EP3609477A1

EP3609477A1 - Pectin microcapsules, method for the manufacture and use thereof

Info

Publication number: EP3609477A1
Application number: EP18719973.2A
Authority: EP
Inventors: Aurélie RIEU; Jean Guzzo; Ali ASSIFAOUI; Arnaud HEUMANN
Original assignee: Universite de Bourgogne; Institut National Superieur des Sciences Agronomiques de lAlimentation et de lEnvironnement
Current assignee: Universite de Bourgogne; Institut National Superieur des Sciences Agronomiques de lAlimentation et de lEnvironnement
Priority date: 2017-04-13
Filing date: 2018-04-12
Publication date: 2020-02-19
Also published as: FR3065162B1; FR3065162A1; WO2018189490A1; US20200155470A1

Abstract

The invention relates to microcapsules intended to protect probiotics, comprising a shell and a core wherein the probiotics are dispersed in the form of biofilms distributed in distinct clusters, characterised in that the shell and the core are composed of pectin. The invention also relates to the method for manufacturing the capsules, the use thereof, and a formulation comprising the microcapsules according to the invention.

Description

"Pectin microcapsules, its manufacturing process and uses"

TECHNICAL FIELD TO WHICH THE INVENTION REFERS

The present invention relates to the field of probiotics. In particular, the present invention relates to pectin microcapsules comprising biofilm probiotics, its method of manufacture and uses thereof.

BACKGROUND

The gastrointestinal tract of man has 10 ¹⁴ micro-organisms in cohabitation, most of which are in the colon with a concentration around 10 ¹¹ -10 ¹² bacteria / mL. Thanks to molecular and culture techniques in constant evolution, to date more than 1000 bacterial species have been identified as resident of the human gastrointestinal tract and an individual would host at least 160 different bacterial species. This intestinal population, known as the gut microbiota, plays an important role in host physiology (digestion and assimilation of nutrients, protection against colonization of pathogens and modulation of immune responses). Dysbiosis of the intestinal microbiota corresponding to an imbalance in its composition could be associated with many human pathologies, such as obesity, Crohn's disease, or ulcerative colitis. The ingestion of probiotics can modify the composition of the intestinal microbiota and thus allow to restore its equilibrium.

The microorganisms most used as probiotics are bacteria belonging to the genera Lactobacillus and Bifidobacterium, natural hosts of the gut microbiota of humans. The effectiveness of the probiotics is strain-specific, and each strain can contribute to the health of the host through different mechanisms. Among these mechanisms, the modulation of immune functions at the intestinal level is sought for probiotics in relation to their anti-inflammatory capacity.

However, after ingestion, probiotics face a number of environmental stresses before arriving at their site of action, such as gastric acidity, the presence of hydrolytic enzymes or bile salts produced by the intestine. hail.

However, to be effective on the intestinal flora, probiotics must achieve this in sufficient numbers. In particular, they must be able to withstand gastric acidity and pancreatic juice during their passage through the stomach to be released alive in the intestines. In fact, about 90% of the probiotics ingested are destroyed in the face of these physicochemical stress conditions (acidic pH and high ionic strength).

To compensate for bacterial mortality and allow bacteria to better withstand the stresses encountered in the gastrointestinal tract, a first solution Provided by the state of the art consists in formulating the probiotics with a large bacterial load. Indeed, the amount of live probiotics during ingestion must be sufficient for them to persist in the gastrointestinal tract.

However, this first solution is not satisfactory insofar as many probiotics do not resist the stress encountered in the gastrointestinal tract before arriving at their site of action. In addition, the cost of production related to this approach is quite expensive and affects the final price of probiotics.

A second solution is to encapsulate the probiotic bacteria in a matrix, usually alginate, and / or to adapt the probiotic bacteria to the stress conditions encountered in the gastrointestinal tract before possibly encapsulating them.

The publication of Cheow et al., 2013, BioMacromolecules, describes, for example, chitosan-coated alginate or carrageenan microcapsules comprising probiotics (Lactobacillus rhamnosus GG) which have been incubated in situ so as to form a biofilm. This publication shows that the encapsulation of probiotics in the form of biofilm makes it possible to improve the viability of probiotics when they are subjected to the stress due to lyophilization or to a prolonged stay in heat (60 to 70 ° C). However, it has been demonstrated that alginate or carrageenan microcapsules comprising probiotics in biofilm form do not make it possible to improve their viability in a simulated gastric liquid compared to microcapsules comprising planktonic probiotics. This publication concludes that chitosan-coated alginate capsules represent the most appropriate probiotic delivery system due to their release profile and viability during storage. It also indicates that there is a need in the state of the art for providing new capsules that incorporate additional protective materials to protect probiotics from thermal and acid exposures.

The publication of Cheow et al., 2014, Carbohydrate Polymers, which is a continuation of the aforementioned publication, discloses microcapsules of alginate / carob seed meal and / or alginate / xanthan gum comprising a biofilm of Lactobacillus rhamnosus GG formed in situ. These microcapsules have a better resistance to stress and in particular to the acid medium than the microcapsules described above.

However, these microcapsules, although satisfactory, can be improved to allow, for example, optimal adhesion of probiotics in the intestines.

Also known from the state of the art, the publication of Nabil Aoudi, and Aurélie Rieu, et al. 2014, Cellular Microbiology, which notably discloses that bacteria, including Lactobacillus casei, in the form of a biofilm, have anti-aging properties. inflammatory bowel movements superior to those same bacteria grown in planktonic form.

Thus, the culture conditions of the probiotics and the microcapsule / capsule formulation methods remain to be defined and optimized, in particular to improve the survival of the probiotic bacteria, as well as their functionality in the intestine.

It is therefore desirable to have a new formulation that can provide probiotic bacteria with a level of survival at the different levels of the gastrointestinal tract that is satisfactory, namely that allows to produce probiotic bacteria in a viable and active form so that it can act at the level of the intestine.

It is also useful to have a new formulation that can be simple to implement, economical and feasible on an industrial scale.

The object of the present invention is thus to provide a new microcapsule avoiding at least partly the aforementioned drawbacks and in particular to improve the survival of probiotic bacteria in the gastrointestinal tract, while being simple to implement.

In particular, an object of the invention is to provide microcapsules for improving the survival of probiotic bacteria in the stomach, to vectorize them into the colon and to release them in a physiological state favorable to their implantation and to their probiotic activity.

OBJECT OF THE INVENTION

Thus, the present invention relates to microcapsules intended to protect probiotics (bacteria or yeasts, for example), comprising an envelope and a core in which said probiotics are dispersed in the form of biofilms distributed in distinct clusters, characterized in that said envelope and said heart are composed of pectin.

