CS235785B1 - Method of specific microbic preparation production against mosquito larvae - Google Patents

Abstract

Method of producing specific microbial preparation against mosquito larvae and molluscs from the crystalline bacilli Baoillus thuringiensis serovar H-14 (israelensis) submersible method with constant agitation and aeration at 29 - 32 ° C by inoculation into sterile liquid soil containing assimilable carbohydrates and assimilable an organic nitrogen source, characterized by. that assimilable carbohydrates and assimilable nitrogen sources with growth factors and vitamins are used in proportion 1: 1-3 with 1 with oligobiogenic addition elements at a starting pH of 7.5, wherein the end of fermentation is on release spores and crystals, and these from fermentation soils are isolated at temperatures up to 80 ° C with the addition of a wetting agent and a preservative

Description

The present invention relates to the production of a novel bacchia preparation as a highly selective biological composition for controlling mosquito larvae and blood sucking larvae.

Fighting mosquitoes and flyflies as trappers and disease carriers is led by the destruction of their larvae in natural waters · Until now, only chemical insecticides have been used (chlorinated hydrocarbons, carbaaates, organophosphates) · Mosquito resistance to these substances has gradually developed and replaced with new substances and high toxicity to humans and animals · They caused long-term illness and irreparable damage · In wide terrestrial applications, these products had an impact on game and wildlife and caused a critical reduction in their number · Residues of these substances were introduced into the animal's food with plants and caused by uncontrolled relocation of toxic substances even outside the place of application · By transfer to running and ground water residues of chemical insecticides entered the drinking and service water supply and disrupted the health and environment of the population.

These serious consequences of the use of mosquito agents and, in particular, their failure in malaria epidemics have led to the search for naturally effective replacements which, with high selectivity, are not alien to the environment, create residues and do not harm other organisms ·

New strains of Bacillus thuringiensis serovar.H · -7 (aizawai), serovar H »13 (pakistani) and serovar EWL4 (isaelensis) were isolated during the search,

235 785 which are highly selectively effective for mosquito and flyworm larvae, disease carriers and harmless to other organisms in the same environment: plants, animals and humans · Monsters in the environment of residues because they are part of the diet of other organisms and are quickly removed from aquatic organisms environment and soil * Even large-area treatment does not harm other organisms ·

The method according to the invention allows the production of a specific bacterial preparation against mosquito and flyworm larvae from the Bacillus thuringiensis serovar. and in a short time of 32 - 70 hours ·

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to methods for producing an insectioid bacterial bacterial mosquito preparation from a crystalline bacillus of the species Bacillus thuringiensis serovar. Israelensis, Pakistan and aizawai by submersing under constant agitation and aeration at characterized in that the assimilable carbohydrates and the assimilable nitrogen source are used in a ratio of 1: 1 to 3: 1, containing growth factors and vitamins at an initial pH of up to 7 »5, wherein the end of the fermentation is conditioned by release of the insecticidal component. isolates and dried fermentation fluids with the addition of wetting agent and preservative ·

Bacillus thuringiensis serovar H-14 (israelensis), serovar.H-7 (aizawai) or H-13 (pakistani) is cultured aerobically under submersive conditions in sterile liquid medium under constant aeration and stirring. The cultivation time is 32 to 7 hours, the optimum temperature is 30-2 ° C, the initial pH can be between 6.5 and 7> 5 and the optimum at 7> 0 to 7.2 ·

During fermentation, the pH is maintained in this range by the addition of a buffer, e.g. KgHPO4 or CaCO4 or NaHCO4.

Various liquid media containing aimitable carbon and nitrogen can be used. The carbon source may be assimilable mono-di- or polysaccharides, e.g. o glucose

235 785 maltose, glyseol, starch. An assimilable nitrogen source is a protein material, preferably containing simultaneously vitamins and growth factors, eg yeast autolysate, yeast extract, dried yeast, peptone, soy flour, wheat flour, corn flour, defatted cotton germ flour, casein hydrolyzate, corn extract , stillage after the production of alcoholic beverages, and the like. It is preferred to add to the culture medium CGobiogenic elements such as magnesium, manganese, iron, zinc, calcium and sodium in the form of salts.

After the development and 90-95% of the bacillary culture had been sporulated while the insecticidal crystals were released at the same time, the effective spore biomass and inclusions were isolated and dried from the fermentation fluid. Lactose or sodium chloride ($ 5 to $ 30) and a wetting agent, such as Triton X 100, Tween 20, are mixed into the fermentation fluid prior to isolation.

