WO1991001803A1

WO1991001803A1 - Multilayer granules containing coated active substances, production method, device for implementing such method and utilization of the granules obtained

Info

Publication number: WO1991001803A1
Application number: PCT/FR1990/000604
Authority: WO
Inventors: Bernard Digat
Original assignee: Institut National De La Recherche Agronomique (Inra)
Priority date: 1989-08-09
Filing date: 1990-08-08
Publication date: 1991-02-21
Also published as: FR2650758A1; EP0437598A1; FR2650758B1

Abstract

The granules (1) are each comprised of a coated core (2), and are characterized in that the coating is comprised of two layers (3, 4), the layer closer to the core (first layer) not providing by itself around the core (2) a unique layer, autonomous and/or capable of keeping its properties, and the layer situated immediately after (second layer) being a protection layer. As an example, the core (2) is comprised of a natural or artificial seed, and a first layer is comprised of beneficial microorganisms such as bacteria and fungi. The granules may be used particularly for the introduction in substrates and soils, particularly for regenerating them, of granules containing bacteria and/or fungi which are beneficial for plants, to provide microbiological fertilizers for plants, to obtain natural or artificial seed coated by bacteria (bacterisation) and/or beneficial fungi (fungisation), or coated by substances which stimulate or delay germination.

Description

MULTI-LAYERED GRANULES CONTAINING ACTIVE COATED SUBSTANCES, METHOD FOR THE PRODUCTION THEREOF, DEVICE FOR CARRYING OUT SAID METHOD, AND USE OF GRANULES

OBTAINED

The present invention relates to multilayer granules containing coated active substances, to a process for manufacturing them, to a device for carrying out this process, and to the use of the granules obtained.

The present invention allows the inclusion of various molecules (metabolites, enzymes, antibodies, polymers, viruses), living matter (plant embryos, nematodes) or individual cells living or killed (bacteria, fungi, plant or animal cells) or specifically natural or artificial seeds in the form of granules with a multilayer structure. The invention relates to a multilayer encapsulation process, as well as a process for coating inert or living material ensuring complete protection thereof.

The core of the granules is constituted by a hard or liquid or liquid material, and it is surrounded by two or more envelopes, of thicknesses ranging for example from 0.1 to 6 millimeters depending on the nature of the material that is used. wants to encapsulate.

These granules find their applications in the fields of agriculture (microbiological fertilizers, granules with phytostimulating and phytoprotective properties, multienrobée seeds, artificial seeds), of breeding (reconstitution of microflora,

RΞMFIACEMEN SHEET encapsulation of hormones, adjuvants in animal feed), pharmacy (inclusions with polyvalent enzymatic and hormonal functions, inclusions of antigens and antibodies), agri-food (temporary protection of biodegradable substances), conservation fragile elements which cannot undergo freezing but only relative dehydration or, on the contrary, in granular form in freezing. In French patent applications no.

77-10 254, 79-28 956, 81-04 474, 81-24 403, 83-02 847, 86-08 842, various methods of direct inclusion of microorganisms have been described for the purpose of constituting inocula microbials in polymers.

In French patent application n ° 87-06667, the applicant claimed the design and the process of double encapsu ation or of indirect inclusion concerning the preparation of inocula made up of microorganisms coated in a nutritive matrix itself surrounded an envelope and a film in the form of biodegradable granules. In this process:

1) the microorganisms are cultivated in a feed matrix composed of a complete nutritive extract and coated with a hydroretenting agent,

2) then this matrix is encapsulated in a porous envelope containing another hydroretenting agent,

3) which envelope can finally be protected by hydrophilic coating.

However, none of these methods envisages the simultaneous inclusion in multilayers characterized in that it allows the obtaining of a

REPLACEMENT SHEET spherical or para-spherical structure in which:

(a) the center or heart is constituted by a living or inert or chemical matter;

(b) the layers enveloping the core are successive, of identical or advantageously different nature;

(vs) . a film-forming agent finally isolates the product;

(d) the final product thus obtained according to the layers (a), (b) and (c) can again undergo one or more encapsulations (which increases its volume accordingly).

A seed could not be encapsulated in a bacterization or mycorrhization medium by a process as described in French patent application No. 87-06 667. Indeed, this would require an intermediate stage of drying the layer of the medium. bacterization or mycorrhization surrounding the seed, such a step leading to the destruction of at least part of the microorganisms.

The method according to the present invention takes advantage of the fact that the immobilized central material can be protected from the effects of the external environment by several layers of different nature, and sufficiently protective or adjuvant or synergistic so that the target is reached more easily and more effectively. The method according to the present invention also makes it possible to conserve the moisture of the core or of an internal layer, which is often desired in order to preserve their properties. Thus, in the case of the bacterization of the seeds, it is possible to retain approximately 15% of the residual moisture e of the layer containing the bacterial inoculum by virtue of the coating by encapsulation for several weeks. The present invention therefore firstly has for

REPLACEMENT SHEET subject of the granules each consisting of a core comprising coated materials of biological origin, characterized in that the coating comprises two layers, the one which is closest to the core (first layer) not providing by itself around the core a single layer, autonomous and / or capable of retaining its properties, and the one located immediately after (second layer) being a protective layer. In accordance with a first embodiment, the core of the granule is constituted by a hard and relatively smooth element, and the first layer, by a material which is not self-supporting and / or sensitive to external agents. In this case, the core 5 preferably has a general spherical or paraspherical shape or else has an i / D ratio close to 1.

