HUT65725A - Process for the production of compositions containing biosynthetic pesticidal products and use of the compositions obtained - Google Patents

Description

The present invention relates to a process for the preparation of biosynthetic pesticidal products obtained from microorganisms of the Bacillaceae family and to the use of the obtained compositions.

- 2 A process for the recovery of biosynthetic pesticide products from micro-organisms generally consists of the following main steps:

- fermentation of the micro-organism for culture,

recovering this culture, and

- finishing the product in the form of pulp, powder, granules or cream.

Culture isolation is a delicate step in this procedure. This step, which essentially consists in separating the culture, in the form of biomass, and the fermentation broth, is one of the reasons for product loss and generally low yields.

In addition, the active ingredient of the pesticide product is a sensitive compound due to its biological structure, which is easily denatured when certain conditions (such as, in particular, temperature, pH, solvents and additives) are neglected.

The low yields observed during harvesting of the culture can be attributed in particular to the following factors:

- difficult separation of many components of a more or less viscous culture,

- mechanical effects during separation, leading to degradation of the active substance in the pesticide product and thus a reduction in biological activity,

- addition of adjuvants that affect the stability and / or physical appearance of the formulation (such as the viscosity of the pulp), the segregation of the pulp or ointment during storage and the dispersion of the formulation in water. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 3 sleepers.

In addition, a marked decrease in biological activity can be observed when the formulation is in the form of a paste. This form of finishing is advantageous in some cases because it avoids the drying step (which causes less damage to the product and which is expensive but is required for longer preservation).

In fact, when calculating the biological activity, there is actually a difference of 3050% between the biological activity of the culture and the pulp, while the difference between the powder obtained by drying the pulp and the biological activity of the culture is smaller. Therefore, it is believed that the biological activity of the pulp is not fully available.

SUMMARY OF THE INVENTION The present invention is directed to formulations which are free of the above drawbacks. More specifically, the present invention relates to a process for the preparation of formulations with maximum biological activity in an economical manner, i.e., to avoid yield and loss of biological activity at all levels (both quantitative and qualitative) while providing a stable and appropriate physical appearance. It has also been observed that the compositions of the present invention have extremely high biological activity.

To this end, the process of the present invention for preparing compositions comprising biosynthetic pesticidal products derived from microorganisms of the Bacillaceae family and tannin or a tannin derivative comprises the following steps:

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i) culturing the microorganism in culture medium to obtain a culture containing a large amount of microorganisms / 1. step ii) adding tannin or a tannin derivative to the resulting culture (step 2). step iii) removing water from a culture containing tannin or a tannin derivative to form a slurry (step 3). and / iv) preparing the pulp thus obtained / step 4. step/.

According to a preferred method of the invention, tannic acid is added to the culture in a second step.

Biosynthetic pesticides from the Bacillaceae family can exist in very different forms in concentrated formulations. Some of these forms are:

- the organisms themselves, at some stage in their development, including possible vegetative forms, in their original form together with the culture medium; fully or partially lysed form; wholly or partially sporulated; a shape from which the spores in some way, e.g. bacterial autolysis, completely or partially liberated, or a product spontaneously selected by organisms / e.g. exotoxins /; a product extracted from such organisms by any known means, including lysis of said organism, e.g. endotoxins; products that can be selected at certain stages of the organism's development (optionally by spores-mixed crystals or endotoxins) or simultaneously several of these different forms.

These various forms may be mixed with the residues from the fermentation broth. Good results have been achieved with mixtures containing spores, crystals or endotoxins and possibly exotoxins spontaneously formed during bacterial autolysis of the bacterium. Such a mixture may contain, in addition to spores, any crystals which may be associated therewith and possible exotoxins, cells or cellular debris and residual solid components of the culture medium used for culture.

Bacillus pesticides belonging to the Baclllaceae family are generally very suitable sources of non-Bacillus microorganisms such as Bacillus. thüringiensis, Bac. sphaericus, Bac. popilliae, Bac. cereus, Bac. larvae, Bac. lentimorbus, Bac. friborgiensis and Bac. penetrans / Pasteuria /.

