CS235785B1 - A method for producing a specific microbial preparation against mosquito and larvae larvae - Google Patents

Abstract

Method of producing a specific microbial preparation against mosquito and midge larvae from the crystallophorous bacillus Bacillus thuringiensis serovar H-14 (israelensis) by submergence method with constant stirring and aeration at a temperature of 29 - 32 °C by inoculation into sterile liquid soil containing assimilable carbohydrates and an assimilable organic nitrogen source, characterized in that assimilable carbohydrates and an assimilable nitrogen source containing growth factors and vitamins are used in a ratio of 1:1 to 3:1 with the addition of oligobiogenic elements at an initial pH of up to 7.5, with the termination of fermentation being when spores and crystals are released and these are isolated from the fermentation soil at temperatures up to 80 °C with the addition of a wetting agent and a protective agent

Description

The present invention relates to the production of a new bacterial preparation as a highly selective biological preparation for the control of mosquito larvae and blood-sucking midges.

The fight against mosquitoes and midges as tormentors and carriers of diseases is conducted by destroying their larvae in natural waters. Until now, only chemical insecticides (chlorinated hydrocarbons, carbonates, organophosphates) have been used for this purpose. Mosquitoes gradually developed resistance to these substances and were replaced by new substances, often with high toxicity for humans and animals. They caused long-term diseases and irreparable damage in them. When widely used in terrariums, these preparations affected game and animals in the wild and caused a critical reduction in their numbers. The residues of these substances got into the food of animals with plants and caused uncontrolled movement of toxic substances even beyond the application site. By passing into running and groundwater, the remains of chemical insecticides got into drinking and utility water supplies and disrupted the health and environment of the population.

These serious consequences of the use of chemical substances against mosquitoes, and especially their failure in malaria epidemics, led to the search for natural effective substitutes that would be highly selective, not foreign to the environment, would not create residues and would not harm other organisms.

During the search, new strains of the bacteria Bacillus thuringiensis serovar.H·-7 (aizawai), serovar H»13 (pakistani) and serovar EWL4 (isřaelensis) were isolated,

235 785 which are highly selectively effective against mosquito and midge larvae, vectors of diseases, and harmless to other organisms in the same environment: plants, animals and humans. Monsters in the environment leave residues because they are part of the food of other organisms and are removed by them in a short time from the aquatic environment and from the soil. Even continuous large-scale treatment does not harm other organisms.

The method according to the invention enables the industrial production of a specific bacterial preparation against mosquito and midge larvae from the crystallophorous bacillus Bacillus thuringiensis serovar.H-14 (israelensis), H-13 (pakistani), H-7 (aizawai) in an economical manner on a large scale using modern equipment and in a short time of 32 - 70 hours.

The invention relates to methods for producing an insecticidal bacterial preparation against mosquitoes from the crystal-forming bacillus of the species Bacillus thuringiensis serovar· israelensis, pakistani and aizawai by the submersible method with constant stirring and aeration at a temperature of 29 to 32°C by inoculation into sterile liquid soil containing assimilable carbohydrates and an assimilable organic nitrogen source, characterized in that assimilable carbohydrates and assimilable nitrogen source are used in a ratio of 1:1 to 3:1, with the content of growth factors and vitamins at an initial pH of up to 7.5 >, whereby the termination of fermentation is conditioned by the release of the insecticidal component, which is further isolated from the fermentation liquid and dried with the addition of a wetting agent and a preservative.

Bacillus thuringiensis serovar H-14 (israelensis), serovar H-7 (aizawai) or H-13 (pakistani), is cultivated aerobically in submerged conditions in sterile liquid medium with constant aeration and stirring. The cultivation time is 32 to 76 hours, the optimum temperature is 30 - 20°C, the initial pH can range between 6.5 and 7.5 and the optimum is 7.0 to 7.2.

During fermentation, the pH is maintained within this range by adding a buffer, e.g. KgHPO^ or CaCO^ or NaHCO^.

Various liquid media containing assimilable carbon and nitrogen can be used. The carbon source can be assimilable mono-, di- or polysaccharides such as glucose.

235 785 maltose, glycerol, starch. An assimilable source of nitrogen is protein material, preferably containing vitamins and growth factors, e.g. yeast autolysate, yeast extract, dried yeast, peptone, soy flour, wheat flour, corn flour, flour from defatted cotton * germs, casein hydrolysate, corn extract, distillates from the production of alcoholic beverages, etc. It is advantageous to add to the culture medium ligobiogenic elements such as magnesium, manganese, iron, zinc, calcium and sodium in the form of salts.

After the development is complete and the bacillus culture is 90-95% sporulated, with the simultaneous release of insecticidal crystals, the effective biomass of spores and inclusions is isolated from the fermentation liquid and dried. Before isolation, lactose or sodium chloride (5 to 30%) and a wetting agent, e.g. Triton X 100, Tween 20, are mixed into the fermentation liquid with the biomass.

Chevron, Slova sol, etc. ($5 to $10) · Isolation of spore material is carried out by centrifugation or filtration or a combination of both. · The fermentation liquid with biomass can be concentrated on a film evaporator or a Laval centrifuge. · Drying can be carried out in several ways: by hot air flow, lyophilization, in a vacuum dryer, in a spray dryer, precipitation with acetone and subsequent drying. · Spore and crystal material in the form of a dry powder retains its insecticidal properties for many years if stored in a dry and cool place.

The activity of the product is determined by a biological test on Culax pipiens tutogenicus or Aedes aegypti mosquito larvae and by comparison with the international IPS standard (value 1000 International Units). The activity is expressed in International Biological Units. The biological test is evaluated after 48 hours.