In particular, the nucleus may consist of a natural or artificial seed, of spherical or paraspheric form, which may be a pre-coated seed. However, the seeds may have a non-spherical or non-spherical shape (lettuce seeds), and then comprise a pre-coating giving them a shape approaching that of the spherical shape.

The nucleus can also be constituted by granules of various natures such as plant embryos or by plant ca s associated in a spherical or paraspheric mass. _Q According to another embodiment, the core consists of a soft, semi-liquid or liquid material, for example an oily substance. The core can also consist of a polymeric substance. 5 According to another embodiment, the

REPLACEMENT SHEET core and / or the first layer can constitute an inclusion structure. ^". at least one active substance, chosen in particular from microorganisms, bacteria, such as Rhizobacteria, and fungi, such as Mycorrhizae, Fusariums and fungi antagonists of pathogenic agents such as uric, living cells of animal origin or plant and cell assemblies constituting functional organisms and individuals, and entomophagous nematodes and their larvae, metabolites, enzymes, viruses, antigens, antibodies and hormones.

In the case where the core and / or the first layer constitute a. inclusion structure of at least one active substance chosen from microorganisms, living cells, cell groups, nematodes or their larvae, said active substance is advantageously coated in an agent consisting of at least one nutritive substance, in particular by a complete nutritive extract, of plant or animal origin, to which is added, if necessary, a hydroretenting agent.

The microorganisms, cells or cell assemblies are brought into the nucleus preferably in the form of a more or less concentrated suspension. The feeder matrix can constitute a culture medium for said microorganisms or others before the formation of the granules. The complete nutritive extract can be an extract of plant origin based on algae, which are chosen in particular from the Fucus, La inaria and Ascophyllum species, said sterile extract being brought to pH 7, for example with 1 magnesium oxide.

According to another embodiment of the present invention, the core consists of

REPLACEMENT SHEET an inclusion structure of at least one active substance comprising:

(a) a core consisting of a matrix coating the active substance, said matrix being composed (1) of at least one coating agent in which said active substance is coated; and

(2) at least one hydroretenting agent; and

(b) a porous protective envelope surrounding said core (a) and composed:, (1) of at least one film-forming agent capable of undergoing biological degradation; and (2) at least one hydroretenting agent. Such an inclusion structure is described in PCT international application No. 88-00239 of May 11, 1988, the content of which is incorporated by reference in the present description.

The hydroretenting agent which can enter into the composition of the granules according to the invention is in particular a clay, such as bentonite, kaolin, talc, vermiculite and montmorillonite.

In the granules according to the invention, the second layer comprises or is composed of at least one film-forming agent. This is chosen in particular from alginates, polypectates, chitosan, carrageenans and other biopolymers.

In the case in particular where the granules according to the invention constitute an inoculum or a coated seed, the film-forming agent is associated with at least one hydroretenting agent, as defined above, and, if necessary, at least one phytostimulating substance. and / or at least one phytosanitary substance compatible with microorganisms, living cells and cell assemblies present in the nucleus and / or the first layer. The phytostimu ante substances are chosen in particular from nitrates, growth substances, various mineral salts, etc. By way of example, mention may be made of indol acetic acid and its analogues, Cytokinin, aluminum phosethyl (aluminum-tris-O-ethyl phosphonate). Phytosanitary substances are chosen from fungicides, insecticides, molluscicides, and herbicides compatible with microorganisms, living cells or coated cell groups. The granules according to the invention can also comprise a hydrophilic protective film surrounding the second layer, and constituted in particular by a very hydrophilic clay, such as bentonite.

In accordance with another embodiment of the invention, the granules were again subjected at least once to the double coating creating first and second layers of a nature similar to, or different from, the first and second layers as defined above . The granules according to the invention can be in the form of paraspherical elements whose average diameter of the core is between approximately 3 and 10 mm, and whose average overall diameter is between approximately 4 and 12 mm. Examples of granules according to the invention are as follows: the nucleus is constituted by a seed, and the first layer by beneficial microorganisms such as bacteria and fungi; - the nucleus is made up of a seed, and the two coating layers are made up of beneficial microorganisms, such as bacteria and fungi, which are different from one layer to another; the nucleus consists of a seed of legumes, such as soybeans, peas and beans, and the first layer is a layer of bacterization by Rhizobium and / or Pseudomonas, or any other genus and species of beneficial microorganisms;

- the nucleus consists of a seed, and the first layer is a mycorrhization layer containing the mychorizal fungus Paxil us involutus;

