BE1024197B1 - Process for coating microorganisms, powder of said coated microorganisms obtained and pharmaceutical, nutraceutical, cosmetic, food or sanitary composition comprising it. - Google Patents

Abstract

The present invention relates to a method of coating microorganisms, preferably probiotic microorganisms and a powder of said coated microorganisms obtained by this method, and compositions comprising this powder.

Description

Process for coating microorganisms, powder of said coated microorganisms obtained and pharmaceutical, nutraceutical, cosmetic, food or sanitary composition comprising it

FIELD OF THE INVENTION [0001] The present invention relates to a process for coating cellular microorganisms, in particular bacteria and fungi, including yeasts, the powder of said coated microorganisms, as well as a pharmaceutical, nutraceutical or cosmetic composition, food or sanitary comprising said coated microorganism powder and the applications of these compositions.

BACKGROUND OF THE INVENTION [0002] Although probiotics can be defined in several ways, depending on the understanding of the mechanisms of action of their effects on human health, it is generally accepted that These are living microorganisms, bacteria or yeasts used as living microbial food supplements that confer a specified and demonstrated beneficial effect on the health of the host that ingests them, for example by improving its intestinal microbial balance. Because of their beneficial effects on health, probiotic microorganisms have been used in the pharmaceutical, cosmetic, nutraceutical and food processing fields, in particular for the preparation of so-called functional foods, that is to say having a beneficial effect on the health or having prophylactic or therapeutic virtues against some disorders or certain diseases, affecting the animals or the human.

However, the ability of microorganisms to survive and multiply in a host strongly influences the effectiveness of their probiotic action. Indeed, the probiotic microorganisms must be metabolically stable and active in the product that contains and must survive during treatment and shelf life, especially during storage, food supplements and / or food passing through the digestive tract superior. In particular, they must withstand the extremely acidic conditions of the stomach, the action of enzymes and bile salts in the small intestine, but also the oxygen content or hydrogen peroxide during storage. This stability and efficacy over time, but also during passage through the digestive tract, must be maintained for beneficial effects once in the host's gut (Anal & Singh, 2007).

As a guide, the standard that any product, sold with health claims from the addition of probiotics must contain at least 106 - 107 viable probiotic bacteria per gram (FAO / WHO, 2001).

[0005] However, the number of viable probiotic bacteria capable of providing a targeted beneficial effect is often too low.

[0006] Different approaches have been used to improve the viability of probiotics, including the selection of strains resistant to acidity and bile salts, adaptation to stress, the use of oxygen impermeable containers, fermentation. continuously-batchwise, the incorporation of trace elements such as peptides and amino acids and microencapsulation.

Microencapsulation of probiotic microorganisms is a physicochemical or mechanical process for trapping a substance in a polymer material to produce microcapsules or microbeads, with a diameter ranging from about 1 μm to about 1000 μm, that can release their content at controlled rates under the influence of specific conditions.

[0008] The encapsulation of probiotics is mainly used to protect the cells against an unfavorable environment, rather than for a controlled release.

Atomization is a well known drying process characterized by high production rates and relatively low operational costs. However, the probiotics subjected to atomization are heat sensitive and drying results in cell death due to dehydration and thermal inactivation of enzymes.

Freeze-drying is traditionally used to dry and store fermentation initiation cultures of a food preparation process and probiotics.

There is known a process for obtaining lactic acid bacteria, in the form of a powder, comprising the steps of culturing the lactic acid bacteria, concentrating the culture and lyophilizing it.

Freeze-drying makes it possible to obtain a thorough dehydration of the microorganisms compatible with very long periods of storage of the products in the form of powder.

This method implements product temperature changes quite aggressive for microorganisms because it requires freezing, which is not without consequences for the cells. In some cases, it causes cellular alterations (by peroxidation of fatty acids, but also genetic by a modification of proteins.

The level of cell viability after lyophilization varies depending on many factors, including the strain of the microorganism and the effectiveness of the protective agent used during lyophilization (Morgan & al., 2006).

The powders obtained are however poorly resistant to air, moisture and / or temperature. They hardly retain a stable cell concentration during storage. In addition, when microorganisms such as lactic acid bacteria pass through the stomach after ingestion, a majority of cells are killed by gastric juice before reaching the intestine.

