FR2615203A1 - INOCULUM IN WHICH THE MICROORGANISMS ARE PACKED IN THE FORM OF BIODEGRADABLE PELLETS, ITS PREPARATION PROCESS AND ITS APPLICATIONS - Google Patents

Abstract

The structure for the inclusion of at least one active substance (microorganisms, cells or cell assemblies) is provided in the form of granules comprising a core (N) consisting of a matrix coating the active substance, said matrix being comprised of: (1) at least one coating agent; and (2) at least one hydroretention agent; and a porous protection envelope (E) comprised of (1) at least one film-forming agent susceptible of undergoing a biological degradation; and (2) at least one hydroretention agent, a hydrophilic protection skin (P) generally surrounding the envelope. Said structure in particular preserves elements included and immediately provides for an immediate multiplication of microorganisms followed by a progressive release of the inoculum since the first contacts with a hydrated medium, thereby producing a good colonization of the target (root system, soil, digestive system according to the particular application of the inoculum).

Description

TNOCULUM IN WHICH MICROORGANISMS ARE CONDITIONED
IN THE FORM OF BIODEGRADABLE GRANULES. ITS PREPARATION PROCESS AND APPLICATIONS
The present invention relates to an inoculum in which the microorganisms are packaged in a formulation allowing their very good conservation in a dry medium and their release in a humid environment.This one finds application particularly in the fields of agriculture, such as microbiological fertilizers and indirectly for the microbiological coating of seeds; horticulture and market gardening, by incorporation into natural and artificial substrates and off-ground crops, to reconstitute the flora of the digestive system of animals; and in animal feed, in the manufacture of silage
Microorganism inocula can be packaged in various formulations.

However, in most cases, the type of formulation chosen largely determines the effectiveness of the inoculum: - Liquid formulations are the easiest to
realize, but they do not always give satisfaction
tion. Indeed, in liquid media, micro
organisms appear naked and in a scattered
inoculation into soil or substrates.

In these conditions, the balance of power is often
favor of resident flora and the results of
inoculation can be all the more disappointing as
the antagonism of the nutochtone flora is powerful. We
know that besides that in one gram of fertile soil, one
can find more than a billion bacteria.

- solid formulations, in the form
powders, sometimes give better results.

In this case, the microorganisms are more or less
dehydrated, sometimes even freeze-dried. The addition of an inert but hydrophilic, clay-like protective support is necessary to obtain good efficacy. One of the problems is that the microorganisms, before entering into activity, must be rehydrated, which generally causes loss of viability by osmotic shock. In addition, clay particles do not appear to offer a sufficient protective bulwark against the multitude of resident telluric aggressors.

In order to avoid these problems, gelled formulations in solid form have been used, in which the microorganisms are immobilized and which have the advantage of being able to be hydrated according to what one wants to obtain. Certain gels, consisting of polymers such as polyurethane, polysaccharides, such as xanthan and cellulose derivatives, or polyacrylamides, have been proposed.

However, the inclusion techniques of microorganisms in the polymer gels appear to be delicate. Thus, in French Patent No. 2,501,229, it is proposed for example to provide a culture medium inoculated with the microorganism or a suspension of microorganisms obtained by centrifugation or filtration of said medium, in a heated solution of polysaccharide, and forming the gel by cooling. This heat treatment ensuring crosslinking of the gel can be replaced by the action of a metal salt or by synergistic treatment with another polymer. In French Patent No. 2 519 022, it is recommended to lower the activity of water in the inoculum below its critical value to a value of less than 0.5. For this purpose, drying is carried out by atomization by hot and dry air flow or a solution saturated with a compound having the activity of the required water evolving towards
balance with the inoculum. Moreover, we can
point out that polyacrylamides are not biodegradable
and have a very bad reputation
concerning toxicity.

 The present invention aims to remedy all of these drawbacks. It has indeed been sought to ensure a good preservation of the inoculum selected in stable structures, easy to manufacture, to market and use in the aforementioned fields, also to ensure an immediate multiplication of microorganisms and a gradual release. of the inoculum from the first contacts with a hydrated medium, and finally to obtain a good colonization of the target <root system, soil, digestive tract following the particular application of the inoculum> and, consequently, the expected beneficial effects .