- the nucleus consists of a tomato seed, and the first layer consists of a medium containing propagules of the fungus Fusarium g oxysporum (non-pathogenic strain) at a rate of 10 propagules / ml of medium, and / or nematode larvae entomophagous Neoplectana carpocapsae, the active substance in the nucleus is Bradyrhizobium japonicum, and the first layer consists of a medium containing Pseudomonas f uorescens-putida, the resulting granules constituting inoculu s making it possible to stimulate the nodulation of legumes and soybeans in particular. The present invention also relates to a process for manufacturing granules according to the invention, in which a film-forming agent capable of passing from the soluble state to the insoluble state is used, characterized in that: (a) a granular material or a viscous material capable of forming droplets to form the core of the granules, or else the components of the core are combined with one another to form grains or a viscous material capable of forming droplets;

(b) a fluid material is chosen to constitute the first layer of the granules, or else the components of the first layer are combined together, so as to obtain a first system of fluid consistency, by adding thereto, where appropriate, a

REPLACEMENT SHEET viscosity regulating agent;

(c) a fluid film-forming material is chosen to constitute the second layer of the granules, or else the components of the first layer are combined with one another, so as to obtain a second system of fluid consistency, of film-forming nature, by adding, if necessary, a viscosity regulating agent;

(d) placing the grains one after the other or dropping droplets of the core material in the central channel of an injection device; said first system is introduced into said central channel upstream from its outlet edge in an annular space, so that the grain or the droplet, when falling, is coated by said first system; and the second system is introduced into a channel opening into an annular space surrounding the outlet of the central channel, so that drops of the core material surrounded by the first and second layers are obtained at this outlet coating;

(e) these drops are dropped into an aqueous medium in which the film-forming agent of the second layer passes into the insoluble state, so as to obtain, as a result of the hardening of said film-forming agent, granules dispersed in said medium;

(f) collecting said granules, and, if necessary, drying them;

(g) if desired, these granules are coated with film material.

In step (c), an alginate in the form of sodium alginate in pure water is used as film-forming agent, and in step (e), an aqueous granule-forming medium is used, a bath of calcium salt, such as succinate or gluconate.

REPLACEMENT SHEET so as to carry out the displacement of Na ions by Ca ⁺⁺ ions.

The present invention also relates to a device for implementing the method as defined above, characterized in that it comprises an injector body comprising: a first element provided with a central bore; a second element disposed around the first and delimiting a second bore coaxial with the central bore, of diameter equal to or slightly greater than this, coming to be in its extension, said first and second elements forming a first annular passage opening out between the central bore and the second bore; a third element disposed around the second and delimiting a third bore coaxial with the previous two, of diameter slightly greater than the second bore, coming to be in its extension, said second and third elements forming a second annular passage opening out between the second bore and the third bore; a material supply channel intended to form the first sewing passing through the third element and the second element and opening into the first annular passage; a material supply channel intended to form the second layer passing through the third element and opening into the second annular passage; said device also comprising means for successively introducing grains or droplets of core material into the central bore, and means for receiving grains or droplets with double coating.

REPLACEMENT SHEET The device according to the invention also comprises means for sending the first and second layer materials under pressure into the corresponding supply channels. The core material in grains or droplets falls into the central channel under the effect of gravity, or else means are provided for controlling the fall of the grains or droplets by pneumatic positive pressure upstream of the injection device 0 or by vacuum downstream of said device.

The means for successively introducing the grains of core material into the central bore can be constituted by a pneumatic device 54 sucking the grains one by one into a grain tank, positioning them each time in the inlet of the bore central, from the body of the injection device.

The applications of the granules obtained according to the invention are as follows: introduction into the substrates and into the soils, in particular for regenerating them, granules containing bacteria and / or fungi beneficial to plants. 5 - introduction into substrates and soils, in particular for biological control against telluric pathogens attacking plants, of granules containing antagonistic microorganisms such as Pseudomonas fluorescens, _Q Fusarium oxysporum non-pathogenic or Trichoderma. constitution of microbiological fertilizers for plants, obtaining granules with phytostimulating and phytoprόtective properties;

- obtaining natural or artificial seeds coated with bacteria

REPLACEMENT SHEET (bacterization) and / or beneficial fungi (fungus), or by stimulating or retarding substances. germination; encapsulation of hormones, viruses, bacteria or yeasts in order to introduce them into agri-food environments or to reconstitute the microflora of the digestive tracts by incorporation in food or by direct ingestion for intestinal vaccination, or even to encapsulate adjuvants for animal feed; making inclusions with polyvalent enzymatic and hormonal functions, inclusions of antigens and antibodies, inclusions made up of immiscible or chemically poorly or not compatible products; realization of the temporary protection of biodegradable substances, and of multiple-structure consumable granules in the field of agrifood. To better illustrate the object of the present invention, the following will be described in more detail, the preparation of granules according to the invention, with reference to the accompanying drawing. In this drawing: - Figure 1 is a sectional view, on an enlarged scale, of a granule according to the invention; Figure 2 is a schematic cross-sectional view of an injection device according to the invention; - Figures 3A to 3C are schematic views illustrating the operation of the granule manufacturing apparatus as a whole; Figure 4 is a photograph (magnification x 10) of seeds and granules according to

REPLACEMENT SHEET 1 'Example ._ ..