In order to overcome these problems, processes for coating microorganisms, in particular lactic acid bacteria, using gelatines, sugars, gums, etc. have been proposed. The conventional method for coating microorganisms, particularly lactic acid bacteria, usually comprises the step of introducing a specific coating on the dried cells or on the concentrated cell culture as shown in Figure 1.

Microorganisms, in particular lactic acid bacteria are cultured in an aerobic or anaerobic bioreactor using a fermentation medium containing assimilable sources of amino acids, peptides, sugars, vitamins and minerals. These nutrients contained in the medium are only used for the proliferation of cells.

Concentrated cultures are obtained by means of separation by centrifugation or ultrafiltration, whose purpose is only the separation and concentration of the cells contained in the culture medium.

A coating composition can be applied to the dry culture of microorganisms, in particular lactic acid bacteria thus obtained, followed by drying, usually by lyophilization.

Alternatively, the culture of microorganisms, in particular concentrated and formulated lactic acid bacteria, can be introduced into an aqueous solution of the coating composition, followed by drying, usually by freeze-drying.

This coating may be of the microsphere type, and applied to microorganisms, in particular to lactic acid bacteria by an injection nozzle in a microencapsulation process.

For microencapsulation of microbial cells, it is essential that the encapsulation process be carried out under relatively mild conditions to ensure high viability of the encapsulated cells.

[0023] There are many microencapsulation techniques. However, the techniques applied to probiotics are generally limited to encapsulation in gel particles, fluidized bed coating, and spray drying.

The encapsulation of probiotic microorganisms in gel particles can be classified into 2 groups, depending on the process used to form the capsules or beads: extrusion and emulsion techniques.

Extrusion is a physical technique that uses hydro-colloids as encapsulating materials. The polymer solution is first mixed with the microbial cells. This mixture is then extruded through a high pressure nozzle in the form of droplets into a solution containing a solidifying agent.

Gelling occurs by contact of the polymer solution with the solidifying agent, by cooling or a combination of both. The factors that influence the size of the microspheres produced are: - the diameter of the dispersion nozzle, - the viscosity and the flow rate of the polymer solution, - the flow distance between the nozzle and the solidification solution, - the concentration and the temperature of the polymer solution.

New techniques have been developed for the formation of droplets and thus microspheres by extrusion: (a) the formation of droplets by an electrostatic force, (b) the formation of droplets by the cut jet device, (c) the formation of droplets by the frequency of the vibrations, (d) the formation of droplets by coextrusion (coaxial flow).

The main advantages of the extrusion method are the simplicity of its operation, at lower cost, and the operating conditions ensuring high cell viability. The major disadvantage of this technique is that it is difficult to industrialize and automate due to the formation of microbeads.

The production of an emulsion is a chemical technique for encapsulating probiotic cells, which also uses hydro-colloids as encapsulating materials. This technique involves the dispersion of a "cell-polymer" suspension (discontinuous phase) in an oily / organic phase (continuous phase). The mixture is then homogenized to form a water-in-oil emulsion using a surfactant and stirring. The gelation of the dispersed phase is initiated by cooling or by the addition of a solidifying agent to the emulsion. The microspheres produced are then collected by filtration or centrifugation.

This technique has the advantage of being easy to industrialize and gives a high survival rate of microorganisms. The major disadvantage of this technique is that the microbeads produced have a wide range of sizes and shapes.

Spray drying is commonly used for the microencapsulation of probiotic microorganisms. It involves atomizing a solution containing the microbial cells and the polymer matrix into the hot drying air, followed by rapid evaporation of the water.

The microencapsulated product is then separated, in the form of a dry powder, from the transport air in a cyclone. Different spray drying parameters such as product feed rate, airflow, product temperature, inlet air temperature, and outlet air temperature need to be optimized to produce well-formed microspheres, capsules or beads.

This technique has the advantage of having high production speeds but also disadvantages such as the costs of industrial installation and operation. But the main problem is the use of high temperatures and osmotic stress due to dehydration, which results in relatively high losses of viability and activity immediately after spraying, in particular the inactivation of essential enzymes which maintain the equilibrium. cellular.