According to the invention, "probiotics" are defined as living microorganisms (yeasts or bacteria) exerting a beneficial action on the health of the host which ingests them by improving the balance of the intestinal flora, beyond the effects traditional nutritional products. This definition has been approved by the United Nations Food and Agriculture Organization (UNAA) and the World Health Organization (WHO).

According to various embodiments of the invention, the following characteristics may be used alone or in any technically possible combination: the biofilms derived from probiotics are formed in situ;

the concentration of probiotics in microcapsules varies from 8 to 1 log CFU / g, preferably varies from 9.5 to 10.5 log CFU / g, and typically is 10 log CFU / g;

said envelope and the core composing the microcapsules are of amidated pectin and methylated having:

a degree of amidation (DA) ranging from 5 to 30%, preferably from 20 to 30%, and

a degree of esterification (DE) ranging from 5 to 50%, preferably from 20 to 30%;

the probiotics are chosen from: a probiotic bacterium, such as Lactobacillus, Bifidobacterium, Enterococcus, Propionibacterium, Bacillus and Streptococcus, etc. or a yeast, such as a yeast of the genus Saccharomyces cerevisiae, Saccharomyces. boulardi, etc., or a mixture thereof;

said heart comprises another non-probiotic active substance (ie different from a probiotic bacterium or a yeast), such as a polyphenol, a vitamin, a prebiotic, such as inulin, fructo-oligosaccharides (FOS ), etc., or a mixture thereof;

the microcapsules are in dehydrated (for example freeze-dried) or frozen form, so as to optimize their preservation.

The present invention also relates to a probiotic formulation, characterized in that it comprises at least the microcapsules as described above.

Another subject of the present invention relates to a method of manufacturing microcapsules as described above, comprising the following steps:

(i) preparing a homogeneous mixture composed of at least: an oil / water pectin solution or emulsion containing a suspension of probiotics;

(ii) encapsulating droplets of this homogeneous mixture in a crosslinking solution, so as to form microcapsules comprising: a pectin shell and a pectin ring forming core, wherein the probiotics are immobilized;

(iii) culturing the microcapsules obtained at the end of step (ii) in a culture medium, so as to form in situ within the microcapsules biofilms distributed in distinct clusters from the immobilized probiotics;

(iv) optionally, the dehydration (such as lyophilization) or the freezing of the microcapsules obtained at the end of step (iii).

According to various embodiments of the invention, the following characteristics can be used alone or in any technically possible combination:

the homogeneous mixture of step (i) comprises a pectin content, by weight, relative to the total volume of the solution, ranging from 2% (w / v) to 10% (w / v), preferably from 3% (w / v) to 8% (w / v) and typically 3.5% (w / v) to 5% (w / v);

the mixture of step (i) comprises a probiotic concentration ranging from 10 ⁵ CFU / ml to 10 ⁹ CFU / ml, preferably from 10 ⁶ CFU / ml to 10 ⁸ CFU / ml and typically of the order of 10 ⁷ CFU / mL;

the encapsulation step (ii) comprises a first substep of injection (iia) by means making it possible to drip the mixture of step (i) into the solution of crosslinking stirred, so that each drop forms a microcapsule when it comes into contact with the crosslinking solution;

the encapsulation step (ii) comprises a second sub-step called crosslinking of the microcapsules (iib) comprising the maturation of the microcapsules obtained during the injection sub-step (iia) for a period of from minus 3 minutes, preferably from 5 to 60 minutes, and in particular from 10 to 25 minutes;

the crosslinking solution is composed of divalent cation, such as Ca ²⁺ , Zn ²⁺ , Ba ²⁺ , Fe ²⁺ in the form of chloride, sulphide, acetate, or a mixture thereof, and is in particular Ca ²⁺ in the form of chloride;

in step (i), another non-probiotic active substance, such as polyphenol, vitamins, minerals, a prebiotic, etc. is added to the homogeneous mixture.

The present invention also relates to the use of the microcapsules as described above, or of the abovementioned probiotic formulation comprising said microcapsules, or microcapsules obtained according to the above-mentioned method, for protecting the probiotics during passage in the stomach and for deliver them in an active form to the intestine in an animal.

By "animal" according to the invention is meant mammals, birds, fish, insects, etc., as well as humans.

The present invention also relates to the microcapsules described above, or the abovementioned probiotic formulation or microcapsules obtained according to the process described above, for its use as a medicament.

The invention also relates to microcapsules described above, or to the abovementioned probiotic formulation or microcapsules obtained according to the method described above for use in animals in order to:

to prevent and inhibit the proliferation of pathogenic agent; or

- to modulate the immune response; or

to influence the production of virulence factors of the pathogenic agents within the host organism; or

to treat a dysbiosis of the intestinal microbiota.

For the remainder of the description, unless otherwise specified, the indication of a range of values "from X to Y" or "between X and Y" in the present invention means as including the X and Y values.

DESCRIPTION OF THE FIGURES

The following description with reference to the accompanying drawings, given as non-limiting examples, will make it clear what the invention consists of and how it can be achieved.

In the accompanying drawings: FIG. 1 is (a) a confocal microscopy observation of a biofilm of Lcasei ATCC334 Example 1 on a flat pectin film according to kinetics of biofilm formation at t = 24 h; and (b) is a 3D representation of this 24 h biofilm (Syto9 / IP tagging); and

FIG. 2 is an observation in confocal microscopy: (a) an overview (730 μηη scale) representing 12 pectin microcapsules according to example 1 of the invention which were incubated in an MRS culture medium in which, within each microcapsule, the various distinct clusters of biofilm comprising the probiotics L. casei ATCC334 are distinguished, (b) an enlarged view (scale 150 μηι) of the microcapsules of FIG. 2 (a) in which one can see 4 microcapsules inside which are arranged the distinct clusters of biofilm included in the pectin network; and (c) an even more enlarged view (scale 5 μηη) of the interior of a microcapsule of Figure 2 (b) and showing two clusters of biofilm;

FIG. 3 is a cryo-scanning electron microscopy observation: (a) an overview (2.0 mm scale) of several pectin microcapsules according to example 1 of the invention which have been incubated in a MRS culture medium; (b) an enlarged view (100 μηι scale) of the inside of a microcapsule of FIG. 3 (a) in which distinct clusters of biofilm (including L. casei ATCC334 bacteria) distributed in the network can be distinguished; pectin; (c) an enlarged view (scale 40.0 μηι) of FIG. 3 (b) in which the clusters of biofilm (two in the figure) comprising the bacteria, (d) and (e) are further distinguished; an even larger view (scale 10.0 μηη) of the interior of a biofilm cluster of FIG. 3 (c) in which the probiotics which secreted a matrix forming a biofilm in the form of clusters are distinguished, the adhering biofilm at the pectin network wall;

FIG. 4 is a graph showing the weight loss of mice over time with DSS treatment (from day 5 to day 11): the condition "physiological water + DSS" corresponds to the mice that have received the DSS treatment and fed with physiological water; the "formulation + DSS" condition corresponds to the mice treated with DSS and gavaged with a formulation comprising the microcapsules according to the invention (pectin microparticles containing L. casei probiotic biofilms);

FIG. 5 and FIG. 6 are graphs representing the quantification over time of the bacteria of the genus Lactobacillus spp (FIG. 5) and L. casei (FIG. 6) in the faeces of the group of mice which have received the formulation according to said invention. as shown in Figure 4 (pectin microparticles containing L. casei probiotic biofilms).