The inoculum according to the invention, which is formed of microorganisms included in a support and which is in the form of granules, meets all of these objectives. This inoculum is characterized in that the granules comprise <a) a core consisting of a coating feeder matrix
the microorganisms to be inoculated, said matrix
feeder being composed
<1) a complete nutrient extract for said micro
organisms in which they are coated; and
<2) at least one hydroretenting agent; and <b> a porous protective envelope surrounding said core
<a) and composed
<i) at least one film-forming agent liable to undergo
biological degradation; and
<2) at least one hydroretention agent.

Preferably, the granules of the inoculum further comprise, according to the invention, a protective hydrophilic film surrounding
the envelope <b).

 The microorganisms are introduced into the core (a) preferably in the form of a more or less concentrated suspension. The feeder matrix may constitute a culture medium for said microorganisms prior to the formation of the granules, in which case the resulting inoculum has a very high concentration of microorganisms, for example, for bacteria, up to 3 x 1010 bacteria per ml.

 The complete nutrient extract (a1) may be of vegetable origin or of animal origin. In the first case, it may be an extract based on algae, which are chosen in particular from the species Fucus, Lamjnarsa and Ascophyl Sum, said extract being brought to pH 7, for example by magnesium oxide.

 The hydroretenting agents (a2) or (b2) advantageously consist of clays, such as bentonite, kaolin, talc or vermiculite.

 As for the film-forming agents (b1), they may in particular be constituted by alginates, chitosan or other biopolymers.

 In accordance with. another interesting feature of the invention, the envelope (b) may further contain at least one phytostimulant substance chosen in particular from nitrates, growth substances, various mineral salts, etc., and / or a phytosanitary substance chosen especially among fungicides, insecticides and herbicides compatible with coated microorganisms. As phytostimulant substances indol acetic acid and Cytokinin may be mentioned. In the case of phytosanitary substances, fungicides and insecticides should be used in the case where the coated microorganisms are bacteria, and bactericides and insecticides when the coated microorganisms are fungi.

 As regards the film (c), it is advantageously constituted by a very hydrophilic clay, such as bentonite.

Concerning the proportions of the various components of the granules which constitute the inoculum according to the invention (after dehydration), it can be indicated that the feeder matrix <a> can comprise from 5 to 10 g of the extract (a1) and from 1 to 5 g of the (or) hydroretenting agent (s) Ca 2), for 10 to 15 ml of water, and that the envelope <b) can comprise from 1 to 5 g of the <or> film-forming agent <s> s) <b1) and from 5 to 20 g of the water-repellent agent (s) <b2>, for 10 to 15 ml of water.This corresponds to the following initial formulation - for the feeder matrix <a>, 5 & 10 g of the extract
(a1) and the 5 g of the hydro agent (s)
retender (s) <a2) per 100 ml of water, the microphone (s)
organism <s) being present in said matrix by
example has a concentration of the order of 107 a.

1010 bacteria per ml of water for bacteria, and
the order of 10 to 106 propagules per ml of water for the
mushrooms - for the shell (b), 1 to 5 g of the agent (s)
film-forming agent (s) (bolus) and s at 20 g of the agent (s)
hydroretenteur (s) (b2) per 100 ml of water.

 In particular, the granules may be in the form of parasphere elements whose core (a) has a mean diameter of between 2 and 6 mm and whose thickness of the envelope (b) is between 0.5 and 3 mm, the outer film, when present, may have a thickness of between 1 and 3 mm.

 there is no particular limitation on the microorganisms that can be introduced into the nucleus. We can mention bacteria such as Rhizobacteria, and fungi such as Xycorrhizae. Particular examples of these bacteria will be given below with reference to the applications of the inoculum according to the invention.

The present invention also relates to a process for the preparation of the inoculum which has just been defined, in which a film-forming agent capable of passing from the soluble state to the insoluble state, characterized by the fact that <A) is associated between they, in an aqueous medium, the
core component (a) granules so as to obtain
a first system of pasty consistency <B) is associated with them, in an aqueous medium, the
components of the envelope tb), so as to obtain a
second system of pitiful consistency, in which
the film-forming agent is in the soluble state tc> the above-mentioned first and second systems are passed through
in two coaxial nozzles, the first system, in
the central nozzle, and the second system, in space
ring delimited by the two aforementioned nozzles,
way that the exit of this double nozzle, one obtains
drops of the core material surrounded by the
envelope material (D> these drops are dropped in a second aqueous medium
in which the film-forming agent goes to the insoluble state,
so as to obtain, as a result of the hardening of said
film-forming agent, granules dispersed in said
medium (E), said granules are collected and, if appropriate,
dry them; and <F) if desired, these granules are coated in
film material.