According to Chavez & Ledeboer, 2007, the logarithmic number of probiotics decreases linearly with the outlet air temperature of the atomizer (in the range of 50 ° C - 80 ° C) and working with lower temperatures leads to an accumulation of powder at inside the cyclone. Spray encapsulation of Lactobacillus acidophilus reduces viability by about 100-fold compared to before encapsulation due to the high temperature used in the process (Zhao et al., 2008). Various factors such as the type and concentration of the support materials, the time-temperature combinations and the heat resistance of the microbial cells influence the viability of the encapsulated cells by atomization. By using a pre-gelatinized modified starch as a support material, O'Riordan (2001) nevertheless obtained a loss of less than one log unit (0.78) when encapsulating Bifidobacterium cells with a temperature inlet temperature of 100 ° C and an outlet temperature of 45 ° C.

During a spray coating, that is to say in the fluidized bed technique, the probiotic cells must be in solid form, but not necessarily completely dried. They are then suspended in the air and a liquid coating material is sprayed onto the probiotic cells. The main advantage of this technique is that it easily allows the addition of an additional layer of targeted molecules for release into the gut, for example.

The purpose of the microencapsulation is to preserve the viability of microbial cells encapsulated adverse environmental conditions encountered during their implementation, when stored before use, but also when added to food and during passage through the digestive tract. However, there is a loss of cell viability during the steps of the microencapsulation process itself or the subsequent drying.

A problem in the approach of coating probiotics is that these technologies stabilize bacteria most often in liquid form. To improve storage, it is convenient to convert these capsules or beads into dry powder using techniques such as atomization, lyophilization or fluidized bed. Nevertheless, it is also known that lyophilization induces a significant mortality of bacterial cells because of the loss of membrane integrity and the denaturation of macromolecules. The components of the medium in which the bacteria are dried may have a greater effect on the stability of the probiotics than the microencapsulation itself.

Double encapsulation in alginate and maltodextrin of Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM showed no difference in the loss of viability observed following lyophilization compared with that observed with cell suspensions prepared with maltodextrin. alone: the log reduction of Lactobacillus GG is 0.63 and 0.84, the log reduction of

Lactobacillus NCFM is 1.89 and 1.95 respectively for doubly encapsulated cells and free cells (Sohail & al., 2013).

The viability of Bifidobacterium longum 15708 microencapsulated by an extrusion and spraying technique (spray) in an alginate matrix after lyophilization was evaluated by Amine et al. (2014) . Viability losses of 2.9 and 2 log were observed, respectively. Freeze-drying of the free cells resulted in a loss of 2.75 log.

The multiplication of these probiotic microorganisms inside capsules or balls composed of a polymer material is already known in the art. Indeed, it has been shown that the production of high populations of probiotics was possible inside alginate beads.

However, in general, such conventional methods for coating lactic acid bacteria which include the steps of introducing the coating composition or microencapsulation after concentration and optionally drying cultures, lead to the freeze-drying step, to cell survival efficiencies that remain low for many probiotics.

In addition, it is known that the probiotic microorganisms show a loss of cell viability during the steps of the microencapsulation process itself and during their drying.

State of the Art [0043] Champagne et al. (2000 & 2007) describe a production of probiotic microorganisms within a capsule composed of a polymeric material, particularly alginate beads. However, this document does not describe or suggest treating these alginate beads obtained by an additional drying step, in particular by lyophilization.

The international patent application WO2015 / 000972 discloses an improved method of lyophilization of encapsulated cells comprising two successive stages of two brief incubations, less than one hour and insufficient to ensure cell multiplication in a medium comprising protective molecules, followed by a drying step of these encapsulated and treated cells.

OBJECTS OF THE INVENTION The object of the present invention is to provide a novel process for encapsulating microorganisms, in particular probiotic microorganisms chosen from the group consisting of bacteria, such as lactic acid bacteria and bifidobacteria, as well as fungi, in particular yeasts, and which does not have the disadvantages of the state of the art.

A particular object of the invention is to obtain an industrializable process which generates powders of these microorganisms, in particular probiotics encapsulated with low mortality of encapsulated cells.