DETAILED DESCRIPTION OF AN EXEMPLARY EMBODIMENT

The following description will make it clear what the invention consists of and how it can be achieved.

The Applicant has focused on the development of new microcapsules including probiotics able to withstand gastric acidity and that of pancreatic juice during their passage in the stomach to release viable probiotics in the intestines where they have particular beneficial action on the health of the host that ingests them.

The Applicant has unexpectedly discovered that microcapsules comprising a pectin shell and a pectin-forming core in which probiotics in the form of a biofilm are immobilized make it possible to obtain this technical effect.

Indeed, the Applicant has shown that such microcapsules pectin could increase the survival of bacteria or probiotic yeasts at different levels of the gastrointestinal tract and release them in viable and active form in the intestine of the 'host. The microcapsules according to the invention, after ingestion, thus make it possible to resist the gastric acidity, to that of the pancreatic juice, as well as to the hydrolytic enzymes and to implant themselves, moreover in the intestine.

Thus, the Applicant has developed a new formulation of microcapsules simple to implement, which does not require additional protective materials to withstand gastric stress as suggested in the publication Cheow et al., 2013.

In fact, surprisingly, the simple use of pectin among all the existing polysaccharides makes it possible to form microcapsules having a pectin shell and a core composed of a pectin network having a similar resistance, or even an improvement in gastric stress compared to Microcapsules of the prior art for which it is necessary to apply at least two layers of polysaccharide (alginate / chitosan) to protect bacteria or probiotic yeasts.

The use of pectin is furthermore not suggested in the prior art insofar as the three-dimensional network formed by pectin in the presence of calcium ions is relatively heterogeneous with respect to the alginate network. This is due to the presence of ester and amide groups and the presence of branched zones in pectin (Assifaoui et al., Soft Matter 2015). However, surprisingly, this heterogeneity of structure is responsible for the development of bacteria and probiotic yeasts in biofilms. Thus, unlike alginate, which has a linear structure, the particular pectin gel network unexpectedly allows for better growth of probiotic bacteria and yeasts within that network.

Thus, the present invention firstly relates to microcapsules intended to protect probiotics, comprising an envelope and a heart in which said probiotics are dispersed in the form of biofilms distributed in distinct clusters, characterized in that said envelope and said core are composed of pectin.

"Biofilm" refers to structured communities of bacteria or yeast enclosed in a self-producing polymer matrix that adheres to a living or inert surface (Costerton et al., 1999).

In particular, biofilms derived from probiotics are formed in situ.

It has indeed been demonstrated by the inventors that the probiotics and in particular the probiotic bacteria of the genus Lactobacillus, grown in biofilm, are more resistant to stress mimicking the conditions encountered in the gastrointestinal tract and moreover exhibit an activity. increased anti-inflammatory.

Thus, the biofilm formation of bacteria and probiotic yeasts combined with the use of pectin, makes it possible to form microcapsules with good resistance to environmental stresses encountered between ingestion and the site of action (colon). The biofilm is also preserved until the place of delivery of the bacteria. In fact, the microcapsules according to the invention are capable of releasing at the level of the colon the bacteria with a biofilm phenotype, that is to say having, on the one hand, adhesion properties which are much greater than the planktonic cells and on the other hand on the other hand, increased probiotic activity (immunomodulation). Indeed, the enzyme that degrades pectin is naturally present in the colon. It is therefore a targeted delivery of bacteria in a selected compartment of the intestine, the colon. Once naturally released from the envelope and matrix pectin, probiotics in the form of biofilm can be fixed in the intestinal villi of the colon and exert their beneficial effects on the health of their host.

Generally, the probiotic bacteria and yeast suitable for the present invention are thus able to form a biofilm and can be chosen from: Lactobacillus, Bifidobacterium, Enterococcus, Propionibacterium, Bacillus and Streptococcus or a mixture thereof.

For example, the probiotic bacteria may be chosen from: acidophilus L. crispatus L. gasseri L. delbrueckii L. salivarius L. casei L. paracasei L. plantarum L. rhamnosus L. reuteri L. brevis, L. buchneri, L. fermentum, B. adolescentis, B. angulation, B. bifidum, B. brief, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophilic, or a mixture thereof, preferably the probiotic bacteria are L. casei and L. rhamnosus, or a mixture thereof.

The probiotic yeasts that are suitable for the present invention can be chosen from: Saccharomyces cerevisiae, Saccharomyces boulardii, etc. or a mixture thereof.

Advantageously, the concentration of probiotics in microcapsules is very high. It varies, for example, from 8 to 1 log CFU / g, preferably from 9.5 to 10.5 log CFU / g, and is typically 10 log CFU / g. In addition, according to the invention, other non-probiotic active substances, namely different from a bacterium or a yeast, can be included in the core of the microcapsules. In particular, they are trapped in the pectin network.

These other active substances may be chosen in particular from: a polyphenol, a vitamin (thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), folic acid (B9) and cyanocobalamin (B12), a mineral (magnesium, calcium, iron, etc.), a prebiotic, such as inulin, FOS, etc., or a mixture thereof.

The addition of these other substances such as polyphenol may allow for example to potentiate the effect of biofilms.

Indeed, among the food compounds, polyphenols of plant origin are very reactive molecules which, according to in vitro and in vivo studies carried out in mice, can have a strong impact on the signaling of the intestinal cells, but also on the bacteria of the gut microbiota.

For example, it has been demonstrated for several polyphenols that they can induce an increase in the adhesion and proliferation of probiotic bacteria such as Lactobacillus rhamnosus on enterocytes (Parkar et al., 2008, The potential influence of fruit polyphenols on microflora colony and human gut health, International Journal of Food Microbiology (124), pp. 295-298). In addition, intestinal bacteria are known to convert polyphenols into metabolites that may have important activities on the modulation of intestinal homeostasis (van Duynhoven et al., 201 1, Metabolic fate of polyphenols in the human superorganism, Proceedings of the National Academy of Sciences of the United States of America (108), 4531-4538).

