PL246674B1 - Method of obtaining microcapsules containing probiotic bacteria - Google Patents

Abstract

The subject of the application is a method of obtaining microcapsules containing probiotic bacteria enclosed inside a capsule made of a natural polymer, alginate from brown algae, in which method a solution of a natural polymer with probiotic bacteria is prepared - component A, component B is prepared in the form of an oil phase containing surfactants, a cross-linking solution is prepared - component C, and then the individual components are mixed. The method is characterized in that the cross-linking agent - component C, which is a 0.1 - 0.5 M aqueous solution containing Ca²⁺ ions is introduced at a rate of 0.3 - 2 ml per minute, maintaining the mass ratio of the alginate solution to the cross-linking agent solution at the level of 1:1, wherein previously an aqueous solution of sodium alginate, with a concentration of 1 - 5% by weight, is mixed with an MRS solution in which the concentration of probiotic bacteria is 10⁸ - 10¹² CFU/ml, and the polymer solution is combined with the probiotic bacteria solution in a 4:1 ratio, at a temperature of 20 to 250°C (component A), then the oil phase is introduced into the system — (component B), the mixture of components A and B is mixed, a cross-linking agent is introduced, the mixture of components A, B and C is cross-linked, at a temperature of 20 - 25°C, for 5 to 30 minutes, the microcapsules are separated, they are washed with 1% aqueous sodium chloride solution and subjected to the lyophilization process.

Description

Description of the invention

The subject of the invention is a method for obtaining microcapsules containing probiotic bacteria enclosed inside a capsule made of a natural polymer, for example alginate from brown algae.

Microencapsulation is a process of coating or enclosing a given material or mixture of materials inside a shell (membrane) which is a specific material or system. Usually these materials differ in their physicochemical properties, therefore as a result of microencapsulation a composite product is obtained - microcapsules, consisting of a core and a shell with different material characteristics. There are many methods of microencapsulation, which can be divided into two main groups: chemical processes (e.g. coacervation, interfacial or in situ polymerization) and mechanical or physical processes (e.g. spray drying, microfluidic encapsulation, spin extrusion, nozzle vibration technology).

The most commonly used biomaterials in encapsulation are resorbable natural and synthetic polymeric materials, among which two groups can be distinguished: polysaccharides, including alginate, chitosan; and aliphatic polyesters, such as polylactide (PLA), polyglycolide (PGA), polylactide-glycolide copolymer (PLGA). Alginate microcapsules are biocompatible, biodegradable and non-toxic systems for delivering active substances. The raw material used to obtain microspheres is alginates, polysaccharides obtained from marine algae, composed of D-mannuronic acid and L-guluronic acid molecules linked by a glycosidic bond. Due to their biocompatibility and non-toxicity, alginates have been used as carriers for active substances, and are used in the form of microgels, microcapsules and microspheres. They are most often used in the food and pharmaceutical industries. Popular methods of obtaining alginate capsules include techniques such as extrusion, emulsification and spray drying.

The prior art includes a solution which is the subject of patent application P.395685, which relates to a method for manufacturing gel microcapsules and is characterized in that at least two gasosol streams are simultaneously introduced into the chamber and sprayed onto each other, one of which is a mixture of the gelling component being the microcapsule wall and the substance being enclosed, and the other contains substances entering into the hardening - gelling reactions of the microcapsule wall, wherein the size of the particles of the phases dispersed in the gas cannot be greater than 100 μm.

From the American patent application US 2009/0098628 also as EP1702058B1 is known a method and device for manufacturing microcapsules containing microorganisms. A characteristic feature of this solution is the formation of drops in contact with the liquid surface by means of a porous partition.

From the American patent description US 6 033 888 is known the process of microencapsulation of microbial cells, which consists in dispersing solidified droplets using ultrasound. The creators of the solution developed a new process, which strengthened alginate microcapsules and their tolerance to contact with another substance - citrate, however, in this process also creates alginate droplets of the appropriate size, which are then introduced to the appropriate depth into the gelling solution.

