in terms of ecologization of agricultural production. - Saint-Petersburg., 1992, pp. 66-70).

However, in practice it turned out that not every clone of S. griseus-420 capable progenitin C and D. In some cases appeared clones synthesizing mainly streptothricin F, low antibacterial and antifungal activity.

The present invention is a method of producing drug for combating plant diseases - phetolamine-300 with the necessary component structure streptothricin complex and high yield of the target product.

This result is achieved by selection at the stage of preparation of inoculum of the strain Streptomyces griseus 420 clones, forming mainly streptothricin C and D, as well as by optimizing the fermentation conditions of the selected clones, contributing to the formation streptothricin necessary part of that is achieved by putting in a fermentation medium lysine in an amount of 0.01 to 0.2%.

The proposed method is as follows:

In the culture of S. griseus 420 on the sloped agar add distilled water and produce wash spore material from the jamb. Obtained after filtration through a glass filter N4 pure suspension of single spores plated on Petri dishes with glucose - potato agar medium and grind it in the Cup. After cultivation at 281oC ticeoC for 10 days, after which the culture test on productivity. For this purpose from each crops take a piece of agar medium with spores and bring in Catalogne bulb, with a capacity of 750 ml, with a medium of the following composition (%): corn flour - 4,0; KH2PO4- 0,03; (NH4)SO4- 0,06; NH4NO3- 0,7; NaCI - 0,2; MgSO4to 0.05, CaCO3- 0,5; tap water up to 100 (the average values of concentrations for each individual component can vary in the range of 15%). The pH before sterilization is 6.6 to 7.2. Growing lead in the rocking chair with a speed of 220 rpm and grow the inoculum at a temperature of 281oC for 24-30 hours. Then inoculate each flask in an amount of 5% is transferred into a flask with a fermentation medium of the following composition (%): corn flour - 3-5; molasses - 1,5-2,0; lysine - 0,01-0,2; (NH4)2SO4or NH4NO3- 0,6-0,7; KH2PO4- 0,01-0,09; NaCI-0,05 - 0,25; MgSO4- 0,005-0,05; CaCO3- 0.5 to 1.5; propanol - 0,1-0,5; tap water. Growing lead in the rocking chair at a temperature of 281oC for 72 hours. The determination of the level of antibioticaccutane and composition of the antibiotic complex is carried out as follows: samples of the culture gigastacey 30 min, filtered through a paper filter. The filtrates determine the content of the antibiotic microbiological method using as the test culture You. subtilis 6633 (GF XI, vol.2).

The composition of the antibiotic fractions determined using thin-layer chromatography on plates with silica gel 60. As the mobile phase, use a solution of the following composition: sodium acetate - 123,03 g, sodium chloride -58,44 g, butyl alcohol tertiary - 100 ml, distilled water to 1000 ml.

Chromatography conduct a bottom-up fashion. After the chromatography was carried out, the plate is dried in the air. Next, the chromatogram is evenly sprayed with 0.2% ninhydrin solution in acetone. The plate is heated in an oven at 105oC for 5 minutes Components streptothricin complex are shown as purple spots on a pink background. The chromatography was carried out samples of native solution are compared with the standard sample of pure phytobacteriology containing (in %):

streptothricin IN - 2,0

streptothricin WITH - 20,0

streptothricin D - 50,0

streptothricin E - 13,0

streptothricin F - 15,0

The results of the chromatography was carried out to select clones that form the antibiotic complex, with the slanted agar. Out of 100 tested clones only 15% form the antibiotic complex of necessary staff.

Productivity of selected clones by antibioticaccutane are 20000-36000 units/ml.

Cooked seed material used for the production of the drug. What initially on the rocking grown seed material into the fallopian tubes in the medium of the following composition, %:

corn flour - 4,0

KH2PO4- 0,03

(NH4)2SO4- 0,06

NH4NO3- 0,7

NaCI - 0,2

MgSO4- 0,05

CaCO3- 0,5

water (water) up to 100

the pH of 6.6 to 7.2

After 24-36 hours of growth at a temperature of 281oC on a rocking chair with 200-220 rpm receive standard vegetative planting material.

This seed material in an amount of 5% sowing sowing machines on a pre-sterilized nutrient medium. Production time is 24-36 hours. Then the seed is passed in industrial fermenters in the amount of 5-10% on the pre-sterilized fermentation medium of the following composition, %: corn flour - 3-5; molasses-1,5-2,0; lysine-0,01 - 0,2; (NH4)2SO4or NH4NO3- 0,6 - 0,7; KH2PO4- 0,01 - 0,09; Pai temperature 281oC, air flow rate of 1.0-1.5 m3/m3min, thereby forming 20000-33000 units /ml antibiotic. The process of biosynthesis can be carried out in basesalary fermenters.

