FR2650758A1 - MULTI-LAYERED GRANULES CONTAINING ACTIVE COATED SUBSTANCES, THEIR MANUFACTURING METHOD, DEVICE FOR CARRYING OUT THIS METHOD AND USE OF THE GRANULES OBTAINED - Google Patents

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

MULTILAYERED GRANULES CONTAINING ACTIVE SUBSTANCES
COATINGS, THEIR MANUFACTURING PROCESS, DEVICE FOR THE
IMPLEMENTATION OF THIS PROCESS, 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 individualized cells living or killed (bacteria, fungi, plant or animal cells) or specifically of 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 soft 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 one 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, encapsulation of hormones, in adjuvants of animal feeds) . pharmacy (inclusions with versatile enzymatic and hormonal functions, inclusions of antigens and antibodies), gro-alimentary (temporary protection of biodegradable substances), conservation of fragile elements which cannot be frozen but only relative dehydration or, on the contrary, in granular form in freezing.

 In French patent applications nc 77-10 254, 79-28 956, Si-04 474, Si24 403, g3-02 847, 86-08 842, various methods of direct inclusion of microorganisms have been described for the purpose of constitution of microbial inocula in polymers.

In French patent application no. 87-06 667, the applicant claimed the design and the process of double encapsulation or of indirect inclusion concerning the preparation 'inoculu .., consisting of microorganisms coated in a nutritive matrix itself surrounded by an envelope and a film in the form of biodegable granules, In this process
1, microorganisms are grown in a matrix
feeder composed of a complete nutritious extract and
coated with a hydroretenting agent,
2) then this matrix is encapsulated in an envelope
porous containing another hydroretenting agent,
3) which envelope can finally be protected by a
hydrophilic lamination.

However, none of these methods envisages inclusion in multilayers, characterized in that it makes it possible to obtain a spherical or paraspherical structure in which
(a) the center or heart is made up of a material
alive or inert or chemical
(b) the layers enveloping the core are successive, of
identical or advantageously different in nature;
(c) a film-forming agent finally isolates the product
(d) the final product thus obtained according to steps (a), (h)
and (c) may again experience one or more
encapsulations (which increases its volume accordingly).

 A seed could not be encapsulated in a bacteria or mycorrhization medium by a process as described in French patent application no. 87-06 667. Indeed, this would require an intermediate step of drying the layer of bacteria medium. 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 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 process according to the present invention also makes it possible to conserve the humidity of the core, which is often desired in order to preserve its properties. In this case, in the case of the bacterization of the seeds, it is possible to retain approximately 15 of the residual moisture of the bacterial circulation due to the coating for several weeks.

 The present invention therefore firstly relates to granules each consisting of a coated core, characterized by the fact 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.

 According to a first embodiment, the core of the granule is constituted by a hard and relatively smooth element, and the first layer, by a non-self-supporting material and / or sensitive to external agents.

In this case, the core preferably has a spherical or para-spherical general drill or else has a 1 / D ratio close to i.

 In particular, the nucleus can consist of a natural or artificial seed, of spherical or paraspherical shape, which can be a pre-coated seed.

However, the seeds can have a non-spherical or non-paraspherical shape (lettuce seeds), and then comprise a pre-coating giving them a shape approaching the spherical shape.

 The nucleus can also be formed by plant embryos or by plant calluses associated in a spherical or paraspheric mass.

According to another embodiment, the core is constituted by a soft, semi-liquid or liquid material, for example an oily substance. The core can also consist of a polymeric substance
According to another embodiment, the core and / or the first layer may constitute an inclusion structure of at least one active substance, chosen in particular from microorganisms - bacteria, such as
Rhizobacteria, and fungi, such as Mycorrhizae, living cells of animal or plant origin and cell groups constituting functional organisms and individuals, and entomophagous nematodes and their larvae, metabolites, enzymes, viruses, antigens, antibodies and hormones.

 In the case where the etycu nucleus the first layer constitutes an 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 by at least one nutritive substance, in particular by a complete nutritive extract, of plant or animal origin, and to which is added, if necessary.

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 feed matrix can constitute a culture medium for said misroorganisms 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 species Fucus, Laminaria and Ascophyllum.

said sterile extract being brought to pH 7, for example by magnesium oxide.