A final object of the invention is to obtain such lyophilized microorganism powders which comprise a high concentration of encapsulated live microorganisms capable of having a probiotic or non-probiotic efficacy in pharmaceutical, cosmetic, agri-food and nutraceutical-type compositions. or sanitary, especially in sanitary compositions used in the purification of pipes or septic tanks, but also to obtain powders preferably having uniform sizes of solid particles consisting of these capsules, microspheres or beads, said powders being able to be stored, handled and stored for prolonged periods.

Characteristic Elements of the Invention The present invention relates to a process for coating or encapsulating microorganisms, preferably probiotic microorganisms, the process comprising the following steps, preferably consecutive: optionally a first stage of cultivation and multiplying said microorganisms, optionally followed by a step of collecting said microorganisms, - a step of coating said microorganisms in polymeric capsules, - a second step of cultivation and multiplication of said microorganisms encapsulated in a suitable nutrient solution for a period of time. between 2 hours and 7 days, - a step of collecting microorganisms multiplied and encapsulated and - a step of drying by lyophilization of microorganisms multiplied and encapsulated to form a powder of encapsulated microorganisms.

The present invention also relates to a powder of microorganisms, preferably probiotics, incorporated in polymeric capsules and preferably obtained by the method of the invention.

Preferably, in the process and the powders of the invention, the microorganisms are chosen from probiotic bacteria and fungi, in particular yeasts.

Advantageously, the probiotic bacteria are chosen from the group consisting of lactic acid bacteria or bifidobacteria, more particularly the lactic acid bacteria are chosen from the group consisting of Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus brevis, Lactobacillus casei, in particular Lactobacillus casei subsp. Casei or Lactobacillus casei Shirota, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, in particular Lactobacillus delbrueckii subsp. lactis or Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus pentosaceus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactococcus lactis, Oenococcus oeni, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus halophilus, Streptococcus thermophilus or their mixture.

In the process and the powders of the invention, the bifidobacteria are chosen from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis including Bifidobacterium animalis subsp. animalis and Bifidobacterium animalis subsp. lactis, Bifidobacterium angulatum, Bifidobacterium bifidum,

Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bididobacterium longum, Bifidobacterium pseudolongum or a mixture thereof. The microorganisms may also be selected from the group consisting of Micrococcus varians, Staphylococcus carnosus, Staphylococcus xylosus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bacillus natto, Bacillus clausii, Bacillus cereus var. toyoi, Bacillus pumilus, Bacillus thuringiensis, Escherichia coli nissle strain, Paenibacillus alvei, Propionibacterium freudenreichii Escherichia coli nissle strain, Propionibacterium freudenreichii and yeasts, preferably Saccharomyces cerevisiae, Saccharomyces boulardii, Yarrowia lipolytica, Brettanomyces bruxellensis, Debaryomyces hansenii, Candida albicans and Kluyveromyces marxianus.

Advantageously in the process and the powders of the invention, the polymeric material coating polymeric capsules is a hydro-colloid selected from the group consisting of alginate, agar, carrageenans, preferably the -carraghenane starch, chitosan, alginate, pectin, pullulan, gelatin, gums, in particular gellan gum or xanthan gum, cellulose acetate phthalate, gelatin, milk proteins, in particular casein or a mixture of them, as described by Burgain & al. (2011) as well as by Rathore & al. (2013); said materials being able to be combined with other elements known to improve this coating, in particular maltodextrins, cellulose, hemicellulose, ethylcelllulose, carboxycellulose and their mixture.

In the method of the invention, the nutrient solution encapsulated microorganisms essentially comprises amino acids, saccharides, minerals and optionally vitamins.

Preferably in the powder of the invention, the microorganism is either Lactobacillus rhamnosus and comprises a Lactobacillus rhamnosus concentration greater than 1.5 × 10 11 (cfu / g), preferably greater than 2 × 10 11 (cfu / g). or the microorganism is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus and Lactobacillus bulgaricus and comprises a concentration of microorganisms per capsule greater than 1 x 109 (cfu / g), preferably between 1 x 109 (cfu / g) and 2 x 1011 (cfu / g).

Another aspect of the invention relates to a pharmaceutical composition comprising a suitable pharmaceutical diluent or carrier, a food composition comprising a diluent or a suitable food vehicle or a nutraceutical composition comprising a suitable diluent or vehicle and the powder of the invention.