Next, the culture fluid is acidified to pH of 5.0-5.5, evaporated in a vacuum evaporator plant to 10-12% of dry substance, dried in the spray dryer with inlet temperature 140-160oC, output 60-70oC. Standardizes chalk or kaolin to obtain a drug containing 300000 unit/g, prepared the drug is Packed in Kraft paper bags of 10, 15, 20 kg of the loss of the antibiotic are 182 %.

Information confirming the possibility of carrying out the invention

Example 1. Prepared as described above seed strain Str. griseus-420 of the tubes on the sloped agar was perestal in the fallopian flask with sterile antifoam and a nutrient medium of the following composition, %:

corn flour - 4,0

KH2PO4- 0,03

(NH4)2SO4- 0,6

NH4NO3- 0,7

NaCI - 0,2

MgSO4- 0,05

CaCO3- 0,5

tap water up to 100

a pH of 6.8 to 7.2

After 24 hours growth at a temperature of 281oC on a rocking chair with 200-220 rpm received standard seed.

realizowany nutrient medium. Production time is 24 hours. Then the seeds sowed industrial fermenters in an amount of 5% on pre-sterilized fermentation medium of the following composition: corn flour - 3%, molasses - 1,5; lysine - 0,01; NH4NO3- 0,7; NaCI - 0,2; MgSO4- 0,05; CaCO3- 0,5; propenol of 0.1. The fermentation time is 50 hours at a temperature of 281oC, air flow rate of 1.0-1.5 m3/m3minutes the activity of the culture liquid was 30000 IU/ml Content of streptothricin With was 22%, streptothricin D - 52%. Next, the culture fluid was acidified with hydrochloric acid to pH of 5.0-5.5, was evaporated on a vacuum evaporator plant to 10% solids, dried on spray when the air temperature at the inlet 150oC, output - 65oC. was Standardized chalk, prepared the drug was Packed in Kraft paper bags of 20 kg Loss antibiotic amounted to 182%.

Example 2. Preparation of inoculum and technology biosynthesis was carried out as in example 1. The cultivation of seed amounted to 36 hours. 10% of seed planted fermentation medium of the following composition, %: corn flour -5,0; molasses - 2,0; lysine - 0,1; KH2PO4- 0,03; NaCI - 0,2; MgSO4- 0,05; CaCO3- 0,5; prod/ml The composition of the antibiotic fractions corresponded to the standard sample phytobacteriology.

Next, the culture fluid was acidified to pH 5.5, was evaporated on a vacuum evaporator plant to 12% of dry substance, dried in the spray dryer at an air temperature of 160oC at the inlet and the 70oC on exit. Was standardized kaolin, prepared the drug Packed in Kraft paper bags of 20 kg Loss antibiotic was 20%.

Example 3. Preparation of inoculum and biosynthesis was carried out as described in example 1. The cultivation of seed was 30 hours. 5% of seed planted fermentation medium of the following composition, %: corn flour - 4,0, molasses - 1,8, lysine - 0,05, KH2PO4- 0,03, NaCI - 0,2, MgSO4to 0.05, CaCO3- 0,5, propanol of 0.1. The fermentation time is 60 hours at a temperature of 281oC. the activity of the culture liquid 32000 units/ml On the component composition of the antibiotic consistent with phytobacterial.

Next, the culture fluid was acidified to pH 5.5, was evaporated on a vacuum evaporator plant up to 10% of dry substance, dried in the spray dryer with inlet temperature 140oC, output 60oC. has Standardized the kaolin, the finished preparation is badanych fermentation nutrient medium (than the one described in example 1) activity in the culture fluid is significantly reduced. When using more enriched fermentation media (than the one described in example 2) have a thick environment and activity of the culture liquid is also greatly reduced.

As seen from the above examples, using this method of obtaining vitulina for 6010 hours of fermentation to achieve activity in the culture fluid 30000 - 33000 units /ml or 10000 - 11000 μg/ml and reach the exit of antibiotic 8-9 kg/m3the culture fluid.

1. The method of producing drug for combating plant diseases containing as an active start complex streptothricin antibiotics, including the preparation of inoculum, the cultivation of a producer in a fermentation medium containing sources of nitrogen, carbon, and mineral salts, concentration and drying of the culture fluid and standardization of the drug, characterized in that as a producer using Streptomyces griseus strain 420, in the preparation of inoculum are the selection of clones with preferential formation of streptothricin C and D, and a growing producer lead to a fermentation medium containing 0.01 to 0.2% of lysine.

2. The method according to p. 1, characterized in that cultivation of the producer are on the enzyme is(NH4)2SO4or NH4NO3- 0,6 - 0,7

KN2RHO4- 0,01 - 0,09

NCl - 0,05 - 0,25

MgSO4- 0,005 - 0,05

Caso3- 0,5 - 1,5

Propanol - 0,1 - 0,5

Water - the Rest

3. The method according to p. 1, characterized in that the selection of clones in the preparation of inoculum are using thin-layer chromatography.

 

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