In particular, in step (B), an alginate in the form of sodium alginate in pure water is used as film-forming agent, and in step (D) the aqueous medium is used. forming granules, a calcium salt bath, such as succinate or gluconate, so as to effect the displacement of Na ions by Ca ions
The applications envisaged for the inoculum according to the invention are the following: Introduction in substrates and in soils of
beneficial populations for plants such as
bacteria (Rhizobacteria, Rhizobium, Agrobacterium
"K84", etc.) or fungi (Hycorhizes,
Trichoderma, etc.), among other things to regenerate
"tired" soils and, correspondingly, constituting
pathogen-resistant soils or substrates.

- Inoculation of soils and substrates with the aid of
pathogenic bacteria (Pseudomona solanacearum,
Agrobacterium tumefaciens, Erwlnia carotova.) Or
pathogenic fungi (Fusarium, Phytophthora,
Verticillium, Fytium> useful in studies of
varietal resistance for example.

- Inoculation of fermentative bacteria in
silage in order to speed up the process of
fermentation.

- Introduction of bacteria and / or yeasts
different types of agri-food environment
reconstitution of microflora of digestive tract
by incorporation in food (very good
compatibility and easy biodegradability) or by
direct ingestion, the inocula of the invention having,
by a judicious choice of its components, a very
good compatibility with food and being
easily biodegradable.

 In accordance with another application of the invention, the nutrient extract, in which the microorganisms are coated, can also be used for coating seeds.

 To better illustrate the object of the present invention, will be described below, in more detail, the preparation of an inoculum according to the invention.

I - Preparation of the feeder matrix loaded with micro
organizations.

 Sterile distilled water is used to prepare a sterile seaweed cream pie. Depending on the algae used, creams with different properties are obtained. It is also possible to use commercially available algae powders. The pH is adjusted to 7 with magnesium oxide.

 In parallel, a concentrated suspension of micro-organisms is prepared, to which a little sterile bentonite is added.

These two preparations are mixed for several hours in the following proportions - seaweed cream at pH 7. ........................ 90 g - bentonite. . ...... .................. 10 g - concentrated suspension of micro-organisms ....... 1000 ml
In the hydrated state, this medium ensures excellent multiplication of microorganisms. In the dehydrated state (10 to 20% residual humidity), it allows a very good survival of the microorganisms, it also rehydrates easily and, during the rehydration, the microorganisms find all the necessary ingredients. multiplication.If we want to obtain a highly concentrated matrix of microorganisms, it is possible to cultivate them in this medium in agitation and at an optimal temperature (25 to 28 C for example for soil bacteria) .

He - Preparation of the protective envelope
A sterile mixture of sodium alginate and kaolin is prepared in the following proportions - sodium alginate ........................... ... 15 g - kaolin, 100 g - distilled water ................................. 1000 ml
III - Obtaining fresh pellets
The device shown in FIG. 1 is used. This device comprises two containers 1, 2 intended to contain respectively the core material and the covering material. Each of these containers 1 and 2, equipped with a magnetic stirring device. respectively lc and 2p, is connected by a pipe, respectively 3 and 4, d an injection device 5 which will be described below and which enters the neck of a container 6 for receiving the granules formed. On each of the lines 3 and 4 are arranged peristaltic pumps respectively 7 and 8 which bring the corresponding materials into the injection device 5.This is constituted by a double nozzle, the first nozzle <5) being formed by end of a tube connected to the pipe .4, and the second (coaxial with the first being connected to the pipe 3.

 In operation, the core material and the shell material reach the injection device 5, so that the first is surrounded by the second. The assembly exits at the base of the injection device 5 in the form of drops 9, which fall into a bath 10 of succinate or calcium gluconate (concentration 0, in) contained in the container 6.

The sodium alginate used in the formulation of the shell material, which is a water-soluble substance, is then converted into calcium alginate, which is insoluble in water, constituting, with the kaolin which also forms the envelope, a resistant layer.

 The longer the residence time of the beads obtained in the calcium salt, the more the envelope is loaded with Ca ++ ions and will be solid.

IV - Pellet coating
On leaving the bath, the granules are allowed to stand for a few hours at room temperature. Then, they are rolled in a very hydrophilic clay such as bentonite, so as to form a film around each granule.

This operation preserves residual moisture and dampens the crushing inside the matrix, which could be caused by a sudden dehydration.