[0057] A final aspect of the invention relates to a sanitary composition, in particular for the purification of waste present in pipes or septic tanks and comprising the powder of the invention.

The present invention will be described in detail below in the description of a preferred embodiment of the invention, with reference to the figures and the example presented by way of non-limiting illustration of the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 represents the steps of a method of the state of the art for coating lactic acid bacteria.

FIG. 2 represents the main steps of the process for obtaining a lyophilized powder of coated lactic acid bacteria according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of coating or encapsulation, in particular microencapsulation of a microorganism, that is to say a living cell selected from the group consisting of bacteria , in particular lactic acid bacteria or bifidobacteria and fungi, preferably an encapsulated probiotic microorganism comprising, as represented in FIG. 2, the following, preferably successive, steps: optionally a first culture 1, multiplication or prior propagation of said cellular microorganisms, preferably probiotics, in particular microorganisms, in particular lactic acid bacteria or yeasts, on a suitable microorganism growth medium, in particular an aqueous solution preferably comprising as nutrients, at least amino acids, saccharides, minerals and possibly vitamins; optionally a step of collecting the multiplied microorganisms called formulation step 2 of said microorganisms to put them in conditions suitable for the subsequent coating step; coating or encapsulation, in particular micro-encapsulation of said microorganisms or cells, preferably probiotic, multiplied in capsules, that is to say microspheres or polymeric beads; a second culture 4, multiplication or propagation of said microorganisms in an adequate nutrient solution, preferably an aqueous solution comprising as nutrients at least saccharides, amino acids, minerals, and possibly vitamins and, during a phase of cell growth in (within) said polymeric capsules, said microspheres or said polymeric beads, to increase the concentration, preferably by a factor of 2, 4, 6, 10, 100 or 1000 or more of said microorganisms, probiotics, within the capsules, microspheres or polymeric beads, for a period of time suitable for this culture, multiplication or propagation, preferably for a duration of more than 2 hours, preferably a duration of more than 4 hours or 6 hours. hours, more than 12 hours or more than 24 hours or 48 hours, in particular for a period of time n 7 days, preferably between about 6 hours and about 4 days, more particularly between about 12 hours and about 48 hours; - harvesting 5, preferably on a sieve of suitable size, said microorganisms, preferably probiotic, encapsulated and - drying 6, preferably by lyophilization microorganisms, preferably probiotic, encapsulated to form a powder of microorganisms preferably encapsulated probiotics.

According to the invention, the culture medium of the encapsulated microorganisms is a nutritive and preferably aqueous solution known for the growth of microorganisms, in particular bacteria and fungi, including yeasts, and preferably comprises amino acids. or source peptides of amino acids, saccharides, that is to say monosaccharides such as glucose or polysaccharides, minerals in particular in the form of halide salts and alkali or alkaline earth metals, optionally vitamins preferably having antioxidant activities and optionally acids, such as ascorbic acid or citric acid.

Preferably, the nutrient solution comprises the following elements: casein peptone, yeast extract, meat extract, polyoxyethylene sorbitan monooleate (polysorbate 80 or tween 80 ®), glucose, minerals (preferably chosen from the group consisting of potassium, sodium, ammonium, magnesium and manganese and possibly zinc, copper, iron, boron, cobalt, sulphates, in particular calcium sulphate and / or sulphate. magnesium, diammonium phosphate, phosphoric acid, possibly meat peptone, tryptone, glucose or other saccharides, vitamins).

The capsules or the beads are polymeric capsules or polymeric beads, the main material (a hydro-colloid) is selected from the group consisting of alginate, agar, carrageenans, preferably the -carraghénane starch, chitosan, alginate, pectin, pullulan, gelatin, gums, in particular gellan gum or xanthan gum, cellulose acetate phthalate, gelatin, milk proteins, in particular casein or a mixture of them, as described by Burgain & al. (2011) as well as by Rathore & al (2013). said materials being able to be combined with other elements known to improve this coating, in particular maltodextrins, cellulose, hemicellulose, ethylcelllulose, carboxycellulose and their mixture. Preferably, the polymeric capsules or polymeric beads are capsules of a material selected from the group consisting of alginate or a mixture of alginate and maltodextrins.