The granule 1 shown in Figure 1 consists of a core 2, for example a substantially spherical seed, comprising a conventional pre-coating 2a_. The diameter d of the pre-coated core is of the order of 3-10 mm.

The pre-coated nucleus is surrounded by a layer 3 of bacteria or mycorrhization medium protecting the seed and allowing the plant to grow. Layer 3 is e itself surrounded by a layer 4 of cured film-forming material and of hydroretenting agent. The overall diameter D is of the order of 4 to 12 mm.

FIG. 2 represents, in section, an injection device 5 making it possible to manufacture granules according to the invention. This device comprises a first element 6 comprising a central trimming 7. The external wall of the element 6 comprises two successively recesses 8, 9 and ends in o ^a frustoconical part 10.

A second element 11, of cylindrical shape ending in a frustoconical shape, comes to be placed around the first by coming into abutment against the recess 8. The element 11 delimits, at the opposite end 5, a bore 12 coming to be located substantially in the extension of the bore 7. The element 11 delimits with the element 6 a passage 13 first cylindrical, then frustoconical, opening between the bores 7 and 12, and it comprises, in its cylindrical part _Q , a groove spherical 14.

^" Finally, the injection device 5 comprises a three element 15 surrounding the second and comprising a bore 16 coaxial with the bores 7 and 12, but with a diameter slightly greater than these. It is also formed between the elements 11 and 15 a

REPLACEMENT SHEET 14

passage 17, first cylindrical, then frustoconical, opening between the two bores 12 and 16.

The injection device is completed by two lateral tubes 18 and 19, arranged in the same cutting plane, but offset in height relative to each other. The tube 18, for supplying the material of the first layer 3, passes through the elements 15 and 11 and opens into the passage 13, between the recess 9 and the frustoconical wall 10. The tube 19, for supplying the material of second layer 4, passes through the element 15 and opens into the passage 17, in line with the groove 14 of the element 11.

To make the coating of the core more effective, the materials arriving through the tubes 18 and 19 can follow a spiral path at the level of the passages 10 and 17 respectively.

A. Aspiration of the nucleus

The core tank 20 being in the high position, a vacuum pump creates a strong depression in the arm 21 which lowers and a core is sucked.

B. Positioning of the core and injection of first and second layer materials

The core reservoir 20 descends to the low position and the arm 21 positions the core in the center of the central bore 7 of the injection device 5.

The first and second layer materials are injected under pressure into the tubes 18 and 19 respectively of the device 5 using a double pump with geared motor and electronic regulator, and they are distributed in the passages 13 and 17 respectively of the injector device 5. In each tube, the pressure can be regulated precisely and separately depending on the viscosity of the materials used as well as on

REPLACEMENT SHEET volume and shape of the cores to be encapsulated. C. Release of the core and encapsulation The core tank 20 is in the low position. The depression which holds the core at the end of the arm 21 is cut off automatically, and the core falls into the central bore 7 of the injection device 5 and instantly undergoes double coating.

The coated core then falls into the crosslinking bath 22 where it will stay for a variable time

(one minute to 30 minutes) until the peripheral envelope is sufficiently calcified to form a flexible and resistant capsule.

The diameter of the central bore 7 and of the injection device varies depending on the volume and the shape of the core to be encapsulated. The degree of viscosity of the materials increases slightly with the dimensions of the core:

Diameter of the diameter of the core channel (mm) central (mm)

4.0 to 4.8 6

4.8 to 5.5 8 5.5 to 8.2 10

6.7 to 9.3 11 to 12

The operation of the injection device which has just been described will be explained with reference to FIGS. 3A to 3C. In these figures, the reference numbers 20, 21 and 22 designate respectively:

- a nucleus tank

- a pneumatic device for sampling and positioning the cores

REPLACEMENT SHEET a reticulation tank. Example I

Bacterization of soybean seeds by

Bradyrhizobium japonicum

(1) The symbiotic bacterium Bradyrhizobium japonicum strain "G 49" is cultivated in a YEM medium based on yeast extract and mannitol:

The pH is adjusted to 6.8. The culture takes place with stirring at 28 ° C. and in the dark. After approximately 4 days of culture, the stationary phase corresponding to

9 1.10 basteries per milliliter is reached.

(2) The viscosity of the bacterial medium is then increased by mixing 9% of sterile algae powder (Laminaria digitata powder, reference ALGOVERT D, Company SOBALG) therein, so as to obtain a viscosity of approximately 70 centipoise, measured at viscometer (TV CONTRAVES viscometer based on the principle of shear speed measurement by a rotary torque).

(3) In addition, a homogeneous mixture based on sodium alginate (1.5%) and kaolin (10%) is prepared so as to obtain a viscosity of 350 to 400 centipoise and will constitute the 'envelope of 1' together.