In accordance with another embodiment of the present invention, the core 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
heart (a) and composed
(1) at least one film-forming agent liable to undergo
biological degradation; and
(2) at least one hydroretenting agent.

 Such an inclusion structure is described in PCT international application no. 88-00239 of 11 October 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, where appropriate, at least one phytostimulating substance. and / or at least one phytosanitary substance compatible with microorganisms, living cells and cell groups present in the nucleus and / or the first layer.

The phytostimulating 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, the
Cytokinin, aluminum phosethyl (aluminum-tris-Oethyl phosphonate). The 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 may also comprise a hydrophilic protective film surrounding the second layer, and constituted in particular by a very hydrophilic clay, this bentonite.

 In accordance with another embodiment of the invention, the granules were again subjected at least once to the double coating creating the 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 an elongated, spherical ents whose average diameter of the core is between approximately 3 and 1 min, and whose overall average diameter is between approximately 4 and 12 min. .

Examples of granules according to the invention are the following
- the nucleus is made up of a seed and the first
layer by beneficial microorganisms such as
bacteria and fungi
- the nucleus consists of a seed, and the two
coating layers are formed by micro
beneficial organisms, such as bacteria and
mushrooms, different from layer to layer
- the nucleus consists of a seed of legumes,
such as soybeans, peas and beans, and the first layer
is a layer of bacteria containing 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 layer of mycorrhization by the
mycorrhizal fungus Paxillus involutus
- the nucleus consists of a tomato seed, and the
first layer consists of a medium containing
Neoplectana entomophagous nematode larvae
carpocapsae
- the active substance in the nucleus is Bradyrhizobium
japonicum, and the first layer consists of a
medium containing Pseudononas fluorescens - tutida,
resulting granules constituting inocula
to stimulate the modulation of legumes
and soy in particular.

The present invention also relates to a method 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 grain or viscous material
able to form droplets to form the
nucleus of the granules, or else we associate them
core components 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 we associate between
them the components of the first layer, so
obtain a first fluid consistency system, by
adding, if necessary, a regulating agent of
viscosity; (c) a film-forming fluid material is chosen to constitute
the second layer of granules, or else we associate
between them the components of the second layer, so
to obtain a second fluid consistency system, to
film-forming character, adding, if necessary, a
viscosity regulating agent (d) the grains are positioned one after the other or
droplets of the core material into the
central channel of an injection device; we
introduces said first system into said channel
central upstream of its exit border following a
annular space, so the grain or the
droplet, falling, is coated by said first
system; and cn introduces the second system in a
channel opening into an annular space surrounding the
exit from the central channel, so that, at
level of this output, drops of the material of
core surrounded by the first and second coating layers (e) these drops are dropped in 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
hardening of said film-forming agent, granules
dispersed in said medium (f), said granules are collected, and, if necessary,
dry them (g) if desired, these granules are coated in
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), as aqueous medium for forming granules, a calcium salt bath, such as succinate or gluconate, so as to effect the displacement of the Na + ions by the 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 with a central bore
- a second element arranged around the first and
delimiting a second bore coaxial with the bore
central, with a diameter equal to or slightly greater than
this one, 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 arranged around the second and
delimiting a third bore coaxial to the two
previous, slightly larger in diameter 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 layer crossing the third element and the
second element and opening into the first passage
annular
- a channel for bringing in material intended to form the
second layer crossing 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 the grains or droplets with double coating.

 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 or. by depression 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 sucking the grains one by one into a grain tank, positioning them each time in the inlet of the central bore of the body of the injection device.

The applications of the granules obtained according to the invention are as follows:
- introduction into substrates and soils,
in particular to regenerate these, granules
containing bacteria and / or fungi
beneficial for plants
- constitution of microbiological fertilizers for
plants, obtaining granules with phvto properties
stimulants and phytoprotective
- obtaining natural or artificial seeds
coated with bacteria (bacteria) and / or
beneficial fungi (fungus), or by
substances stimulating or delaying germination
- encapsulation of hormones, viruses, bacteria or
yeasts so that they can be introduced into approved media
feed or tower replenish the microflora of
digestive systems by Incorporation in food
or by direct ingestion for vaccination
intestinal, or to encapsulate adjuvants
for animal feed
- realization of inclusions with versatile functions
enzymatic and hormonal, d inclusions of antigens
and antibodies, with inclusions consisting of products
immiscible or chemically poorly or not compatible
- realization of the temporary protection of substances
biodegradable, and consumable granules
multiple structure in the field of agro
food.