Capsules, microspheres or beads are almost exclusively produced using water-soluble polymers that provide a high degree of permeability to low molecular weight nutrients and metabolites, thereby providing the optimal conditions necessary for the proper functioning of the cells. , preferably probiotic, immobilized. In addition, preferably, the capsule, microsphere or bead materials are selected to provide enteric release of microorganisms, i.e., into the mammalian gut, including the man to whom they are administered.

Both natural and synthetic water-soluble polymers have been used for microencapsulation of microbial cells. Although synthetic polymers offer greater mechanical strength and chemical stability, natural polymers are preferred to their synthetic counterparts because they are less harmful to the integrity and cell viability of encapsulated or coated microorganisms.

The microspheres, capsules or beads obtained by the process of the invention are of a size generally of between about 1 μm and about 1000 μm, preferably between about 5 μm and about 800 μm, more particularly between about 10 μm. and about 500 μm.

Advantageously, according to the invention, the microorganisms, preferably probiotics, in particular lactic acid bacteria are propagated a first time in a bioreactor under aerobic conditions or anaerobic conditions using a fermentation medium containing adequate nutrients, in particular one or more assimilable source (s) of amino acids, peptides, saccharides, vitamins and minerals.

This multiplied probiotic microorganism culture is then introduced into a coating composition, without prior concentration; the culture of probiotic microorganisms being preferably mixed with a solution of between about 1% and about 10% sterile sodium alginate, preferably about 5% sterile sodium alginate.

Said mixture is added dropwise by extrusion into a sterile solution containing between about 3% and about 1% CaCl 2, preferably about 2% Cacl 2 and between about 0.1% and about 5% glycerol, preferably about 1%. % glycerol, at room temperature while stirring continuously and these capsules, microspheres or these beads obtained are then cured in this solution for a period of between about 30 minutes and about 2 hours, preferably for about 1 hour.

The cell viability of the coated probiotic cell powder thus obtained is surprisingly superior to that of a coated probiotic cell powder obtained from capsules, microspheres or beads containing a concentrated culture of these probiotic microorganisms.

Preferably, the microorganisms are gram-positive bacteria, in particular species of lactic acid bacteria mainly used as probiotics and treated by the steps of the process of the invention are chosen from the group consisting of Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus brevis, Lactobacillus casei, in particular Lactobacillus casei subsp. Casei or Lactobacillus casei Shirota, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, in particular Lactobacillus delbrueckii subsp. lactis or Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus pentosaceus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactococcus lactis, Oenococcus oeni, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus halophilus, Streptococcus thermophilus, other bacteria described by Makinen & Bigret, 1993 or by Anal & Singh, 2007 or their mixtures.

Lactic acid bacteria, which are among the most important probiotic microorganisms generally associated with the gastrointestinal tract, have numerous beneficial effects on the human intestinal flora, including for immune stimulation, for the reduction of cholesterol level, inhibition of pathogen growth, maintenance of a healthy intestinal microflora, prevention of cancer, improvement of lactose utilization, prevention of diarrheal diseases or constipation, absorption of calcium, vitamin synthesis and protein predigestion (Li & al., 2009).

These gram-positive, rod-shaped, non-sporulating, catalase-negative bacteria, which are generally anaerobic but aerotolerant, are acid-tolerant and can be fermentative; lactic acid is the main end product of sugar fermentation (Axelsson, 1993).

In addition, other microorganisms commonly used as probiotics are bifidobacteria, also treated by the process steps of the invention, preferably selected from the group consisting of Bifidobacterium adolescent, Bifidobacterium animalis, including Bifidobacterium animalis subsp. and Bifidobacterium animalis subsp.lactis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bididobacterium longum and Bifidobacterium pseudolongum.

Bifidobacteria are also Gram-positive and are rod-shaped, but ensure their growth in anaerobic. These bacteria can grow at a pH in the 4.5-8.5 range. Bifidobacteria ferment carbohydrates, producing mainly acetic acid and lactic acid in a molar ratio of 3: 2 (v / v), but not carbon dioxide, butyric acid or acid propionic acid.