(4) Thanks to a double pump with cyclic and adjustable injection rate, the materials (2) and

(3) are respectively injected into the two channels

REPLACEMENT SHEET of the injection device of FIG. 1.

{. ^' . ) Simultaneously, a pneumatic device, the suction source of which is supplied by a compressor-depressor, makes it possible to position the seeds one after the other at the level of the central bore of diameter 8 mm of the injection device ( figure 2).

(6) In the latter, each soybean is coated by the bacterial medium (2), then by the envelope medium (3).

(7) The doubly coated seed leaves the lower opening of the central channel of the injection device and falls into a tank this saturated solution of calcium gluconate, which leads to hardening of the envelope.

(8) i.after about a minute, the encapsulated seed is taken out of the bath and immediately dried.

From a bacterial medium containing g l., _ 0 Bradyrhizobium paponicum per milli itre, the

Q encapsulated soybean seed will carry approximately 1.10 bacteria (immediate numbering by the indirect immunofluorescence technique) before drying and

7 approximately 1.10 bacteria after drying for one hour at

50 ° C (see Table 1). In FIG. 4 of the centered drawing, is designated by 23 a soya bean and by 24 a granule obtained according to this example. Two of these granules were cut in half to reveal the interior. Example 2

Bacterization of soybean, pea and beet seeds by Pseudomonas fluorescens

Pseudomonas fluorescens bacteria strain "L26-1" and "M3" are cultivated in a medium

REPLACEMENT SHEET succinate:

Succinic acid 4 g / 1

K ₂ HP0 ₄ 6 g / 1 KH ₂ P0 ₄ 3 g / 1

(NH ₄ ) S0 ₄ 1 g / 1

MgS0 ₄ .7H ^ O 0.2 g / 1

The pH is adjusted to 7.0. Culture takes place in agitation? at 26 ° C and in the dark. After approximately 48 hours of culture, the stationary phase corresponding to g

1.10 bacteria per milliliter is reached.

As in Example 1, steps (2) to (8) are then followed. Bean and pea seeds are used. The beet seeds are pre-coated.

Whereas, for coated soybean and beet seeds, the diameter of the central channel of the injection device is 8 mm, for wrinkled pea seeds, it is 10 mm.

From a bacterial medium containing g 1.10 Pseudomonas fluorescens per milliliter, the soya, pea and beet seed encapsulated will carry after each drying about 1.10 bacteria (counting by the automatic dilution technique with a spiral sower on agar medium) ; cf. Table 1. Example _3

Bacterization of tomato seeds by avirulent Pseudomonas solanacearum.

The Pseudomonas solanacearum strain 2515 which is used is an avirulent mutant resistant to kanamycin induced by the TM 5 transposon derived from the strain GMI 1229 (BOUCHER et al, Journal of General Microbiology, 131, 2449-2457 (1985)) , that comes from

SHEET ^E REPLACE MENT itself a spontaneous derivative resistant to streptomycin and rifamycin from the wild strain GM 1000 (= GUY TO 3) isolated from tomatoes in KOUROU, French Guiana (DIGAT B., hy and ho to distinguish Pseudomonas solanacearum strains, causal agent of the bacterial wilt of Solanaceous and Musaceous crops in the Caribbean zone. Proceedings of Caribbean Food Crops Society Vol VI: 86-91, 1968).

The culture is carried out with stirring at 28 ° C. and in the dark in an LPG medium:

Yeast extract (Difco) 5 g / 1

Pancreatic tryptic peptone (Difco) 5 g / 1

Glucose 10 g / 1

The pH is adjusted to 7.0. After approximately 48 hours, the stationary phase corresponding to 1.10 9 bacteria per milliliter is reached.

As in Example 1, steps (2) to (8) are followed. Pre-coated tomato seeds are used. From a bacterial medium containing

Q

1.10 Pseudomonas solanacearum cells per milliliter, encapsulated tomato seeds

Q will carry approximately 1.10 bacteria (counting by the automatic dilution technique with a spiral inoculator on agar medium) (cf. Table 1).

REPLACEMENT SHEET TABLE 1

Bacterization of seeds by encapsulation

Number ^* of bacteria present on seeds before and after drying (1 h at 50 ° C)

Type of Concentration Type of Before After seed bacteria bacteria medium drying drying

(bact / ml)

Pseudomonas 4,67.10 ⁹ 2,63.10 ^s 2,35.10 ^e fluorescens M3 SOJA Pseudomonas 2,43.10 ³ 2,82.10 ^s 2,47.10 ⁸ fluorescens L 26.1

Bradyrhizo- 6.97.10 ⁹ 4.07.10 ^e 3.58.10 ⁷ dium japonicum G49

PEAS Pseudomonas 4,67.10 ⁹ 6,00.10 ^e 5,81.10 ^v fluorescens M3

BEET- Pseudomonas 1.40.10 ⁹ 1.11.10 ^e 5.03.10 ^v RAVE fluorescens M3 (pre-coated) 2.43.10 ⁹ 2.04.10 ^e l, 17.10 ^v

TOMATO Pseudomonas 3,21.10 ⁹ 2,75.10. ⁸ 1.62.10 ⁷ solanacearum 2515 (pre-coated)

^* The bacterial numbering is carried out by the automatic dilution technique with spiral inoculator on agar medium and by the serological technique of indirect immunofluorescence (for Bradyrhizobium). The numbers shown represent the average of three repetitions. Example 4

Mycorrhization of beet seeds by the mycorrhizal fungus Paxillus Involutus.