 Figure 2 illustrates the release of microorganisms in the inoculated medium.

 The dry granules (FIG. 2A) are introduced into a humid medium (soil, substrate, digestive system, etc.) In contact with this medium, the film P of the granules absorbs the available water, which then passes through the envelope. porous E. The rehydration of the matrix, then the micra-organisms of nucleus N <Figure 2B> is then slowly and progressively, then the envelope E tears under the pressure of the matrix and the contents of the granule are released in the medium <Figure 3C> Firstly, the rehydrated micro-organisms absorb the nutrients from the matrix that are nearby, then they acquire their autonomy and disperse in the medium.

To illustrate the good preservation of the inoculum according to the invention, it has been manufactured, by the process just described above, bacterial granules based on
Pseudomonas f2 uorescens.

 During manufacture, the viability of Feudomonas fluorescens cells decreases to about 50,000 to 100,000 viable cells per dry granule when a starting concentration of 10 bacteria per milliliter is used. This is illustrated by FIG. 3, which is the 25 * Ci2-C viability curve of these cells (strain L 26-1), the granules having been dissolved at 4 ° C. in phosphate buffer 0, 02M; pH 6.4.

Legend of Figure 3
A: before mixing
B: after mixing
C. after formation of the beads
(core + envelope> and
D: after filming and drying.

 Subsequently, the conservation of the inoculum appears to be satisfactory since, on the 60th day, at the laboratory temperature 25 ° C.sub.2 ° C., the number of bacteria per granule is still 5000 to 10,000, as illustrated by FIG. FIG. 4, the granules being in the same conditions as in the case of FIG.

Certain strains of Rhizobacteria (group Pseudomonas fluorescens - P. putida> have a stimulating effect on the growth and protection of plants.When bacteria root system lettuce for example, we can obtain a significant gain in growth and The results obtained for various experimental plants are indicated in the table.
BOARD
BACTERIZATION OF SUBSTRATES
Improved yields according to the age of the plant
SUBSTRATE PLANT *% GAIN AGE
MAX./TEMOIN (days)
PELARGONIUM
Fresh weight of the plant T66% + S33 16.0 100
Number of cuttings id 31,4 100
Number of cuttings T20'h + E60 t HF20% 47,4 100
LETTUCE
Fresh weight of the sheet T100% 21.0 45
Fresh weight of the sheet Te 50h + Tr 50% 19,0 53
Precocity of the apple head id 113,0 53 CHRYSANTHEXE
Height of the plant E45% + t 35% + T20% 30.4 36
Number of leaves spread id 26,9 36
APPLE (Malus x 'Evereste'>
Plant height T 75% + S 25% 44.0 45
Height of the plant id 73,0 102
Height of the plant id 12,0 480
Number of branches 20.4 480
APPLE 'Golden Smothee'
Fruit weight natural soil 11.5 1460 t LEGEND: T: Brown peat E: Pine bark
S: Loire sand t: Blonde peat
Te: Terra maraicher HF: Wood fiber ("Hortifibre")
Tr: Frank earth.

Claims (23)