In addition to lactic acid bacteria, other bacteria such as Micrococcus varians, Staphylococcus carnosus, Staphylococcus xylosus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bacillus natto, Bacillus clausii, Bacillus cereus var. toyoi, Bacillus pumilus, Bacillus thuringiensis, Escherichia coli Nissle strain, Paenibacillus alvei, Propionibacterium freudenreichii

Escherichia coli strain nissle, Propionibacterium freudenreichii have also been identified as having probiotic effects (Holzapfel, Haberer, Geisen, Bjorkroth, & Schillinger, 2001; Anal & Singh, 2007) and are treated by the successive preference stages of the method of the invention.

In addition to bacteria, other microorganisms such as fungi may also be coated by the method of the invention and present in the powders of the invention. Among the preferred fungi, mention may be made of yeasts, such as Saccharomyces cerevisiae, Saccharomyces boulardii, Yarrowia lipolytica, Brettanomyces bruxellensis, Debaryomyces hansenii, Candida albicans and Kluyveromyces marxianus. The invention also relates to the pharmaceutical composition, preferably enteric, nutraceutical, food or sanitary optionally comprising a diluent such as a solvent, or pharmaceutical, cosmetic or food vehicle suitable and the powder of microorganisms, preferably probiotic, encapsulated and dried of the invention, as well as applications, particularly therapeutic (including for maintaining or restoring the health of a mammalian animal, including humans, but also for the treatment or prevention of inflammatory infectious diseases, such as gastroenteritis, particularly the newborn, or the disease of Crohn) and cosmetics of these compositions. In particular the present invention relates to the application of the sanitary composition of the invention for the purification of pipes or septic tanks.

The food, nutraceutical or pharmaceutical compositions of the invention are preferably enteric compositions present in the form of a hard capsule or soft capsule, tablets, sachets or appropriate formulations containing materials such as starch or cellulose easily administered per os.

Example: Obtaining alginate capsules with Lactobacillus (Lb.) cream rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus or Lactobacillus bulgaricus [0080] Microspheres or capsules of Lb. rhamnosus, Lb. acidophillus, Lb. helveticus or Lb. bugaricus in a matrix made of alginate were prepared according to the following protocol: [0081] 250 g or 50 g of a sterile solution of alginate at 5% by weight were added to equivalent amounts of 250 g of Lb cream. . rhamnosus (7 x 109 cfu), at 50 g of Lb. helveticus (8.73 x 109 cfu), 250 g of Lb. acidophilus (1.14 x 109 cfu), or 50 g of Lb. bulgaricus (8.03 x 108 cfu).

A dropwise drip with a drop rupture apparatus in a laminar flow regime was performed to produce microspheres by solidification in a solution composed of CaCl 2 at 2% by weight and glycerol at 1% by weight. agitation. The separation of the microspheres was then carried out using a sterile agent.

250 g or 50 g of the microspheres or capsules thus produced were cultured in a bioreactor in order to increase the Lb cell concentration. rhamnosus, Lb. acidophilus, Lb. helveticus or Lb. bulgaricus inside the microspheres before lyophilization.

A dry, fluid powder of microspheres or capsules was obtained.

Under the same conditions, a more concentrated control in Lb cells. rhamnosus, Lb. acidophilus, Lb. helveticus or Lb. bulgaricus was performed in order to have a concentration in the microspheres equivalent to the Lb concentration. rhamnosus Lb. acidophilus, Lb. helveticus or Lb. bulgaricus obtained after the cell growth phase in the microspheres.

The levels of viable bacteria in the microspheres or capsules (cfu) were analyzed before and after the growth phase in the microspheres or capsules and after freeze-drying. These were compared with the levels of viable bacteria obtained in more concentrated microspheres or capsules before and after lyophilization (control). Similar unexpected results have been obtained with other microorganisms strains of lactic acid bacteria, bifidobacteria or yeasts. Therefore, these different types of microorganisms can find at these higher and more viable concentrations advantageous applications in food, nutraceutical, cosmetic or pharmacy as well as in the sanitary field, in particular for the treatment of waste in pipelines and septic tanks.