A mycorrhizal fungus strain

Paxill.us involutus, isolated from birch (Betulus alba) selected for its stimulating effects on plant growth, is cultivated in a nutritive medium "MMN" (MARX 1969).

The culture is carried out with stirring (100 t / minute). After 21 days of culture at 20 ° C, the mycelial suspension is ground for 10 seconds in an "aring Blender" mixer. The viscosity of the mycorrhizal medium is then increased by mixing 7.5% of sterile seaweed powder (Laminaria digitata powder, reference "Algovert" Company SOBALG) so as to obtain a viscosity of approximately 65 centipoise (measured with the TV viscometer CONTR VES).

As in Example 1, steps (3) to (8) are followed. The beet seeds used were previously coated. The diameter of the central channel of the injection device is 8 mm. Example 5

Encapsulation of tomato seeds with larvae of the entomophagous nematode Neoaplectana carpocapsae (≈ Steinernema feltiae)

During seed germination, some radicles can be attacked by larvae of Lepidopteran insects present in the soil.

One of the possibilities of combating them consists in encapsulating the seeds with entoiaophagous nematodes so as to develop a method of iological control. Before being encapsulated, the

REPLACEMENT SHEET tomato seeds (Lycopersicon esculentum) are coated according to a conventional process.

Larvae of the ento ophagous nematode Neoaplectana carpocapsae are obtained from the larvae of Galleria mellonella. They are harvested and washed for 10 minutes in a Merseptyl bath (ethyl-mercuri-thiosalicy ate of sodium at 1 ° / ₀₀ ) and rinsed in the sterile RINGER liquid. The concentration of the suspension is approximately 5,000 nematodes per milliliter.

The viscosity of the suspension is then increased by adding 9% of sterile algae powder (Laminaria digitata powder, reference "Algovert", Company SOBALG), so as to obtain a viscosity of approximately 70 centipoise (measured using a TV viscometer CONTRAVES).

As in Example 1, steps (3) to (8) are then followed. The diameter of the central channel of the injector is 8 mm. The survival of nematodes is checked after 48 hours storage at + 4 ° C by dissection of the encapsulated seeds and observation at laboratory temperature with a stereoscopic magnifying glass.

₃ From a suspension containing 5.10 Neoaplectane carpocapsae larvae per milliliter, the sseemmeennccee ddee tomato will carry approximately 5.10 2 nematodes.

Example 6

soy nodulation.

The symbiotic bacterium Bradyrhizobium japonicum strain "G 49" 'causes in soybeans the appearance of root nodules which allow the fixation of nitrogen and air.

REPLACEMENT SHEET Canadian Patent Application No. 227,336 describes the stimulation of this nodulation caused by certain bacterial strains of the Pseudomonas fluorescens-putida group. It therefore seems advantageous to obtain granular structures associating in separate multilayers these two types of bacteria, in order to bacteriaize the soils suitable for the cultivation of soybeans. on the one hand, a granule of Bradyrhizobium japonicum with a diameter of 6 is first formed

Q mill imeters containing about 10 bacteria according to the process already described by the applicant in French patent application No. 87 06 667.

- on the other hand, one obtains a medium with a viscosity of 70 centipoises containing the bacteria

Pseudomonas fluorescens strain "M 3", at a rate of 10 bacterial cells per milliliter by following Steps (1) and (2) of 'Example 1.

- then, as in Example 1, we follow Steps (3) and (4). The bacterial granules containing the Bradyrhizobium japonicum are positioned one after the other at the level of the central channel with a diameter of 10 mm. in the injector, each Bradyrhizobium granule is coated with the bacterial medium with Pseudomonas and then with the envelope. then as in Example 1, steps (7) and (8) are followed.

From a bacterial medium containing 1.10 g Pseudomonas per milliliter, the granule of

Q

Bradyrhizobium will carry approximately 1.10 Pseudomonas.

The granules obtained can be buried in the ground when sowing soybeans.

REPLACEMENT SHEET

Claims

1. Granules each consisting of a core comprising materials of biological origin (2) coated, characterized in that the coating comprises two layers (3, 4), the one (3) which is closest to the core (2 ) (first layer) does not by itself provide around the core (2) a single layer, autonomous and / or capable of retaining its properties, and that (4) located immediately after (second layer) being a protective layer.

2. Granules according to claim 1, characterized in that the core (2) consists of a hard and relatively smooth element, and the first layer (3), by a non-self-supporting material and / or sensitive to external agents .