It also appears that the bioavailability of minerals such as calcium, iron, zinc, manganese, copper and phosphorus is increased in fermented dairy products relative to milk. Therefore, the microcapsules according to the invention advantageously contain a high amount of vitamins and minerals that will be easily assimilated by the body.

According to the invention, the "prebiotics" are short chain oligosaccharides or polysaccharides consisting of approximately two to twenty units of sugar. They escape digestion in the small intestine and are potential substrates for hydrolysis and fermentation by intestinal bacteria.

It is known that prebiotics can increase the absorption of minerals (especially calcium and magnesium) in the colon, decrease bone loss, and have an effect on immune function. For example, the administration of inulin, oligofructose and trans-galactooligosaccharides leads to a selective increase in faecal concentration of bifidobacterial populations. As mentioned above, the envelope and the core of the microcapsules according to the invention are pectin.

In particular, the microcapsules according to the invention comprise or may consist of an envelope and a heart in which said probiotics are dispersed in the form of biofilms distributed in distinct clusters, characterized in that said envelope and said core are composed of pectin. According to this mode, the microcapsules are only composed of pectin to form the envelope and the heart, that is to say that the microcapsules do not include other polysaccharides to form their heart and their envelope.

In addition, in general, the microcapsules according to the invention do not comprise a coating, ie they are not covered by another protective material, such as a polysaccharide, such as chitosan.

According to the invention, pectin is a polysaccharide of vegetable origin characterized by an α-D-galacturonic acid backbone and small amounts of α-L-rhamnose more or less branched. In particular, it comprises a sequence of two majority structures: a homogalacturonic main chain (or "smooth zone", called HG) and a rhamnogalacturonic chain (or "spiky zone", called RG).

Homogalacturonans are the main chain that makes up pectins (generally representing more than 60% of pectins). These are α-D-galacturonic acid polymers bound in (1 → 4). The length of these chains can range from 70 to 100 galacturonic acid residues in lemon, sugar beet or apple, that is to say having molecular weights of about 12 to 20 kilodalton (kDa). .

The carboxylic function of α-D-galacturonic acids may be in acid form, or ionized by different cations including calcium, or esterified with methanol. Furthermore, the galacturonic acids can be acetylated at 0-2 and / or O-3. Depending on these esterifications, the pectins are characterized by a degree of methylation (DM) and a degree of acetylation (DAc) which correspond to the ratio esterified galacturonic acids (respectively methylated or acetylated) on the total galacturonic acids.

From a functional point of view, three categories of pectins stand out:

if the degree of methylation is less than 5% (DM <5), it is pectic acids;

- if the degree of methylation is less than 50% (DM <50), it is poorly methylated pectins;

if the degree of methylation is greater than 50% (DM> 50), it is highly methylated pectins (HM).

The degree of esterification of pectins has an impact on the flexibility of the molecule: the lower the degree of esterification, the more rigid the pectin. He also has a strong impact on their gelation properties.

In general, said envelope and the core composing the microcapsules are in low methylated pectin which has a degree of esterification (DE = DM) ranging from 10 to 50%, preferably from 20 to 30% and typically of the order of 28. %.

The carboxylic acid function of α-D-galacturonic acids may, during an industrial demethylation treatment in an ammoniacal medium, be amidated. In this case, the pectin is amidated and has a degree of DA amidation.

Preferably, said envelope and the core comprising the microcapsules are amidated pectin having a degree of amidation (DA) ranging from 3 to 30%, preferably from 20 to 30% and typically of the order of 24%.

The degrees of amidation (DA) and esterification (DE) are determined by the titration method (Food Chemical Codex, 1981) (Washington, DC: National Academy). of Sciences.

Advantageously, the microcapsules according to the invention have a mean diameter ranging from 100 μηη to 5,000 μηι, preferably ranging from 250 μηη to 1,000 μηη and in particular ranging from 400 μηη to 800 μηι.

According to one characteristic of the invention, the microcapsules are in dehydrated form (for example freeze-dried), or frozen, so as to optimize their preservation.

Then, the present invention also relates to a probiotic formulation, in particular for animals, characterized in that it comprises at least the microcapsules as described above.

In particular, the probiotic formulation may be in various galenical forms, preferably for oral administration, such as a capsule, a soft capsule, a tablet, a beverage, an ampoule, a powder or any other ingestible dosage form. by a host or allowing the preparation of drinks or nutritional dishes.

For example, the drinks or nutritional dishes may be fermented milk, an ice dairy product, a cereal bar, a non-alcoholic beverage, dietary supplements, a nutritional supplement, croquettes and treats for pets, etc.

As mentioned above, the present invention also relates to a method of manufacturing microcapsules as described above, comprising the following steps:

(i) preparing a homogeneous mixture comprising a solution or an emulsion oil / water of pectin, preferably sterilized, containing a suspension of probiotics;

(ii) the encapsulation of droplets of this mixture in a solution of crosslinking, so as to form microcapsules comprising: a pectin shell and a pectin network core, wherein the probiotics are immobilized;

Steps (i) to (iv) will be described below.

Firstly, a solution or an oil / water pectin emulsion is prepared on the one hand and a suspension of probiotics on the other hand; then the two preparations are mixed until homogenization and the homogeneous mixture is obtained.

Of course, all the features described above for the microcapsules according to the invention also apply to the process of the invention and vice versa.

In particular, the pectin included in the homogeneous mixture is preferably weakly methylated and in particular has a degree of esterification (DE = DM) ranging from 10 to 50%, preferably from 20 to 30% and typically of the order of 28. %. The pectin may also be amidated and has a degree of amidation (DA) ranging from 3 to 30%, preferably from 20 to 30% and typically of the order of 24%.

According to a first characteristic of the process, the homogeneous mixture comprises a pectin content, by weight, relative to the total volume of the mixture, ranging from 2% (w / v) to 10% (w / v), preferably 3% (m / v) at 8% (w / v) and typically 3.5% (w / v) to 5% (w / v).

According to a second characteristic of the process, the homogeneous mixture of step (i) comprises a probiotic concentration ranging from 10 ⁵ CFU / mL to 10 ⁹ CFU / mL, preferably from 10 ⁶ CFU / mL to 10 ⁸ CFU / mL. and typically of the order of 10 ⁷ CFU / mL.