The results for the 4 strains described above are presented in the following Table 1:

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Zhao, & al. (2008). World Journal of Microbiology and Biotechnology 24 (8), 1349-1354. LEGEND Fig. 1

Claims (17)

1. A method of coating microorganisms comprising the following steps: - optionally a first step of cultivation (1) of said microorganisms optionally followed by a step of collecting (2) said microorganisms, - a step of coating (3) said microorganisms in a polymeric capsule, - a second step of culture and multiplication (4) of said microorganisms encapsulated in a nutrient solution for a duration greater than 24 hours, - a step of collection (5) of the multiplied and encapsulated microorganisms and - a step of drying (6) by lyophilization of the microorganisms multiplied and encapsulated to form a powder of encapsulated microorganisms. The method of claim 1 wherein the microorganisms are probiotic microorganisms. 3. The method of claim 1 or claim 2 wherein the microorganisms are selected from bacteria and fungi, particularly yeasts. 4. The method of claim 3 wherein the bacteria are selected from the group consisting of lactic acid bacteria or bifidobacteria. The method according to claim 4, wherein the lactic acid bacteria are selected from the group consisting of Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus brevis, Lactobacillus casei, in particular Lactobacillus casei subsp. Casei or Lactobacillus casei Shirota, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, in particular Lactobacillus delbrueckii subsp. lactis or Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus pentosaceus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, Lactococcus lactis, Oenococcus oeni, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus halophilus, Streptococcus thermophilus or their mixture. 6. The method according to claim 4 wherein the bifidobacteria are selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidibacterium lactis, Bididobacterium longum, Bifidobacterium pseudolongum or a mixture thereof. 7. The process according to claim 3, wherein the microorganism is selected from the group consisting of Micrococcus varians, Staphylococcus carnosus, Staphylococcus xylosus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bacillus natto, Bacillus clausii, Bacillus cereus var. . toyoi, Bacillus pumilus, Bacillus thuringiensis, Escherichia coli nissle strain, Paenibacillus alvei, Propionibacterium freudenreichii Escherichia coli nissle strain, Propionibacterium freudenreichii and yeasts, preferably Saccharomyces cerevisiae, Saccharomyces boulardii, Yarrowia lipolytica, Brettanomyces bruxellensis, Debaryomyces hansenii, Candida albicans and Kluyveromyces marxianus or their mixture. The process of any of the preceding claims wherein the polymeric material coating polymeric capsules is a hydro-colloid. The process according to claim 8, wherein the hydro-colloid is selected from the group consisting of alginate, agar, carrageenans, preferably κ-carrageenan, starch, chitosan, alginate, pectin, pullulan, gelatin, gums, in particular gellan gum or xanthan gum, cellulose acetophthalate, gelatin, milk proteins, in particular casein or a mixture of them, preferably alginate or a mixture containing alginate and other elements known to improve this coating, in particular maltodextrins, cellulose, hemicellulose, ethylcelllulose, carboxycellulose and their mixture preferably maltodextrins. 10. The method according to any one of the preceding claims wherein the nutrient solution of encapsulated microorganisms comprises amino acids, saccharides, minerals and optionally vitamins. 11. Microorganism powder, preferably probiotic, incorporated in polymeric capsules and preferably obtained by the process according to any of the preceding claims. 12. The powder according to claim 11, wherein the microorganism is Lactobacillus rhamonosus and comprising a concentration of lactobacillus rhamnosus greater than 1.5 x 1011 (cfu / g), preferably greater than 2 x 1011 (cfu / g). 13. The powder of claim 11, wherein the microorganism is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus helveticus and Lactobacillus bulgaricus and comprising a concentration of microorganisms per capsule greater than 1x 109 (cfu / g), preferably between 1 x 109 (cfu / g) and 2 x 1011 (cfu / g). A pharmaceutical composition comprising a suitable pharmaceutical diluent or carrier and the powder of any of the preceding claims 11 to 13. A food composition comprising a diluent or a suitable food vehicle and the powder of any of the preceding claims 11 to 13. 16. A nutraceutical composition comprising a diluent or a suitable vehicle and the powder according to any one of the preceding claims 11 to 13. 17. Sanitary composition, in particular for the purification of waste present in pipes or septic tanks and comprising the powder according to any one of the preceding claims 11 to 13.