Good results were achieved for all Bac serotypes. sphaericus and Bac, Thuringiensis, including bacteria resulting from genetic transformation, in particular Bac, serotypes la, 2a2b, 2a2c, 5a5b, 6, 25, 26a26b, 26a26c. sphaericus and Bac., serotypes 1, 3a, 3a3b, 4, 6, 7, 8a8b, 9, 10 and 14. thuringiensis-winds.

The best results were obtained with:

- Bac. sphaericus 5a5b and 25 serotypes and

- Bac. Thuringia serotype 1 / Thuringia variety /, Sera 3a3b / Kurstaki /, Serotype 6 / Subtoxicus and Entomocidus /, Serotype 7 / Aizawai variety /, Serotype 8a8b / Morrison species / and 14 serotypes / Israeliensis • · · «··· ·· • · «· · · · · · · · · · · · · · · · · · · · · · · · · · ·

The different Bac. Thuringia and Bac. sphaericus species may be present directly in the compositions of the invention in the form of microorganisms, in their original or lysed form, as spores, endotoxins or as crystals optionally associated with spores, or more. These different shapes can be mixed with media residues. Good results have been achieved using mixtures containing spores and crystals (crystals are formed spontaneously during bacterial autolysis in the final stage of sporulation). Such mixtures may contain, in addition to spores and optionally spore-associated crystals, cells or cellular debris as well as residual solid components of the culture medium used for culture.

Formulations of the compositions of the present invention are characterized in that the biosynthetic pesticidal products are suspended in solid form in a liquid or in solid form, e.g. in the form of powder or granules.

In addition, the compositions of the present invention may contain various other additives, the quality and function of which are dependent on the use of the compositions and are known to those skilled in the art.

The compositions of the present invention may also contain residues from the preparation of the biosynthetic pesticide.

When the compositions of the present invention are present as a substance suspended in a liquid, these suspensions will generally be in the form of suspensions. · ···

7b contain 10-45% by weight and most often 15-40% by weight of a biosynthetic pesticide product. The liquid must be inert to the biosynthetic pesticide: in particular, it must not inactivate the pesticide or cause precipitation or any other physical alteration. The liquid may be a single solvent or a mixture of solvents. The solvent usually contains water.

Good results were obtained when the solvent contained at least 80% by weight and most preferably at least 95% by weight of water. The liquid present in the composition of the invention is generally substantially water.

In the second step, the required amount of tannin or tannin derivatives is generally 0.05 to 4% by weight of the culture; this value is usually from 0.1 to 3% and preferably from 0.25 to 2%.

The third step, which consists of dewatering the culture, is carried out by conventional methods for removing water, such as centrifugation, ultrafiltration, precipitation, a non-solvent / e.g. acetone / addition, filtration or precipitation; this gives a pulp. The preferred method is centrifugation and / or ultrafiltration.

Possible additives are then added to this paste during preparation.

Bac. Thuringia and Bac. in the case of sphaericus, culturing e.g. at the end of the sporulation phase.

Another method is to isolate the biosynthetic pesticide-producing microorganisms from the fermentation broth at the end of the sporulation phase in the endotoxin or in the spores. · ··· • · · · · · · · · · · · · · · · · ···

- fractions rich in 8 or both of these products. These fractions are hereinafter treated by the same methods as mentioned for the treatment of pulps.

For preparing the compositions of the invention in the form of a dry powder, the pulp obtained above is obtained in the usual manner, e.g. spray drying, freeze drying or tumble drying / drying to produce solid particles which generally contain less than 12% water, and preferably less than 7% water.

Another method consists of adding the possible additives to the pulp before drying and then performing the drying using one of the methods mentioned.

For preparing the compositions of the invention in suspension, the solid pesticidal particles may be dispersed in a liquid phase, preferably containing water, tannin or tannin derivatives, and other possible additives, to form a cream-like substance.

The method of applying the compositions is also an object of the present invention.

The compositions of the invention are useful as pesticides, and more particularly as agents for killing insects at any stage of development. To this end, the compositions are sprayed onto the infected surfaces, plants, rivers, streams and any water surface. Any known method can be used for this purpose, such as manual spraying, mechanical spraying and, in particular, air spraying. Spraying, especially by insects, in particular Lepidopter, Dipter, Coleopter, Aphanipter, Orthopter, Isopter and Homopter target surfaces.