To better illustrate the subject of the present invention, the preparation of granules according to the invention will be described below, in more detail, with reference to the accompanying drawing. On this drawing
FIG. i is a sectional view, on an enlarged scale,
of a granule according to the invention
- Figure 2 is a schematic sectional view
cross-section of an injection device according to the invention
- Figures 3A to 3C are schematic views
illustrating the operation of the switchgear
manufacture of granules as a whole
- Figure 4 is a photograph (magnification x 10)
seeds and granules according to Example 1.

 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 itself surrounded by a layer 4 of hardened film-forming material and of hydroretenting agent. The overall diameter D is of the order of 4 to 12 nm.

 Figure 2 shows, in section, an injection device 5 for manufacturing granules according to the invention. This device comprises a first element 6 comprising a central bore 7. The external wall of the element 6 comprises two successively recesses 8, 9 and ends in a frustoconical part 10.

 A second element 11, of cylindrical forehead ending in a frustoconical forehead, is placed around the first one bearing against the recess 8. The element 11 delimits, at the opposite end, 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, a groove device 14.

 Finally, the injection device 5 comprises a third element 15 surrounding the second and comprising a bore 15 coaxial with the bores 7 and 12, but with a diameter slightly greater than these. I1 is also formed between the elements II and 15 a 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 first layer material 3, passes through the elements 15 and 11 and opens into passage 13, between the recess 9 and the frustoconical wall 10. The tube 19, for supplying the second material 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 efficient, the materials arriving via the tubes 18 and 19 can follow a spiral path at the level of the passages 10 and 17 respectively.

A. Aspiration of news
The core tank 2C 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 new and invention of materials
first and second layers
The core reservoir 20 descends to the low position and the arm 21 positions the core at 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 gear 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 according to the viscosity of the materials used as well as according to the volume and the shape of the cores to be encapsulated.

C. Releasing the nucleus and ancapsulation
The core tank 20 is in the low position.

The depression which maintains the core at the end of the arm 21 is cut off automatically, and the core falls into the central bore 7 of the device. 5 and instantly undergoes double coating.

 The coated core then falls into the crosslinking bath 22 or it will stay 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
Channel Diameter Diameter
core (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 the
Figures 3A to 3C. In these Figures, the reference numbers 20, 21 and 22 respectively denote
- a nucleus tank
- a pneumatic device for sampling and
core positioning
-a crosslinking tank.

Example 1
Bacterization of sota tar seeds
Bradyrhizobium japonicum.

(1) The symbiotic bacterium Bradyrhizobium japonicum strain
"G 49" is cultivated in a YEM medium based on extract
yeast and mannitol
Mannitol 10 g / l,
DIFCO yeast extract 1 g / l,
K2HPO4 0.5 g / l,
MgSO4 # 7H2O 1 g / l,
FeCl3 0.004 g / l,
0.2 g / l NaCl.

The pH is adjusted to 6.8. Culture takes place in agitation
at 280C and in the dark. After 4 days of culture
approximately, the stationary phase corresponding to
1.109 bacteria per milliliter is reached.

(2) The viscosity of the bacterial medium is then increased in v
mixing 9% sterile seaweed powder (
Laminaria diaitata, reference ALGOVERT D, Society
SOBALG), so as to obtain a viscosity of approximately
70 centipoise, measured with a viscometer (viscometer
TV CONTRAVES based on the principle of measuring
shear speed by rotary torque).

(3) Furthermore, a homogeneous mixture based on alginate
sodium (1.5%) and kaolin (10%) is prepared to
obtain a viscosity of 350 to 400 centipoise and
will constitute the envelope of the whole.

(4) Thanks to a double cyclic injection rhythm pump
and adjustable, materials (2) and (3) are respected
injected into the two channels of the device
Figure 2.