 1 - Inoculum which is formed of microorganisms included in a support and which is in the form of granules, characterized in that the granules comprise <a) a core consisting of a coating feeder matrix  the microorganisms to be inoculated, said matrix  feeder being composed   <1) a complete nutrient extract for said micro  organisms in which they are 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 liable to undergo  biological degradation; and  (2> at least one hydroretenting agent.  2 - Inoculum according to claim 1, characterized in that the granules further comprise (c> a protective hydrophilic film surrounding  the envelope <b>.  3 - Inoculum according to one of claims 1 and 2, characterized in that the microorganisms are brought into the core <a> in the form of a suspension.  4 - Inoculum according to one of claims 1 to 3 having a very high concentration of microorganisms, characterized in that the matricenourricière has formed a culture medium of said microorganisms before the formation of granules.  5 - Inoculum according to one of claims l to 4, characterized in that the complete nutrient extract (al) is of plant origin.  6 - Inoculum according to claim 5, characterized in that the complete nutrient extract "al" is an extract based on algae, which are chosen in particular from the species Fucus, Laminarla and Ascopbyllum, said extract being brought to pH 7 .  7 - Inoculum according to one of claims 1 to 4, characterized in that the complete nutrient extract <ai> is an extract of animal origin.  8 - Inoculum according to one of claims 1 to 7, characterized in that the hydrorétenteurs agents (a2) and <b2> consist of clays, such as bentonite, kaolin, talc and vermiculite.  9 - Inoculum according to one of claims 1 to 8, characterized in that the film-forming agents (b1) are chosen from alginates, chitosan and other biopolymeries.  10 - Inoculum according to one of claims 1 to 9, characterized in that the envelope <b) further contains at least one phytostimulant substance and / or at least one phytosanitary substance compatible with the microorganisms present in the nucleus (a ).  11 - Inoculum according to one of claims 2 to 10, characterized in that the film (c> is constituted by a very hydrophilic clay, such as bentonite.  12 - Inoculum according to one of claims 1 to 11, characterized in that in the granules, after dehydration, the feeder matrix comprises 5 to 10 g of the extract (a1) and 1 to 5 g of (or a) hydroretentering agent (s) (a2), for 10 to 15 ml of water, and that the envelope <b) comprises from 1 to 5 g of the film-forming agent (s) <s> (b1) and from 5 to 20 g of the hydro-retaining agent (s) (b2) for 10 to 15 ml of water.  13 - Inoculum according to claim 12, characterized in that its initial formulation is as follows - for the feeder matrix <a), 5 to 10 g of the extract  (a1) and 1 to 5 g of the agent (s) hydrorétenteureur (s)  (a2) per 100 ml of water, the micro-organisms being  present in said matrix, in particular at a  concentration in the range of 107 to 1010 bacteria per ml  of veal for bacteria, and of the order of 103 S  Propagules per ml of water for fungi - for the <b> shell, 1 to 5 g of the agent (s)  film-forming agent (s) (b1) and 5 to 20 g of the agent (s)  hydroretenteur (s) <b2) per 100 ml of water.  14 - Inoculum according to one of claims 1 to 13, characterized in that the granules are in the form of parasphere elements whose average core diameter <a> is between 2 and 6 mm and the thickness of which the envelope <b) is between 0.5 and 3 mm, the outer film, when present, having a thickness of between 1 and 3 mm.  15 - Inoculum according to one of claims 1 to 14, characterized in that the microorganisms are selected from bacteria, such as Rhizobacteria, and fungi, such as Mycorrhizae.  16 - Inoculum according to one of claims 1 to 15, characterized in that the nutrient extract in which the microorganisms are coated, also serves as a coating to seeds.  17 - Process for preparing the inoculum as defined in one of Claims 1 to 16, in which a film-forming agent capable of passing from the soluble state to the insoluble state, characterized by the fact that (A ) in an aqueous medium, the  core components <a> way pellet & get  a first system of pasty consistency <B> is associated with each other, in an aqueous medium, the  components of the <b> envelope, so as to obtain a  second system of pasty consistency, in which  the film-forming agent is in the soluble state (C) the first and second systems mentioned above are passed through  in two coaxial nozzles, the first system, in  the central nozzle, and the second system, in space  ring delimited by the two aforementioned nozzles,  way that at the exit of this double nozzle, we get  drops of the core material surrounded by the  CD casing material> these drops are dropped into an aqueous medium in  the film-forming agent goes into the insoluble state,  to obtain, as a result of the hardening of said agent  film-forming, granules dispersed in said medium (E) said granules are collected and, if appropriate, dried; and <F> if desired, these pellets are coated in  film material.  18 - Process according to claim 17, characterized in that in step <B), is used ,. as the film-forming agent, an alginate in the form of sodium alginate in pure water, and that in step (D), a granular bath of a calcium salt is used as an aqueous medium for granule formation. , as succinate or gluconate, so as to carry out the displacement of Na ions by Ca ions  19 - Method according to one of claims 17 and 18, characterized in that before carrying out the combination of step <A), the microorganisms are cultured in the medium consisting of the nutrient extract (a1), in agitation, and at an optimum temperature for the microorganisms considered.  20 - Use of the inoculum as defined in one of claims 1 to 15, for introducing into the substrates and in the soil, in particular to regenerate, bacteria and fungi beneficial for plants.  21 - Use of the inoculum as defined in one of claims 1 to 15, for inoculating soil and substrates with pathogenic bacteria or fungi, for example to study varietal resistance.  22 - Use of the inoculum as defined in one of claims 1 to 15 for inoculating fermentative bacteria in the silage to accelerate the fermentation process.  23 - Use of the inoculum as defined in one of claims 1 to 15, for introducing bacteria or yeasts into agri-food environments or for reconstituting microflora of the digestive tract by incorporation into food or by direct ingestion .