The method of manufacturing core/shell type microcapsules modified with graphene oxide, known from Polish patent description PL235870, by coaxial extrusion technology using nozzle vibration technology and electrostatic dispersion of formed droplets, is characterized in that an aqueous solution of alginate is prepared with a concentration of 0.01% (w/o) to 30.0% (w/o), with the addition of graphene oxide in an amount of 0.01% (w/o) to 10.0% (w/o) in relation to the volume of the alginate solution, and an emulsion of an aqueous solution of a biologically active substance and a solution of polyester in an organic solvent, wherein the polyester is selected from the group consisting of: lactic acid polymers, glycolic acid polymers, ε-caproic acid polymers, and their copolymers, and the solvent for the biologically active substance is a physiological saline solution or a biologically acceptable buffer, and the ratio of the organic phase to the organic phase is water solution is from 1:1 to 20:1, then microcapsules are formed from this aqueous solution and this emulsion, which are cross-linked in an aqueous solution of calcium chloride or strontium chloride with a concentration of 1 mM to 1 M, for 0.1 hour to 24 hours, after which the microcapsules are rinsed with water, frozen and lyophilized.

Application WO2015019307 discloses microcapsules for protecting probiotics and methods of making the same. In some embodiments, the microcapsules of the application are used to deliver probiotics, combinations of probiotics and prebiotics, and/or synbiotics through the stomach of a mammal to its lower gastrointestinal tract. In some embodiments, the microcapsules comprise a biopolymer and a plant-derived protein. In some embodiments, the microcapsules comprise alginate, iota-carrageenan, or deacylated gellan gum as the biopolymer. In some embodiments, the microcapsules comprise chickpea protein, pea protein, or soy protein as the plant-derived protein. An emulsion comprising a biopolymer, a plant-derived protein, and a probiotic encapsulation material may be cross-linked to form a microcapsule.

From the United States patent specification US 11123384 are known microencapsulated probiotic bacteria protected from degradation by acidic aqueous solutions, high concentrations of bile salts, elevated temperatures and long-term storage and exhibiting increased antibacterial activity compared to their non-microencapsulated counterparts. Microencapsulated probiotic bacteria include probiotic bacteria encapsulated in microcapsules. The probiotic bacteria are live cells of Lactobacillus plantarum. Each of the microcapsules contains a matrix of gelatinized alginate. The matrix completely surrounds the probiotic bacteria in the matrix. The outer surface of the matrix has a coating consisting essentially of one vegetable oil selected from the group consisting of olive oil and rapeseed oil or the outer surface of the matrix is treated with sodium chloride. The microencapsulated probiotic bacteria have an average particle size of less than 1000 microns (pm).

There are no precise indications in the literature as to what input parameters for the microemulsion encapsulation process using ultrasonic or mechanical homogenization significantly influence the output parameters - the size and stability of alginate microcapsules obtained in this process.

According to the invention, a method for obtaining microcapsules containing probiotic bacteria enclosed inside a capsule made of a natural polymer of alginate from brown algae, in which a solution of a natural polymer with probiotic bacteria - component A is prepared, component B is prepared in the form of an oil phase containing surfactants, a cross-linking solution - component C is prepared, and then the individual components are mixed, is characterized in that the cross-linking agent - component C, which is a 0.1-0.5 M aqueous solution containing Ca2 ⁺ ions, is introduced at a rate of 0.3-2 ml per minute, maintaining the mass ratio of the alginate solution to the cross-linking agent solution at a level of 1:1, wherein previously an aqueous solution of sodium alginate, with a concentration of 1-5% by weight, is mixed with a MRS solution, in which the concentration of probiotic bacteria is ^108-1012 CFU/ml, and the polymer solution is combined with the solution of probiotic bacteria in a ratio of 4:1, at a temperature of 20 to 25°C (component A), then the oil phase is introduced into the system - (component B), which is a mixture of capric-caprylic triglycerides with a surfactant, the mixture of components A and B is subjected to mixing, using a mechanical stirrer, at a speed in the range of 400-800 rpm, for 5-10 minutes, maintaining the temperature of the dispersion system at 25°C. The obtained pre-emulsion is subjected to a homogenization process to obtain an emulsion, and then a cross-linking agent is introduced into the obtained system, with continuous mixing at a speed of 400-800 rpm. The mixture of components A, B and C is subjected to cross-linking, at a temperature of 20-25°C, for a period of 5 to 30 minutes, preferably 10 minutes. In order to separate the microcapsules, the oil emulsion is subjected to a centrifugation process, stabilized at a temperature of 5-7°C for 24 hours, and then, after separation of the oil phase, the isolated microcapsules are washed with 1% aqueous sodium chloride solution and subjected to a lyophilization process.