Example 1.

The nutrient medium on the shaker, in the pre-inoculation tank and the farm tank has the following composition: 1.3% soybean meal, 1.4% corn starch, 0.03% 0.002% FeSO^THgO,

$0.002 ZnS0 ₄ »7H ₂ $0.0.002 C^Cl^ and $0.1 NaHCOy Banks

233 785 on a 500 ml shaker is filled with 50 ml of soil and inoculated for 20 hours with a culture of the production strain grown on a slant agar with the following composition: agar 2.0%, starch 2%, peptone 0.75%, yeast autolysate 2%, K ₂ HP0 ₄ 0.68%, oligobiogenic elements 0.1% of the basic solution (H^SO^ 17.4 g, MgS0 ₄ «7H ₂ 0 12.3 g, MnSO^I^O 0.22 g, FeS° ₄ .7H ₂ 0 2.0 g, ZnS0 ₄ .7H ₂ 0 1.44 g, CaCl ₂ *6H ₂ 0 18^3 g, H ₂ 0 1000 ml) Inoculated with a culture from 1 slant agar 3 flasks each The pre-inoculation tank is inoculated with 1 to 3% of the volume of the 12-hour culture from the shaker, the fermenter is inoculated with 2-10% of the volume of the 20-hour culture from the pre-inoculation tank. Cultivation in the fermentation tank takes place at 28 to 32*C with aeration of 1/2 the volume of air and stirring at 300 rpm 32-64 hours, until spores and crystals are released from 95% of the culture cells After fermentation is complete, lactose (5 to 10% calculated on dry matter) and Slovesol (10-15%) are mixed into the liquid in the tank with B. thuringiensis biomass, stirred for 30 min while cooling and then the entire volume is dried in a spray dryer (inlet 150 - 200*C, outlet max.

80*C) The activity of the dry matter is determined by testing on Culex pipiens autogenicus larvae and the number of units of activity of the preparation is determined.

Example 2.

The nutrient medium for the pre-inoculation tank and fermenter consists of 1.5% wheat flour, 1.0% glucose, 0.5% yeast extract, 0.1% HHgPO^I^O, 0.05% MgS0 ₄ .7H ₂ 0,

0.3% NaCl, 0.01% Pe ₂ (S0 ₄ )^· pH adjusted to 7.0 - 7.2· The pre-inoculation tank is inoculated with 1-3% vegetative inoculum of a 12-hour-old production strain, grown on a rotary shaker on medium containing 1% starch,

0.79% peptone, 1.5% yeast autolysate, _0.68 % _K2HPO4 and 0.1% oligobiogenic elements (see example 1) and cultured at 29-30°C with aeration of 1/2 volume of air and stirring at 200 rpm for 20 hours. The fermenter is inoculated with this inoculum using the same medium. Cultivation in the fermenter is carried out at 30°C with aeration of 1/2 volume and stirring at 300 rpm for up to 64 hours, until spores and crystals are released in 95% of the culture.

235,785

To the biomass of spores and crystals in the fermentation liquid, 5-106 NaCl and the wetting agent Syntapon IQ% are added, concentrated on a film evaporator and dried in a spray dryer as in Example 1.

The number of activity units in the obtained preparation is determined by a bioassay on Aedes cantans mosquito larvae.

Example 3·

The nutrient medium in the pre-inoculation and fermentation tank has a composition of 0.75% peptone, 0.5% yeast extract, 1.2% glucose,

0.5% KH ₂ P0., 0.05 36 MgS0 ₄ .7H ₂ 0, 0.3% NaCl, 0.01%

Fe ₂ (S0 ₄ )j· The nutrient medium in flasks on a shaker has the composition of 1*0 56 glucose, 1.0% yeast autolysate, 0.75% peptone,

0.5% KgHPO^ and 0.1% oligobiogenic elements (see example 1)

The inoculation and fermentation procedures are the same as in Example 1.

The biomass of spores and crystals in the fermentation broth is cooled to about 20*C and concentrated about 6 times on an Alfa Laval type separator. Lactose (5 to 15% on dry matter) and the wetting agent Triton X 100 (5 to 10 56) are added to the concentrate, stirred for about 30 min. and precipitated with 2 volumes of acetone. The precipitate is separated by filtration and dried in a stream of air. The activity is determined by a bioassay on mosquito larvae and compared with the activity of an international standard. Example of the effectiveness of a bacterial preparation on L3 larvae of the mosquito Culex pipiens autogenicus mortality

conc. prep. 24 hours 48 hour 0.3 mg/liter 100 56 0.2 mg/liter 90 56 100% 0.1 mg/liter 10 56 60 56 0.05 mg/liter 10 56 50 % 0.025 mg/liter 0 56 10 56 control mm mm

Claims (2)

Method for producing a specific micnobic preparation against mosquito and flyworm larvae from the cryatalophore bacillus Baeilue thuringiensis eerovar H-14 (israelensis), H-13 (pakistani) and H-7 (aizewai) by the Aubus method under constant agitation and aeration at 29 ° 32 ° C by inoculation into a sterile liquid soil containing assimilable carbohydrates and an assimilable organic nitrogen source, characterized by an assimilable carbohydrate and an amiable source Nitrogen containing growth factors and vitamins is used in a ratio of 1: 1 to 3: 1 with the addition of oligobiogenic elements at an initial pH of up to 7 »5, with the end of fermentation releasing spores and crystals, these are isolated from the fermentation broth. and preservatives are dried at temperatures up to 80 ° C · Method according to claim 1, characterized in that lactose or sodium chloride is used as the protective substance. Printed by Moravian Printing Works,