(5) Simultaneously, a pneumatic device, the
suction source is provided by a compressor
depressant, allows positioning one after the other
seeds at the center bore of diameter
8 mm from the injection device (Figure 2).

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

(7) The double coated seed comes out through the orifice
lower of the central channel of the injection device and
falls into a container of saturated gluconate solution
calcium, which leads to hardening of the envelope.

(8) 'About one minute later, the encapsulated seed is
out of the bath and immediately dried.

 From a bacterial medium containing 1.109 Bradvrhizobium iaDonicum per milliliter, the encapsulated soybean seed will carry approximately 1.108 bacteria (immediate count by the indirect immunofluorescence technique) before drying and approximately 1.107 bacteria after drying for one hour at 500C (cf. Table 1).

In Figure 4 of the accompanying drawing, is designated by 23 a soybean 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
beets by Pseudomonas fluorescens.

The bacteria Pseudomonas fluotescens strain "L26-1" and "M3" are cultivated in a succinate medium
Succinic acid 4 g / l,
K2HPO4 6 g / l,
KH2PO4 3 g / l,
(NH4) SO4 1 g / l,
MgSO4 # 7H2O, 0.2 g / l.

 We adjust the p to 7 ,. The culture takes place in agitation at 26 ° C. and in the dark. After approximately 48 hours of culture, the stationary phase corresponding to 1.109 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 min, for wrinkled pea seeds, it is 10 min.

 From a bacterial medium containing 1.109 Pseudomonas fluorescens per milliliter, the seeds of encapsulated soybeans, peas and beets will carry, after each drying, approximately 1.107 bacteria (counting by the automatic dilution technique with a spiral seeder on agar medium); cf. Table 1.

Example 3
Bacterization of tomato seeds by Pseudomonas
avirulent 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 og Generall Microbiology, 131, 2449-2457 (1985)) , which itself comes from 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., why and ho to distinguish Pseudomonas solanacearum strains, causal agent of the bacterial wilt of Solanaceous and Musaceous crops in the Caribbean zone.
Caribbean Food Crops Society Vol VI: 86-91, i968).

The culture is carried out with shaking at 28 ° C. and in the dark in an LPG medium:
Yeast extract (Difco) 5 g / l
Pancreatic tryptic peptone (Difco) 5 g / l,
Glucose 10 g / l.

 The pH is adjusted to 7.0. After approximately 48 hours, the stationary phase corresponding to 1,109 bacteria per milliliter is reached.

As in Example 1, steps (2) to (8) are followed. We use pre-coated tomato seeds. From a bacterial medium containing 1,109 cells of
Pseudononas solanacearun per milliliter, the encapsulated tomato seeds will carry approximately 1,108 bacteria (counting by the technique of automatic dilution with a spiral sower on agar medium) (cf. Table 1).

TABLE 1
Bacterization of seeds by encapsulation
Number * of bacteria present on seeds before and after drying (1 h at 50 C).

Type Type Concentration of medium Before After seedling of bacteria (bacter / ml) Drying Drying
Pseudomonas fluorescens M3 4.67 # 109 2.63 # 103 2.35 # 108
SOIA Pseudomonas fluorescens G26 2.43 # 109 2.82 # 108 2.47 # 108
Statertrizobuten jasoni @@ sum G49 6.97 # 109 4.07 # 108 3.55 # 107
SOIA Pseumdomonas fluorescens M3 4.67 # 109 6.00 # 108 5.81 # 107
BATTERY Pseudotinas fluorescens M3 1.40 # 109 1.11 # 108 5.03 # 107 (pre-coated) 2.43 # 109 2.04 # 108 1.17 # 107
TOMATO Pseudomonas solanateuam 2515 3.21 # 109 2.75 # 108 1.62 # 107 (pre-coatedI
* The bacterial numbering is carried out by the
automatic dilution technique with sowing
spiral on agar medium and by technique
indirect immunofluorescence serology (for
Bradyrhizobium)
The figures shown represent the average of
three rehearsals.

Example 4
Mycorrhization of beet seeds by the
mvcorrhizal fungus Paxillus Involutus.

 A strain of mycorrhizal fungus Paxillus involutus, isolated from birch (Betulus alba) selected for its stimulating effects on plant growth, is cultivated in a nutritive medium "MMM" (MARX 1969).