Preferably, the Ca2 ⁺ ions are derived from a calcium salt solution, such as a 0.1-0.5M calcium chloride solution.

Preferably, component A and component B are combined in a weight ratio of 1:5.

Preferably, the aqueous solution of sodium alginate - the polymer solution - is prepared by dissolving from 1 to 5 parts by weight of sodium salt of alginic acid from brown algae in water in an amount of from 95 to 99.0 parts by weight, preferably 98 parts by weight.

Preferably, the surfactant content is 0.5 to 2.0% by weight relative to the weight of the aqueous phase.

Preferably, surfactants from the group of non-ionic surface-active compounds are used as emulsifiers, such as ethoxylated derivatives of sorbitan esters and lauric, palmitic, oleic fatty acids, and sorbitan esters and lauric, palmitic, oleic fatty acids and mixtures thereof.

Preferably, strains belonging to the genus Lactobacillus or Bifidobacterium are used as probiotic bacteria, preferably Lactobacillus casei, preferably in the amount of ^{108 -1012} CFU/ml.

Preferably, the homogenization process is carried out using a rotor-stator homogenizer at a speed of 7000-12000 rpm, preferably at a speed of 8000 rpm. Preferably, the homogenization process is carried out using an ultrasonic homogenizer and an amplitude of from 50 to 90%, preferably from 65 to 70%.

Preferably, the centrifugation process to separate the microcapsules is carried out at a speed of 500 to 3,000 rpm.

Advantageously, the process according to the invention produces capsules of controlled size, ranging from 10 to 500 micrometers in size.

The greatest influence on the size of alginate microcapsules obtained by the method according to the invention is exerted by the rate of addition and the concentration of the cross-linking agent.

Using ultrasonic homogenization using the method of the invention, stable alginate capsules with sizes ranging from 10 to 500 micrometers were obtained.

The obtained capsules can be used as a raw material carrying probiotics for use in preparations intended for skin care and/or treatment.

The invention is presented in embodiments.

Example 1

The initial emulsion - pre-emulsion, which was directed to cross-linking, was prepared by mixing the water phase and the oil phase. The water phase was an aqueous solution of sodium alginate at a concentration of 2% by weight with MRS broth, in which the concentration of Lactobacillus casei bacteria, as a result of multiplication of ATCC 334 lyophilisate, was 10 ¹⁰ CFU/ml. The ratio of sodium alginate solution to bacterial broth was 4:1. The oil phase was a mixture of capric-caprylyl triglycerides with a surfactant - polyoxyethylene sorbitan esters and oleic acid. The mass ratio of the water phase to the oil phase was 1:5. The surfactant content in the dispersion, in relation to the mass of the water phase was 1%. After combining the oil phase and the water phase, the pre-emulsion was mixed with a magnetic stirrer at a speed of 500 rpm for 10 minutes at room temperature - T=25°C. Then, the mixture constituting the pre-emulsion was subjected to a homogenization process using a rotor-stator homogenizer at a speed of 8000 rpm.