It is possible to add to the homogeneous mixture of step (i), another non-probiotic active substance, such as polyphenol, vitamins, minerals, a prebiotic, or a mixture thereof. In general, this other active substance represents, by weight, relative to the total mass of the homogeneous mixture of from 0.1 to 30%, preferably from 0.1 to 20% and typically from 5% to 15%.

Then, proceed to the encapsulation step (ii).

Generally, this encapsulation step (ii) comprises a first injection sub-step (iia) by means making it possible to drop the homogeneous mixture of step (i) into the stirred crosslinking solution, so that each drop forms a microcapsule when it comes into contact with the crosslinking solution.

For example, this injection step (iia) can be performed by means of a hand shower. In this case, the cutting of the drops is done by gravity and allows in particular to obtain microcapsules having a mean diameter ranging from 1000 to 5000 μηη.

This step (iia) can also be performed by means of an encapsulator.

In this case, the cutting of the drops may be, according to one embodiment, be performed by applying an electric field. The applied electric field ranging from 0.1 kv to 10 kv, preferably from 2 kv to 10 kv and typically from 5 kv to 8 kv. This technique generally makes it possible to obtain microcapsules having a mean diameter ranging from 400 to 1000 μηι.

According to another embodiment, the cutting of the drops can be performed by vibration. This technique generally makes it possible to obtain microcapsules having a mean diameter ranging from 100 to 1000 μηη.

Whatever the means for drip, the drip rate generally varies from 20 μs to 300 μ-ds, preferably ranges from 40 μs to 100 μs, and typically ranges from 40 μs to 80 μs. Us; while the injection height of the homogeneous mixture of step (i) in the crosslinking solution varies from 1 cm to 15 cm, preferably ranges from 2 cm to 10 cm and typically ranges from 2 cm to 6 cm.

Preferably, the crosslinking solution is composed of divalent cation, such as Ca ²⁺ , Zn ²⁺ , Ba ²⁺ , Fe ²⁺ , for example in the form of chloride, sulphide or acetate, or is composed of one of their mixtures. Typically, the crosslinking solution is composed of Ca ²⁺ ions in chloride form.

In particular, the divalent ion concentration of the crosslinking solution ranges from 25 to 750 mM, preferably from 200 to 750 mM, and in particular from 500 to 750 mM.

Following this first substep (iia), the encapsulation step (ii) comprises in particular a second sub-step called crosslinking microcapsules (iib). This second substep comprises the maturation of the microcapsules obtained during the injection sub-step (iia) for a period of at least 8 minutes, preferably ranging from 0 to 60 minutes, and in particular ranging from at 25 minutes.

Advantageously, this substep of crosslinking (iib) is carried out at a temperature below 40 ° C, preferably below 30 ° C and in particular ranging from 4 ° C to 25 ° C.

Following this encapsulation step, the microcapsules thus comprise a pectin envelope and a heart formed of a pectin network and in which the probiotics are immobilized.

Advantageously, the microcapsules obtained according to the process of the invention have a mean diameter ranging from 100 μηη to 5000 μηη, preferably ranging from 250 μηη to 1,000 μηη and in particular ranging from 400 μηη to 800 μηι.

Then, prior to step (iii) of culturing, the microcapsules obtained at the end of step (ii) are preferably recovered, generally by gravitational sedimentation, and then rinsed, generally with distilled water. Then, the step of culturing the microcapsules (iii) in a culture medium, so as to form in situ within the microcapsules biofilms in separate clusters from immobilized probiotics.

By way of example, the culture medium during this culturing step (iii) is chosen from: MRS (deMan, Rogosa, Sharpe), AOAC (Association of Official Analytical Chemists), LB (Lysogeny Broth), TSB (Tryptic Soy Broth), TPY (Tryptone Phytone Yeast), BSM (Bifidus Selective Medium), Enterococcosel Broth (Bile Esculin Azide Broth), Nutrient Broth # 4, CA SO Broth (Casein Peptone Soybean), AC Broth (AH Culture Broth) ), Reinforced Clostridial Medium, BHI (Brain Heart Infusion), Bifidobacterium Broth, Tomato Juice Broth, or a mixture thereof, and is preferably selected from: MRS (deMan, Rogosa, Sharpe), AOAC (Association of Official Analytical Chemists) ).

Preferentially, the pH of the culture medium varies from 3 to 8, preferably from 4 to 7 and typically from 5 to 7.

The temperature of the culture medium varies, for example, from 20 to 40 ° C, preferably from 25 ° C to 37 ° C and generally from 25 to 30 ° C.

This step is generally carried out for a duration greater than or equal to 12 hours, preferably ranging from 12 to 72 hours and generally from 20 to 48 hours.

Finally, the microcapsules obtained at the end of step (iii) are optionally frozen or dehydrated.

One of the methods of dehydration is lyophilization. This freeze-drying step comprises, for example, the freezing of the microcapsules to about -80 ° C., for example in 10 mL vials, ie about 1 g of samples per vial, with a ramp of 8 ° C./min. . Before freezing, cryo-protectants (glycerol type, sugars, antioxidants, etc.) can be added with microcapsules in vials. The samples are then lyophilized, for example 20 hours at -55 ° C. with a pressure of 0.05 mbar, applying 5 steps (-40, 10, 0, 20 and 30 ° C.). After lyophilization, the samples are stored for example at room temperature.

Preferably, the freezing step is carried out by freezing the microcapsules, for example in 10 mL vials, ie about 1 g of samples per vial. Before freezing, cryo-protectants (glycerol type, sugars, antioxidants, etc.) can be added with microcapsules in vials. The freezing temperatures are for example between -20 ° C and -80 ° C, and the temperature decrease is for example with a ramp of 8 ° C / min.

Then, the present invention relates to the use of the microcapsules as described above, or the abovementioned probiotic formulation comprising said microcapsules, or microcapsules obtained according to the aforementioned method, for protecting the probiotics during passage in the stomach and delivering them in an active form to the gut in an animal. Finally, the present invention relates to microcapsules described above, or the abovementioned probiotic formulation or microcapsules obtained according to the process described above, for its use as a medicament.

Of course, all the features described above for the microcapsules according to the invention or for the process according to the invention also apply for the aforementioned medicament.

The invention also relates to the microcapsules described above, or the aforementioned probiotic formulation or microcapsules obtained according to the method described above, for use in animals to:

to prevent and inhibit the proliferation of pathogenic agent; or

- to modulate the immune response; or

to treat a dysbiosis of the intestinal microbiota.

Of course, all the features described above for the microcapsules according to the invention or for the process according to the invention also apply for the aforementioned uses.

EXAMPLES EXAMPLE 1 Preparation of PECTIN MICROCAPSULES AND A FLAT FILM OF PECTIN

For this test, two types of formulations have been developed:

(a) a form of pectin microcapsules according to the invention;

(b) a flat film form in order to better characterize the interactions between the bacterial biofilm and the pectin gel.