 The culture is carried out in agitatioh (100 t / minute). After 21 days of culture at 200C, the mycelial suspension is ground for 10 seconds in a "Waring Blender" mixer.

We then increase the viscosity of the mycorrhizal medium by mixing 7.5% of sterile algae powder (Laminaria digitata powder, reference "Algovert" Societe SOBALS) so as to obtain a viscosity of approximately 65 centipoise (measured with a viscometer TV CONTRAVERS)
As in -Example 1, we follow steps (3) to (8). The beet seeds used were previously coated. The diameter of the central channel of the injection device is 8 min.

Example 5
Encapsulation of tomato seeds by larvae from
entomophaq nematode Necaplectana carpooapsae
(= 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 is to encapsulate the seeds with entomophagous nematodes so as to develop a biological control method. Before being encapsulated, the tomato seeds (Lycopersicon esculentum) are coated according to a conventional process.

 Larvae of the entomophagous nematode Necaplectana carpocapsae are obtained from the larvae of Galleria mellonella. They are harvested and washed for 10 minutes in a Merseptyl bath (1 # sodium ethyl-mercuri-thiosalicylate) and rinsed in sterile RINGER liquid. The concentration of the suspension is approximately 5,000 nematodes per milliliter.

 The viscosity of the suspens ion 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 by viscometer TV CONTRAVERS).

 As in Example 1, steps (3) to (85) are then followed. The diameter of the central channel of the injector is 8 mm.

Nematode survival is checked after 48 hours storage at -4c by dissection of the encapsulated seeds and observation at laboratory temperature under a stereoscopic magnifying glass
From a suspension containing 5.103 Naoaplectane carpocapsse larvae per milliliter, the tomato seed will carry approximately 5 # 10 nematodes.

Example 6
Obtaining multilayer granules containing two
different bacterial species usable for
stimulate self-nodulation a.

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

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 bacterize the soils suitable for the cultivation of soybeans.

- On the one hand, we first form a granule of
Bradvrhizobium iaponicum with a diameter of 6 millimeters
containing around 108 bacteria according to the process already
described by the applicant in the patent application
French n 87 06 667.

- On the other hand, cn obtains a medium with a viscosity of
70 centipoises containing the Pseudomonas bacteria
fluorescens strain "1 3", at a rate of 109 cells
bacteria per milliliter by following Steps (1)
and (2d 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 of diameter 10 mm.

- In the injector, each Bradvrhizobium granule is
coated by the bacterial medium with Pseudomonas then by
the envelope
- Then as in Example 1, we follow steps (7) and
(8).

From a bacterial medium containing 1.109 Pseudomonas per milliliter, the granule of
Bradyrhizobium will carry approximately 1 # = 18 Pseudomonas.

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

Claims (36)