3. Granules according to claim 2, characterized in that the core (2) is of general spherical or paraspherical shape or having a 1 / D ratio close to 1.

4. Granules according to one of claims 2 and 3, characterized in that the core (2) consists of a natural or artificial seed.

5. Granules according to claim 4, characterized in that the seed is a pre-coated seed.

6. Granules according to one of claims 4 and 5, characterized in that the seed has a non-spherical or non-spherical shape and comprises a pre-coating giving it a shape approaching the spherical shape.

7. Granules according to claim 1, characterized in that the core (2) is constituted by plant embryos or by plant calluses associated in a spherical or pa r a spherical mass.

REPLACEMENT SHEET

8. Granules according to claim 1, ca¬ characterized by the fact that the core (2) consists of a soft, semi-liquid or liquid material.

9. Granules according to claim 8, ca¬ characterized by the fact that the core (2) consists of an oily substance.

10. Granules according to claim 1, ca¬ characterized in that the core (2) consists of a polymeric substance.

11. Granules according to one of claims 1 to 3, characterized in that the core (2) and / or the first layer (3) constitute an inclusion structure of at least one active substance, chosen in particular among microorganisms, -bacteria, such as Rhizobacteria, and fungi, such as Mycorrhizae, and fungi antagonistic to telluric pathogens, living cells of animal or vegetal origin, the cell assemblages constituting organisms and individuals functional, the né¬ nematodes entomophages and _^ rs larvae, metabolites, enzymes, via.as, antigens, antibodies and hormones.

12. Granules according to claim 11, ca¬ characterized in that the core (2) and / or the first layer (3) constitute an inclusion structure of at least one active substance chosen from microorganisms - bacteria, such as Rhizobacteria, and fungi, such as Mycorrhizae, living cells of animal or vegetable origin and cell assemblies constituting organisms and functional individuals, and entomophagous nematodes and their larvae, said active substance being coated in an agent consisting of at least one harmful substance, in particular a nutritive extract

REPLACEMENT SHEET complete, of plant or animal origin, and to which is added, if necessary, a hydroretenting agent.

13. Granules according to claim 1, characterized in that the core (2) consists of an inclusion structure of at least one active substance comprising:

(a) a core consisting of a matrix coating the active substance, said matrix being composed

(1) at least one coating agent in which said active substance is coated; and

(2) at least one hydroretenting agent; and

(b) a porous protective envelope surrounding said core (a) and composed:

(1) at least one film-forming agent capable of undergoing biological degradation; and

(2) at least one hydroretenting agent.

14. Granules according to one of claims 12 and 13, characterized in that the hydroretenting agent is a clay, such as bentonite, kaolin, talc, vermiculite and montmori lonite.

15. Granules according to one of claims 1 to 14, characterized in that the second layer (4) comprises or is composed of at least one film-forming agent.

16. Granules according to claim 15, characterized in that the film-forming agent is chosen from alginates, polypectates, chitosan, carrageenans and other biopolymers.

17. Granules according to claim 16, constituting in particular an inoculu or a coated seed, characterized in that the film-forming agent is associated with at least one hydroretenting agent, chosen among others from clays such as bentonite, kaolin, talc, vermiculite and montmorillonite, and, if applicable, at least

REPLACEMENT SHEET a phytostimulating substance and / or at least one phytosanitary substance compatible with micro¬ organisms, living cells and cell groups present in the nucleus and / or the first layer.

18. Granules according to one of claims 1 to 17, characterized in that they also comprise a protective hydrophilic film surrounding the second layer, and constituted in particular by a very hydrophilic clay, such as bentonite.

19. Granules according to one of claims 1 to 17, characterized in that they have been subjected at least once to the double coating creating first and second layers of a nature similar to, or different from, the first and second layers (3 and 4) as defined in claim 1.

20. Granules according to one of claims 1 to 18, characterized in that they are in the form of paraspherical elements whose average diameter of the core is between about 3 and 10 mm, and whose average diameter d the whole is between approximately 4 and 12 mm.

21. Granules according to claim 6, characterized in that the core (2) consists of a seed, and the first layer (3) by beneficial microorganisms such as bacteria and fungi.

22. Granules according to claim 6, characterized in that the core (2) consists of a seed, and the two coating layers (3 and 4) consist of beneficial microorganisms, such as bacteria and fungi, different from layer to layer.

23. Granules according to claim 6, characterized in that the core (2) is

REPLACE SHEET consisting of a seed of legumes, such as soybeans, peas and beans, and the first layer (3) is a layer of bacterization by Rhizobium and / or Pseudomonas, or any other genus and species of beneficial microorganisms.

24. Granules according to claim 6, characterized in that the core (2) consists of a seed, and the first layer is a layer of mycorrhizal fungus by the mycorrhizal Paxillus involutus.

25. Granules according to claim 6, characterized in that the core (2) consists of a tomato seed, and the first layer (3) consists of a medium containing larvae of entomophagous nematode Neoplectana carpocapsae.