RAW MATERIALS

TABLE 1

^* These bacteria originate from a 1% revitalized culture for 24 h in MRS 5.8 culture medium from a 24 hr cryotube output culture in MRS pH 5.8. B) PROTOCOLS FOR THE PRODUCTION OF MICROCAPSULES OF PECTIN (a)

-i) preparation of a homogeneous mixture

First, powdered pectin, sterile, is dissolved in 4% (m / v) in autoclaved ultra pure water under a sterile atmosphere; then 10 ⁷ CFU / mL of bacteria are added. The whole is stirred magnetically so as to obtain a homogeneous mixture.

- ii) the encapsulation of droplets

A Nisco Var 1 type encapsulator was used (parameters: 8 kV, tip 0.7 mm) with a syringe pump (parameters: flow 98.4 μί / h, volume 0.20 mL). The homogeneous mixture obtained above is introduced into a 20 ml syringe before being injected into the encapsulator using the syringe pump. The 8 kV electric field will force the formation of microdroplets that will fall into a stirred CaCl ₂ crosslinking bath, each of which will form a microcapsule. The average diameter of the wet microcapsules thus produced is of the order of 1000 μηη.

Then, the gelation is carried out under a sterile atmosphere for 20 minutes by contact of the pectin solution with the sterile solution of CaCl ₂ at 0.75 M.

Finally, the gel is washed three times in autoclaved ultra-pure water to remove excess CaCl ₂ .

At the end of this step, microcapsules are obtained comprising: a pectin shell and a heart forming a pectin network, in which the probiotics are immobilized.

iii) culturing the microcapsules

The pectin microcapsules containing the immobilized bacteria (1 batch of bead = 4 ml of pectin) are incubated at different times (24 h - 48 h - 72 h) at 28 ° C. in 20 ml of culture medium, so as to form in situ in the microcapsules of biofilms divided into distinct clusters from immobilized probiotics.

Two different culture media are used to allow the growth of biofilm bacteria within pectin microcapsules, MRS medium pH = 5.8 (Conda) or AOAC medium pH 6.8 (Difco).

After incubation, the pectin microcapsules containing the bacterial biofilms are washed three times in ultra pure water (15 mL).

-iv) enumeration of bacteria in the pectin gel

The bacteria are liberated from the pectin gels. For this, the pectin gels (1 batch of microcapsules of 4 mL of pectin) are broken up using a buffer solution (50 mL) of 0.1 M sodium citrate, which allows to release and suspend in solution the bacteria. The re-suspended bacteria are then counted according to two methods: the method of the units forming the colonies or flow cytometry. ^* Colonies Forms Method

The solution of re-suspended bacteria is diluted in a cascade of 10 ^"1 to 10 ^{" 5} with physiological saline and 3 drops of 10 μί are deposited at 10 ^"5 to 10 ° dilutions on an MRS medium agar at pH 5, 8. The petri dish is then incubated for 48 hours at 28 ° C. This method makes it possible to measure the bacterial cultivability.

^* Flow cytometry

The solution of re-suspended bacteria is first washed: centrifugation of 1 ml at 10 000 g and recovery of the bacterial cells in 1 ml of 1 × filtered PBS. Then this bacterial suspension is diluted to be from 10 ^5/10 ⁶ bacteria / mL. Finally, the bacteria are labeled with cFDA (carboxy-Fluorescein DiAcetate) and with ΙΊΡ (Propidium lodide). The labeled bacterial solutions are then analyzed by flow cytometry (BD Acuri, C6). The cFDA labeling makes it possible to measure the bacterial viability while the marquage labeling makes it possible to measure the membrane integrity.

C) PROTOCOLS FOR MANUFACTURING BIOFILM ON FLAT PECTIN FILM (b)

-i) To develop flat films, the protocol consists in introducing a solution of pectin at 4% (m / v) in a petri dish before covering it with a dialysis membrane (Thermofisher, Snakeskin Dialysis Tubing, 10 kDa) . In particular, the pectin solution is obtained by dissolving powdered pectin, sterile in autoclaved ultra-pure water and in a sterile atmosphere so as to obtain a pectin content of 4% (w / v).

-ii) This box is then introduced upside down (membrane down) for 20 minutes in a bath of CaCl ₂ . The calcium ions will diffuse via the dialysis membrane into the pectin solution to allow gelation of the pectin. The gel is then dried to form a film.

-iii) To allow biofilm growth of the probiotic bacteria on the surface of the pectin film, it is introduced for different times (24 h or 48 h) at 28 ° C in culture medium (MRS medium or AOAC medium). ) inoculated with 10 ⁷ CFU / mL of bacteria. After these incubation times, the films are washed three times with ultra pure water.

-iv) Enumeration of bacteria released from pectin gels: the same protocols as those described in paragraph B) .- iv were applied. D) RESULTS

The results of the counts obtained are presented in Table 2 below and are expressed in Log (CFU) for 1 batch of microcapsules, which corresponds to the same amount of pectin as 1 flat film (ie 4 mL of 4% pectin ( m / v)). The gels are so-called "wet" when they do not undergo drying after gelation and that their water content is high.

"renewal mid to 24 h

TABLE 2

Two methods were compared for counting bacteria in pectin gels: the CFU method and flow cytometry. Both methods showed closely similar results, which show a good correlation between cultivability and viability of the bacteria in pectin gels.

This test shows that flat pectin films allow the biofilm growth of the probiotic bacteria tested L. casei. In particular, as shown in Figure 1, a uniform biofilm on the surface of the pectin film was observed by confocal microscopy.

In addition, biofilm growth of L. casei was also observed inside pectin microcapsules from bacteria immobilized in the pectin network (average increase of the initial population of 2 Log).

By confocal microscopy as shown in FIG. 2, it appears that the probiotics developed into spherical microcolonies (biofilms) distributed homogeneously and distinctly within the microparticles of pectin cultured in the MRS medium. Moreover, by cryo-scanning electron microscopy and as shown in FIG. 3a) to FIG. 3e), it also appears that L. casei probiotics have developed into spherical microcolonies (biofilm clusters) within "niches" formed by the pectin network within each microcapsule. Probiotics seem to adhere to this dit-network and form a true three-dimensional structure in the form of microcolonies (Figures 3 (d) and 3 (e)). A matrix secreted by probiotic bacteria exopolymeric substance) and binding them to each other is also visible in Figures 3 (d) and 3 (e).

In addition, the Applicant also tested under the same experimental conditions and successfully another strain Lactobacillus rhamnosus GG in the MRS culture medium for 24 hours. Indeed, biofilms were formed on a flat film of pectin, as well as inside the microcapsules of pectin.