 1 - Granules each consisting of a coated core (2), characterized in that the coating comprises two layers (3, 4), the one (3) which is closest to the core (2) (first layer) providing not by itself 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 paraspheric mass.  8 - Granules according to claim I, characterized in that the core (2) consists of a soft, semi-liquid or liquid material.  9 - Granules according to claim 8, characterized in that the core (2) consists of an oily substance.  10 - Granules according to claim 1, 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 from microorganisms - bacteria, such as Rhizobacteria, and fungi, such as Mycorrhizae -, living cells of animal or vegetable origin, the cell assemblies constituting functional organisms and individuals, entomophagous nematodes and their larvae, metabolites, enzymes, viruses, antigens, antibodies and hormones.  12 - Granules according to claim 11, characterized in that the core (2) and / or the first layer (3J constitute an inclusion structure of at least one active substance chosen from bacteria microorganisms, such as Rhizobacteria, and fungi, such as Mycorrhizae, living cells of animal or vegetable origin and cell groups constituting functional organisms and individuals, and entomophagous nematodes and their larvae, said active substance being coated in an agent consisting of at least one substance nutritive, in particular by a complete nutritive extract, of vegetable 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 protective-porous envelope surrounding said  heart (a) and composed  (1) at least one film-forming agent liable to undergo  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 montmorillonite.  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, chltosan, oarragenans and other biopolymers.  17 - Granules according to claim 16, constituting in particular an insculum 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, verniculite and montmorillonite, and, where appropriate, at least one phytostimulating substance and / or at least one phytosanitary substance compatible with microorganisms, living cells and the cellular assemblies present in the nucleus and / or the first layer.  18 - Granules according to one of claims 1 to 17, characterized in that they further 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 spherical 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) are constituted by beneficial microorganisms, such as bacteria and fungi, different from layer to layer.  23 - Granules according to claim 6, characterized in that the nucleus (2) consists of a seed of legumes, such as soybeans, peas and beans, and the first layer (3r is a layer of Rhizobium bacteria 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 myoorhization by the nycorrhizal fungus 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 the entomophagous nematode Neotlectana carpocapsae.  26 - Granules according to claim 13, characterized in that the active substance of the nucleus (2) is Bradvrhlzobium japonicum and that the first layer (3) consists of a medium containing Pseudomonas fluorescens  Dutida, the resulting granules constituting inoculums making it possible to stimulate the nodulation of legumes and of soy in particular.  27 - Process for preparing the granules as defined in one of claims 1 to 26, in which a film-forming agent capable of going from the soluble state to the insoluble state is used, characterized in that (a) takes a grain or viscous material  able to form droplets to form the  core (2> of granules (1), or else we associate between  them the core components to form grains  or a viscous material capable of forming sharp droplets (b) a fluid material is chosen to constitute the  first layer (4> granules (1) or else  combines the components of the first layer,  so as to obtain a first consistency system  fluid, adding, if necessary, an agent  viscosity regulator (c) a film-forming fluid is chosen to constitute  the second layer (4) of the granules (1), or else  combines the components of the second layer,  so as to obtain a second consistency system  fluid, film-forming, adding, if necessary  if necessary, a viscosity regulating agent (d) the grains are positioned one after the other or  droplets of the core material into the  central channel of an injection device (5); we  introduces said first system into said channel  central upstream of its exit border following a  annular space, so that the grain or the drop  lette, when falling, is coated by said first  system; and we introduce the second system in a  channel opening into an annular space surrounding the  exit from the central channel, so that you get  level of this output, drops of the material of  core surrounded by the first and second layers  of 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), said granules (1) are collected, and, if applicable,  we dry them; (g) if desired, these granules (1) are coated in  the film material.  28 - Process according to claim 27, characterized in that in step (c), an alginate in the form of sodium alginate in pure water is used as film-forming agent, and that in l step (e), as an aqueous medium for forming the granules, a calcium salt bath, such as succinate or gluconate, is used, so as to effect the displacement of the Na ions by the caf ions  29 - Device for implementing the method as defined in one 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) arranged around the first (16)  and delimiting a second bore (12) coaxial with  the central bore (7), of equal or slightly diameter  higher than this, coming to be in its  extension, said first and second elements (6 and  11) forming a first annular passage (10)  opening out between the central bore (7) and the second  bore (12)  - a third element (15) arranged around the second (11)  and delimiting a third bore (16) coaxial with  two previous ones (7 and 12v, slightly in diameter  greater than the second bore (12), coming to be located in  its extension, said second and third elements  (11 and 15) forming a second annular passage (17>  opening between the second bore (12) and the third  bore (16)  - a channel (18) for feeding the material intended to form  the first layer (3), crossing the third element  ment (15! and the second element (11) and opening into  the first annular passage (10)  - a channel (19) for feeding the material intended to form  the second layer (4), crossing the third element  ment (15> and emerging in the second passage  annular (17) said device also comprising means (21j for successively introducing grains or droplets of core material into the central bore, and means i22j 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 in 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 in the central channel under the effect of gravity, or else means are provided to control the fall of 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 aspirating the grains one by one in 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, to constitute microbiological fertilizers for plants, to obtain natural or artificial seeds coated with bacteria (bacteria) and / or beneficial fungi (fungus; or by 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 tracts by incorporation in 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 antigens and antibodies, inclusions consisting of immiscible or chemically poorly or not compatible products.  36 - Use of granules as defined in claim I, to achieve temporary protection of biodegradable substances, and consumable granules with multiple structure in the field of food processing.