Before homogenization, the combined phases: water (A) and oil (B), as a result of mixing with a mechanical stirrer, create a system called a pre-emulsion (such a system is not yet stable, among other things, because the drops of the internal phase, in this case water, have different sizes, which promotes coalescence, and only after homogenization do we obtain a more homogeneous and therefore more stable internal phase - and thus the system can be called an emulsion.

In the next step, a 0.3 M calcium chloride solution was added dropwise at a rate of 1 ml per minute to the homogenized W/O emulsion containing the probiotic bacterial strain while continuously stirring (magnetic stirrer, rotation speed at 500 rpm) to form alginate microcapsules. The mass ratio of the alginate solution to the calcium chloride solution was 1:1. The mixing of the alginate microcapsule dispersion was continued after the addition of the CaCl2 solution was completed for another 10 minutes. In the next step, in order to separate the microcapsules obtained in the process, the oil dispersion was centrifuged at a speed of 500 rpm for 20 minutes, left for 24 hours at 5°C, and then, after separating the oil phase, the microcapsules were subjected to the lyophilization process. Alginate microcapsules with sizes d = 10-500 micrometers were obtained, containing probiotic bacteria - Lactobacillus casei.

Example 2

A pre-emulsion was prepared by mixing the water phase and the oil phase. The water phase was a 2% aqueous solution of sodium alginate with MRS broth, in which the concentration of Lactobacillus plantarum bacteria as a result of multiplication of ATCC 8014 lyophilisate was ¹⁰⁹ CFU/ml. The ratio of sodium alginate solution to bacterial broth was 4:1. The oil phase was a mixture of capric-caprylyl triglycerides with surfactant - ethoxylated sorbitan esters and oleic acid. The mass ratio of the water phase to the oil phase was 1:5. The surfactant content in the dispersion, in relation to the mass of the water phase was 1.0%. After combining the oil phase and the water phase, the pre-emulsion was mixed using a magnetic stirrer at a speed of 600 rpm, for 10 minutes, at room temperature T=25°C. Then, the mixture constituting the pre-emulsion was subjected to the homogenization process using the ultrasonic homogenization process and the sonication amplitude - 69%. In the next step, a 0.3 M calcium chloride solution was added dropwise at a rate of 1 ml per minute to the homogenized W/O emulsion containing the probiotic bacterial strain, while continuously stirring using a magnetic stirrer at a rotational speed of 600 rpm, while maintaining the mass ratio of the alginate solution to the calcium chloride solution at 1:1. After the addition of the CaClb solution was completed, the mixing of the alginate microcapsule dispersion was continued for another 10 minutes. In order to separate the obtained microcapsules, the dispersions were centrifuged at a speed of 500 rpm and then left for 24 hours at 5°C. After separation from the oil phase, washing with NaCl solution, the obtained microcapsules were subjected to the lyophilization process. Lyophilized alginate microcapsules with sizes d = 10-500 micrometers containing Lactobacillus casei were obtained.

The method of obtaining microcapsules in a W/O emulsion, which is the subject of the solution, is a simple process that does not require specialist equipment (e.g. equipped with special partitions). The parameters of the homogenization process and the change of the rate of dripping and the concentration of the cross-linking agent (calcium chloride) allow obtaining stable microcapsules with controlled sizes from 10 to 500 micrometers with high precision. The developed technique of producing microcapsules is economical and ecological, it does not require the use of large amounts of expensive raw materials, e.g. organic solvents. The main advantage of the solution is the high efficiency of encapsulation of probiotic bacteria in microcapsules.

Symbols and abbreviations:

MRS - "De Man, Rogosa and Sharpe" culture media for the multiplication and cultivation of bacteria;

CFU - Colony Forming Unit - a unit defining the number of microorganisms or cells in the tested material.