The dose to be used will depend on the properties of the biosynthetic pesticide used and the target organism to be killed.

Bac. Thuringia and Bac. In the case of sphaericus, generally 50 to 5000 g of solid particles / ha are sprayed. The compositions of the present invention, when in suspension form, can be sprayed directly. However, they may be dispersed, diluted or emulsified with a diluent, e.g. water or an organic diluent. In this case, the dilution is 1: 300, usually 2 to 120, and preferably 4 to 60 v / v. diluent.

The following examples serve to illustrate the invention.

Example 1

450 g soy flour / protein content approx. 52% /, 250 g hydrolyzed starch, 20 g KH ₂ PO ₄ , 20 g K ₂ HPO ₄ , 3.2 g MgSO ₄ .7H ₂ O, 0.12 g MnSO ₄ .H ₂ O, 0. , 14 g of ZnSO ₄ ~, 0.2 g of Fe ₂ / SO _4/3 -t, 1.5 g of CaCl ₂ O ₂ .h and a stirrer was charged with 10 liters of water, pH pipe oxygen tube and mounted levegőztetőrendszerrel , can be sterilized in a 15 liter BIOLAFFITE fermenter.

This medium is mixed by homogenization followed by pH

Set to 7.

The medium is sterilized for 30 minutes at 120 ° C.

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- 10 Media at 30 ° C. cooled and sterile inoculated with 1 liter of Bac. thuringia 14 serotypes / israeliensis cultures / cultures.

The fermenter was stirred at 450 rpm and

Aeration of 0.4 volumes of air per volume of fermentation broth.

hours after the Bac. Thuringia is present as a partially lysed spore bacterium. This culture contains 1.1 x 10 ⁹ spores / ml spore, pH 7.5 and dry matter content 3 g / liter.

The insecticidal activity of the culture was determined using Aedes aegypti, H. d Barjac-I. Larget Biological Titration Method (WHO-VBC-79.774). This value is 300 units / ml ± 20%.

To this product was added 2,000 parts of powdered citric acid.

The pH of the mixture was adjusted to 4 by addition of phosphoric acid.

To 500 g of the product thus obtained is added 5 g of tannin (TANIN PB5 ^R , PHARMACHEMIC, 99% tannic acid content).

The insecticidal activity of the product obtained is within the limits of the measurement error. The added acids and tannin are thus non-toxic to the target insects.

The product is concentrated by centrifugation. thus approx. A pulp containing 12% solids was obtained.

This paste has an insecticidal activity of 2800 units / mg ± 20%.

This paste is liquid and stable over time.

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- 11 2R / comparator / example

In Example 1, Bac. Thuringia serotype 14 cultures were prepared. In the same manner as in Example 1 except that no tannin was added to the pulp.

The resulting pulp had an insecticidal activity of 1500 units / mg ± 20%.

A comparison of the results of Example 1 and Comparative Example 2R 'of the process of the invention shows that the addition of tannic acid to the culture prior to centrifugation facilitates separation, improves the yield of the centrifugation, and renders the active ingredient content of the product more accessible.

3R / comparator / example

As described in Example 1, Bac. Thuringia cultures of 14 serotypes were prepared. Next, a slurry was prepared as described in Example 1 except that tannin was replaced with 0.1% silica gel (AEROSIL R ^R , DEGUSSA).

The resulting pulp had an insecticidal activity of 1300 units / mg ± 20%. The pulp is compact and non-dispersible in water.

Claims (5)

A process for the preparation of a composition comprising a biosynthetic pesticide product from a microorganism of the Bacillaceae family and a tannin or a tannin derivative comprising: - culturing the microorganism in culture medium to produce abundant cultures, - adding tannin or a tannin derivative to the resulting culture, - removing the water from the culture containing the tannin or tannin derivative to form a pulp, and - forming the pulp thus obtained. 2. The method of claim 1, wherein tannic acid is added. Process according to any one of the preceding claims, characterized in that Bac. Thuringia or Bac. sphaericus-type microorganisms were cultured. Method according to any one of the preceding claims, characterized in that from 0.005 to 4% by weight of the culture, tannin or tannin derivative is added to the culture. 5. A method of controlling insects, wherein the method of any one of claims 1-4. The composition of any one of claims 1 to 4 is sprayed onto the infected surface.