26. Granules according to claim 13, characterized in that the active substance of the nucleus (2) is Bradyrhizobium japonicum and that the first layer (3) consists of a medium containing Pseudomonas fluorescensputida, the resulting granules constituting inoclulums making it possible to stimulate nodulation of legumes and soybeans in particular.

27. A process for preparing the granules as defined in one of claims 1 to 26, in which a film-forming agent capable of passing from the soluble state to the insoluble state is used, characterized in that: (a) one takes a granular material or a viscous material capable of forming droplets to constitute the core (2) of the granules (1), or else the components of the core are combined with one another to constitute grains or a viscous material capable of forming droplets;

REPLACEMENT SHEET (b) a fluid material is chosen to constitute the first layer (4) of the granules (1), or else the components of the first layer are combined together, so as to obtain a first system of fluid consistency, by adding thereto, if necessary, an age t viscosity regulator;

(c) a film-forming fluid material is chosen to constitute the second layer (4) of the granules

(1), or else the components of the second layer are combined with each other, so as to obtain a second fluid consistency system, of film-forming character, by adding thereto, if necessary, a viscosity regulating agent;

(d) the grains are positioned one after the other or droplets of the core material are dropped into the central channel of an injection device (5); said first system is introduced into said central channel upstream from its outlet edge in an annular space, so that the grain or the droplet, when falling, is coated by said first system; and the second system is introduced into a channel opening into an annular space surrounding the outlet of the central channel, so that drops of the core material surrounded by the first and second layers are obtained at this outlet coating;

(e) these drops are dropped into an aqueous medium in which the film-forming agent of the second layer passes to the insoluble state, so as to obtain, as a result of the hardening of said film-forming agent, granules dispersed in said medium;

(f) collecting said granules (1), and, if necessary, drying them;

(g) if desired, these granules (1) are coated with film material.

REPLACEMENT SHEET

28. The method of claim 27, characterized in that in step (c), as film-forming agent, an alginate in the form of sodium alginate in pure water is used, and that at 'step (e), as an aqueous medium for forming the granules, a bath of calcium salt, such as succinate or gluconate, is used, so as to effect the displacement of the Na ions by the Ca ⁺⁺ ions.

29. Device for implementing the method as defined in either of claims 27 and 28, characterized in that it comprises an injector body comprising:

- a first element (6) provided with a central bore (7); a second element (11) disposed around the first (16) and delimiting a second bore (12) coaxial with the central bore (7), of diameter equal to or slightly greater than this, coming to be in its extension, said first and second elements

(6 and 1U) forming a first annular passage (10) opening between the central bore (7) and the second bore (12); a third element (15) disposed around the second (11) and delimiting a third bore (16) coaxial with the two preceding ones (7 and 12), of diameter slightly greater than the second bore (12), coming to be in its extension, said second and third elements (11 and 15) forming a second annular passage (17) opening out between the second bore (12) and the third bore (16);

- A channel (18) for feeding the material intended to form the first layer (3), passing through the third element (15) and the second element (11) and opening into the first annular passage (10); a channel (19) for supplying the material intended to form the second layer (4), passing through the third element (15) and opening into the second annular passage (17); said device also comprising means (21) for successively introducing grains or droplets of core material into the central bore, and means (22) for receiving grains or droplets with double coating.

30. Device according to claim 27, characterized in that it comprises means for sending under pressure the first and second layer materials into the corresponding supply channels (18 and 19).

31. Device according to one of claims 27 and 28, characterized in that the core material in grains or droplets falls into the central channel under the effect of gravity, or else means are provided to control the fall of the grains or droplets by pneumatic positive pressure upstream of the injection device (5) or by vacuum downstream of said device (5).

32. Device according to one of claims 27 to 29, characterized in that the means for successively introducing the grains of core material into the central channel are constituted by a pneumatic device sucking the grains one by one into a grain tank , by positioning them each time in the inlet of the central bore of the body of the injection device (5).

33. Use of the granules as defined in claim 1, to introduce into the substrates and into the soils, in particular to regenerate them, granules containing bacteria and / or fungi beneficial for plants, for

REPLACEMENT SHEET constitute microbiological fertilizers for plants, to obtain natural or artificial seeds coated with bacteria (bacterization) and / or beneficial fungi (fungization), or with substances stimulating or delaying germination.

34. Use of the granules as defined in claim 1, for the encapsulation of hormones, viruses, bacteria or yeasts, in order to introduce them into agri-food environments or to reconstitute the microflora of the digestive systems by incorporation into food or by direct ingestion for intestinal vaccination, or to encapsulate adjuvants for animal feed.

35. Use of the granules as defined in claim 1, for producing inclusions with polyvalent enzymatic and hormonal functions, inclusions of angitenes and antibodies, inclusions consisting of immiscible or chemically poorly or not compatible products.

36. Use of granules as defined in claim 1, for providing temporary protection of biodegradable substances, and consumable granules with multiple structures in the field of agrifood.

REPLACEMENT SHEET