Chevron, Words sol ap. ($ 5 to $ 10) · Spore material is isolated by centrifugation or filtration, or a combination of both. · Biomass fermentation fluid can be concentrated on a film evaporator or Laval centrifuge. , in a spray dryer, with acetone precipitation and subsequent drying · Spore and crystal material in the form of dry powder retains its insecticidal properties for many years if stored in a dry and cool place ·

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

Example 1.

The nutrient medium on the shaker, in the pre-booster tank, and on the nt and front tank has the following composition: 1.3% soy flour, $ 1.4 maize starch, 0.03% 0.002 $ FeSO 4 THgO,

$ 0.002 ZnSO ₄ 7H ₂ $ 0.0002 C ^ Cl ^ and $ 0.1 NaHCOy Banks

233 785 per 500 ml shaker is filled with 50 ml of soil and inoculated for 20 hours with a culture of a production strain grown on slant agar of composition: $ 2.0 agar, starch 2%, peptone $ 0.75, yeast autolysate $ 2, K ₂ HP0 ₄ 0.68 $, oligobiogenic elements 0.1 $ base solution (H 2 SO 4 17.4 g, MgSO ₄ · 7H ₂ 0 12.3 g, MnSO ₄ H ₂ O 0.22 g, FeS ° ₄ .7H ₂ 0 2.0 g, ZnSO ₄ .7H ₂ 0 1.44 g, CaCl ₂ * 6H ₂ 0 18 ^ 3 g, H ₂ 0 1000 ml) Inoculate 1 to 3 volume with a 12 hour shaker culture, the fermenter is inoculated with 2-10% volume with a 20 hour old culture from a pre-seed tank. Cultivation in the fermentation tank is carried out at 28-32 ° C under aeration with 1/2 volume of air and stirring at 300 rpm · 32-64 hours until spores and crystals are released from the $ 95 culture cells. of the B. thuringiensis biomass tank mixed with lactose (5-10% calculated on dry matter) and Slovesol (10-15 $), stirred for 30 min · while cooling, and then the entire volume was dried on a spray dryer (input 150-200 ° C) , output max ·

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

Example 2.

The broth for fermentation tank and fermenter consists of 1.5 $ wheat flour, 1.0 $ glucose, 0.5 $ yeast extract, 0.1% HHgPO 4 I 4 O, 0.05 $ MgSO ₄ .7H ₂ 0,

0.3 $ NaCl, 0.01 $ Pe ₂ (SO ₄ ) ^ · pH adjusted to 7.0 - 7.2 · The inoculation tank is inoculated with 1-3 $ of vegetative inoculum of a 12 h old production strain grown on a rotary shaker machines on soil containing $ 1 starch,

0.79 $ peptone, 1.5 $ yeast autolysate, 0.68 K ₂ HPO ₄ and 0.1 $ oligobiogenic elements (see Example 1) and cultures ee at 29-30 ° C under aeration with 1/2 volume of air and agitation 200 rpm 20 hrs. This inoculum seeded a fermentor with the same soil. Cultivation in the fermenter is carried out at 30 ° C under aeration 1/2 volume and stirring 300 rpm · up to 64 hrs. $ 95 culture ·

235 785

5-106 NaCl and Syntapon IQ% are added to the spores and crystals in the fermentation fluid, concentrated on a film evaporator and dried in a spray dryer as in Example 1.

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

Example 3 ·

The culture medium in the pre-seed 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 MgSO ₄ .7H ₂ 0, 0.3% NaCl, 0.01%

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

0.5% KgHPO4 and 0.1% oligobiogenic elements (see Example 1) ·

Inoculation and fermentation procedures are the same as in Example 1 ·

The spore and crystal biomass in the fermentation fluid is cooled to about 20 ° C and concentrated about 6 times on an Alfa Laval separator. Lactose (5-15% on dry weight) and Triton X 100 (5-1056) are added to the concentrate, stirred for about 30 minutes. and precipitated with 2 volumes of acetone. The precipitate was collected by filtration and dried in a stream of air. The activity is determined by a bioassay on mosquito larvae and compared with that of an international standard · Example of the effectiveness of a bacterial preparation on L3 mosquito larvae Culex pipiens autogenicus

conc. prep. 24 hours 48 throw 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,