Claims (15)

1. A method for obtaining microcapsules containing probiotic bacteria enclosed inside a capsule made of a natural polymer, alginate from brown algae, in which a solution of a natural polymer with probiotic bacteria is prepared - component A, component B is prepared in the form of an oil phase containing surfactants, a cross-linking solution - component C is prepared, and then the individual components are mixed, characterized in that the cross-linking agent - component C, which is a 0.1-0.5 M aqueous solution containing Ca ²⁺ ions, is introduced at a rate of 0.3-2 ml per minute, maintaining the mass ratio of the alginate solution to the cross-linking agent solution at a level of 1:1, wherein previously an aqueous solution of sodium alginate, with a concentration of 1-5% by weight, is mixed with a MRS solution, in which the concentration of probiotic bacteria is 108-1012 CFU/ml, and the polymer solution is combined with the solution of probiotic bacteria in a ratio of 4:1, at a temperature of 20 to 25°C (component A), then the oil phase is introduced into the system - (component B), which is a mixture of capric-caprylic triglycerides with a surfactant, the mixture of components A and B is mixed using a mechanical stirrer at a speed in the range of 400-800 rpm for 5-10 minutes while maintaining the temperature of the dispersion system at 25°C, the obtained pre-emulsion is subjected to a homogenization process to obtain an emulsion, and then a cross-linking agent is introduced into the obtained system, with continuous mixing at a speed of 400-800 rpm, the mixture of components A, B and C is cross-linked at a temperature of 20-25°C for a period of 5 to 30 minutes, preferably 10 minutes, the microcapsules are separated by subjecting the oil emulsion to a centrifugation process, stabilized at a temperature of 5-7°C for a period of 24 hours, and then after separation oil phase, the isolated microcapsules are washed with 1% aqueous sodium chloride solution and subjected to the lyophilization process. 2. The method according to claim 1, characterized in that the Ca2 ⁺ ions come from a calcium salt solution, such as a calcium chloride solution with a concentration of 0.1-0.5 M. 3. The method according to claim 1, characterized in that component A is combined with component B in a weight ratio of 1:5. 4. The method according to claim 1, characterized in that the aqueous solution of sodium alginate - polymer solution - is prepared by dissolving from 1 to 5 parts by weight of sodium salt of alginic acid from brown algae in water in an amount of from 95 to 99.0 parts by weight. 5. The method according to claim 4, characterized in that the aqueous solution of sodium alginate - polymer solution - is prepared by dissolving from 2 parts by weight of sodium salt of alginic acid from brown algae in water in an amount of 98 parts by weight. 6. The method according to claim 1, characterized in that the surfactant content is 0.5 to 2.0% by weight relative to the weight of the aqueous phase. 7. The method according to claim 1, characterized in that surfactants from the group of non-ionic surface-active compounds, such as ethoxylated derivatives of sorbitan esters and lauric, palmitic, oleic fatty acids and sorbitan esters and lauric, palmitic, oleic fatty acids and mixtures thereof, are used as emulsifiers. 8. The method according to claim 1, characterized in that the probiotic bacteria used are strains belonging to the genus Lactobacillus or Bifidobacterium, preferably Lactobacillus casei. 9. The method according to claim 8, characterized in that the probiotic bacteria used are the Lactobacillus casei strain and the concentration of bacteria is ^108-1012 CFU/ml. 10. The method according to claim 1, characterized in that the homogenization process is carried out using a rotor-stator homogenizer at a speed of 7000-12000 rpm. 11. The method according to claim 10, characterized in that the homogenization process is carried out using a rotor-stator homogenizer at a speed of 8000 rpm. 12. The method according to claim 1, characterized in that the homogenization process is carried out using an ultrasonic homogenizer and an amplitude of 50 to 90%. 13. The method according to claim 12, characterized in that the homogenization process is carried out using an ultrasonic homogenizer and an amplitude of 65-70%. 14. The method according to claim 1, characterized in that the centrifugation process for separating the microcapsules is carried out at a speed of 500 to 3,000 rpm. 15. The method according to claim 1, wherein capsules of controlled size are obtained, having a size of 10 to 500 micrometers.