EXAMPLE 2: Lyophilization of the Microcapsules of Example 1

Drying tests were conducted using freeze-drying, a method widely used in the probiotic and ferment industry, but which has high mortality rates.

The freeze-drying protocol is as follows:

the freezing of the microcapsules from -20 ° C. to -80 ° C. in 10 ml vials, ie about 1 g of samples per vial, with a ramp of 8 ° C./min;

the samples are then lyophilized for 20 hours at -55 ° C. with a pressure of 0.05 mbar, applying 5 steps (-40, 10, 0, 20 and 30 ° C.);

- After lyophilization, the samples are stored at room temperature.

In order to verify the survival of the bacteria in the pectin gels after freeze-drying, a protocol to re-suspend but also to revive the bacteria was put in place: a batch of lyophilized pectin microcapsules (ie 163 mg of microcapsules +/- 1 1 mg) is dissolved in 50 ml of 0.1 M sodium citrate buffer solution + MRS pH 5.8 diluted 1/4 and incubated for 2 h at 28 ° C.

The counts were carried out on agar culture medium and in flow cytometry.

The results of the counts obtained are shown in Table 3 below:

TABLE 3

After lyophilization, the highest mortality was observed for planktonic bacteria, a loss of - 3.02 log CFU. For bacteria immobilized in pectin microcapsules or for bacteria cultured in biofilm inside pectin microcapsules in MRS culture medium, the bacterial mortality after Freeze drying is similar to knowing respectively 1, 12 UFC Log and 1, 54 UFC Log. The highest survival was observed for biofilm bacteria cultured in AOAC medium in pectin microcapsules, ie 0.4 log of CFU loss.

However, the results presented here are comparable to those found in the literature. For wet gels, it is not relevant to compare biomass values in CFU / g because according to the gelation protocol and the polysaccharides used, the gels obtained may have different water contents which will be taken into account in the process. weight of the gels. In the same way, depending on the drying method, the water content will be taken into account in the weight of the gels (the water contents after drying are generally not indicated). On average in the literature, quantities of bacteria encapsulated in polysaccharides at around 10 log CFU / g are found with drying methods optimizing bacterial survival. For example, for the encapsulation of Lactobacillus rhamnosus GG in biofilm in alginate / carob microcapsules, the biomass obtained is 9.7 Log CFU / g (Cheow, Kiew, and Hadinoto 2014) and 9.38 Log CFU / g for Bifidobacterium bifidum in alginate microspheres (Châvarri et al., 2010).

The results without optimization of the survival of the bacteria after freeze-drying for the "24 h biofilm AOAC" condition are 10.24 log CFU / g and 9.86 for the "24 hr biofilm in MRS" condition.

EXAMPLE 3: ACID STRESS RESISTANCE TEST OF THE MICROCAPSULES OF EXAMPLE 1 IN COMPARISON WITH PLANCTONIC PROBIOTIC BACTERIA

A solution mimicking the stress encountered in the stomach was used (gastric solution). This solution is composed of NaCl (0.2% w / v) and the pH is adjusted to pH = 2 with 1N hydrochloric acid (according to Cook et al; 201 1).

At t = 0, planktonic bacteria or microcapsules (0.8 g) were introduced in a known quantity, ie 8.5 log of CFU, in 5 ml of gastric solution at 37 ° C. After 2 hours of incubation, the planktonic bacteria, the biofilm bacteria in the pectin microcapsules and the bacteria re-released in the medium were counted on MRS agar according to the UFC method (previously described). Beforehand, the microcapsules were recovered and disintegrated in 10 ml of sodium citrate (0.1 M) in order to suspend the bacteria in solution.

With this protocol, the bacteria are subjected to a high stress, which causes in planktonic bacteria a significant mortality, of the order of 4.6 Log of CFU loss. In contrast to this, probiotic biofilm bacteria in the pectin microcapsules according to the invention have a relatively low mortality compared to planktonic bacteria namely on average 1, 15 log CFU loss.

TABLE 4

EXAMPLE 4: TEST OF THE ADHESION PROPERTIES OF THE MICROCAPSULES OF EXAMPLE 1 IN COMPARISON WITH PLANCTONIC PROBIOTIC BACTERIA Test 1

The adhesion of Lactobacillus casei bacteria was studied in vitro in a model of intestinal epithelial cells of the Caco-2 line. The cells are cultured in microplate (24 wells) at the concentration of 10 ⁵ cells per well and maintained for 15 days to obtain a differentiated cell mat (with a brush border).

The biofilm Lactobacillus casei bacteria released from the pectin microcapsules according to the invention are brought into contact with the epithelial cells for 1 h 30 at 37 ° C., at a known concentration (100 times more bacteria than epithelial cells (ME). 100), or 10 times more bacteria than epithelial cells (MOI 10)). After this incubation time, the epithelial cells are washed and the bacteria adhered to the epithelial cells are counted on MRS agar plates.

The same test is carried out with probiotic bacteria of Lactobacillus casei in planktonic form. These bacteria originated from a 1% revitalized culture for 24 h in MRS culture medium at pH 5.8 from a 24 hour cryotube output culture in MRS pH 5.8. These bacteria are contacted with epithelial cells as previously described.

The adhesion rate of planktonic bacteria Lactobacillus casei is similar to that already described in the literature namely 0.68% at MOI 100.

Biofilm bacteria released from pectin microcapsules and grown in MRS or AOAC media had a similar adhesion rate of 0.7%. On the other hand, surprisingly, when these two formulations were lyophilized, the adhesion rate increased considerably, namely to 20%. The biofilm bacteria in the pectin microcapsules thus showed a similar adhesion capacity to bacteria in planktonic form. The freeze-drying of biofilm bacteria in pectin microcapsules has increased their adhesion, and initial results suggest that pectin plays a role in promoting adhesion to the intestinal mucosa.

TABLE 5

^■ s Trial 2

In this test, the same protocol as for Test 1 was used, unlike the addition of pectin in solution to the epithelial cell contacting step.

TABLE 6

^* According to the lyophilization method described in Example 2

This test thus demonstrates that planktonic bacteria have an adhesion capacity to epithelial cells increased by pectin. Indeed planktonic bacteria without pectin have an average adhesion rate of 3.32% and in contact with pectin (4 mg) this rate increases to 44.22%. EXAMPLE 5 IN VIVO TEST WITH DSS (DEXTRAN SODIUM SULFATE)

^■ Experimental protocol The experimental trial was conducted over 16 days.

Male C57BL mice from Charles River Laboratories were divided into two groups:

a group "physiological water + DSS" whose mice were force-fed daily with physiological saline;

a "formulation according to the invention + DSS" group whose mice were fed daily with the formulation according to said invention containing the probiotic species Lactobacillus casei, at a dose of 10 ⁹ CFU / mouse per day and prepared according to the formulation (a) of Example 1 above.

For these two groups, mice received 2% (w / v) DSS (Dextran Sodium Sulfate) treatment in drinking water from day 5 to day 11 of the experiment. DSS caused inflammation in the intestines of the mice.

Every other day, the mice of each group were weighed to observe the evolution of the weight of the mice as a function of time (Figure 4).

Moreover, every other day, the feces of the mice were recovered in order to quantify the bacteria of the Lactobacillus genus on the one hand (Figure 5) and the L. casei species on the other hand (Figure 6). For this, 140 mg of faeces from each group of mice were diluted in physiological saline, and disintegrated with stirring with glass beads, giving a liquid solution (in triplicate). Finally, the solution containing the bacteria was counted according to the method of colony-forming units: the solution of re- suspended bacteria was diluted in cascade io ^th with physiological water and dilutions were plated on MRS agar medium at pH 6 containing 20 mg / ml of vancomycin (medium allowing the growth of bacteria of the genus Lactobacillus) and on an M-RTLV medium agar at pH 6 (medium allowing the growth of bacteria of the L. casei species). The agar plates were then incubated for 48 h at 37 ° C. This method measures bacterial cultivability. Result

It emerges first from this test that the mice which have received the pectin microparticles comprising the probiotic bacteria in biofilm form according to the invention have an improved state of health compared to the other mice which have received physiological saline. Indeed, the DSS caused in the different groups of mice tested intestinal inflammation (diarrhea, weight loss, etc.). However, the weight loss was lower in the group having received the formulation according to the invention as this is illustrated in Figure 4.

Then, as illustrated in FIGS. 5 and 6, this test has demonstrated that the bacteria present in the biofilm contained in the microcapsules of pectin according to the invention are well salted out at the intestinal level and in particular at the level of the colon (presence of probiotic bacteria in the feces (8 log CFU / gr).

Thus, these results demonstrate that the microcapsules according to the invention allows the vectorization and release of probiotics in viable form and allows to retain the functionality of probiotic bacteria.

Claims

1. Microcapsules intended to protect probiotics, comprising an envelope and a heart in which said probiotics are dispersed in the form of biofilms distributed in distinct clusters, characterized in that said envelope and said core are composed of pectin.

Microcapsules according to claim 1 or claim 2, wherein the probiotic concentration in microcapsules varies from 8 to 1 log CFU / g, preferably ranges from 9.5 to 10.5 log CFU / g, and typically is 10 log CFU / g.

3. Microcapsules according to any one of the preceding claims, wherein said envelope and the core composing the microcapsules are amidated pectin and methylated having:

a degree of amidation (DA) ranging from 5 to 30%, preferably from 20 to 30%;

a degree of esterification (DE) ranging from 5 to 50%, preferably from 20 to 30%.

4. Microcapsules according to any of the preceding claims, wherein the probiotics are selected from: a probiotic bacterium, a yeast, or a mixture thereof.

Microcapsules according to any one of the preceding claims, wherein said core comprises another non-probiotic active substance, such as a polyphenol, a vitamin, a prebiotic, or a mixture thereof.

6. Microcapsules according to any one of the preceding claims, characterized in that they are in dehydrated or frozen form, so as to optimize their conservation.

7. Probiotic formulation, characterized in that it comprises at least the microcapsules according to one of the preceding claims.

8. A process for manufacturing microcapsules according to any one of the preceding claims 1 to 6, comprising the following steps:

(ii) encapsulating droplets of this homogeneous mixture in a crosslinking solution, so as to form microcapsules comprising: a pectin shell and a pectin lattice core, wherein the probiotics are immobilized;

(iv) optionally, the dehydration or freezing of the microcapsules obtained at the end of step (iii).

The method of manufacturing microcapsules according to claim 8, wherein the homogeneous mixture of step (i) comprises a pectin content, by mass, based on the total volume of the solution, ranging from 2% (w / v). ) at 10% (w / v), preferably from 3% (w / v) to 8% (w / v) and typically from 3.5% (w / v) to 5% (w / v).

The method of manufacturing microcapsules according to claim 8 or claim 9, wherein the mixture of step (i) comprises a probiotic concentration ranging from 10 ⁵ CFU / ml to 10 ⁹ CFU / ml, preferably 10 ⁶ CFU / ml at 10 ⁸ CFU / mL and typically of the order of 10 ⁷ CFU / mL.

1 1. A method of manufacturing microcapsules according to any one of claims 8 to 10, wherein the encapsulation step (ii) comprises a first substep of injection (iia) by means allowing the drip of the mixture of step (i) in the crosslinking solution stirred, so that each drop forms a microcapsule when it comes into contact with the crosslinking solution.

12. A method of manufacturing microcapsules according to claim 10, wherein the encapsulation step (ii) comprises a second substep called microcapsule crosslinking (iib) comprising the maturation of the microcapsules obtained during the sub- injection step (iia) for a period of at least 3 minutes, preferably from 5 to 60 minutes, and in particular from 10 to 25 minutes.

A process for producing microcapsules according to any one of claims 8 to 12, wherein the crosslinking solution is composed of divalent cation, such as Ca ²⁺ , Zn ²⁺ , Ba ²⁺ , Fe ²⁺ in the form of chloride, sulphide, acetate, or a mixture thereof, and in particular Ca ²⁺ in the form of chloride.

14. A method of manufacturing microcapsules according to any one of claims 8 to 13, wherein in step (i), another non-probiotic active substance, such as polyphenol or vitamins, is added to the homogeneous mixture.

15. Use of the microcapsules according to one of claims 1 to 6, or the probiotic formulation according to claim 7 comprising said microcapsules or microcapsules obtained by the process as defined according to any one of claims 8 to 14, to protect probiotics during passage into the stomach and deliver them in an active form to the intestine in an animal.

16. Microcapsules according to one of claims 1 to 6, or the probiotic formulation according to claim 7 or microcapsules obtained by the process as defined in any one of claims 8 to 14, for use as a medicament.

17. Microcapsules according to one of claims 1 to 6, or the probiotic formulation according to claim 7 or microcapsules obtained by the process as defined according to any one of claims 8 to 14, for use in animals to : to prevent and inhibit the proliferation of pathogenic agent; or

- to modulate the immune response; or

to influence the production of virulence factors of the pathogens within the host organism; or

to treat a dysbiosis of the intestinal microbiota.