METHOD OF ENCAPSULATION OF AN ACTIVE PROTEIN USING ELECTRODEPOSITION TECHNIQUES, AN ANTIBACTERIAL COMPOSITION CONTAINING THE ACTIVE PROTEIN AND A POLYMER, AND ITS USE FOR THE PRODUCTION OF MEDICATIONS INTENDED FOR HUMANS

Abstract

The method of encapsulating an active protein includes (a) establishing a primary mesenchymal cell culture; (b) maintaining the cell culture until the culture surface is fully covered by the cultured cells; (c) obtaining a culture fluid from the cultured cells; (d) purifying the culture fluid from cell debris and suspended cells; (e) transferring the upper liquid phase to a new vessel; (f) gently mixing the purified liquid phase with an aqueous solution of polyvinyl alcohol; (g) adding ethyl alcohol to the mixture while stirring continuously; and (h) depositing the material on the collector surface by electro spinning or electrospraying. The invention includes an antibacterial composition containing an active protein and a polymer and ethyl alcohol. The active protein is a fibrous, fully water-soluble material containing proteins released by mesenchymal cells, including cathelicidin.

Claims

1. A method of encapsulating an active protein using electrodeposition techniques, wherein the active protein is comprised of a fibrous, fully water-soluble material containing proteins released by mesenchymal cells and cathelicidin at an amount from 990 to 1230 pg/100 mg of dry weight of the composition, the method comprising the following steps: (a) establishing a primary mesenchymal cell culture, obtained directly from frozen tissue isolate or from direct inoculation of the tissue isolate, and not subjected to further culture passages and containing 2,000-5,000 source tissue cells and a serum-supplemented culture medium; (b) maintaining the cell culture established in step (a) for 280-340 hours until the culture surface is fully covered by the cultured cells; (c) obtaining a culture fluid from the above of the cultured cells; (d) purifying the culture fluid obtained in step (c) from cell debris and suspended cells by centrifuging said fluid with a force of 300 to 1200×g; (e) transferring the upper liquid phase from above the sediment to a new vessel; (e′) purifying liquid phase from proteins greater than 50 kDa by filtering; (f) gently mixing the purified liquid phase obtained in step (e) with an aqueous solution of polyvinyl alcohol; (g) adding ethyl alcohol to the mixture obtained in step (f) while stirring continuously; and (h) depositing the material obtained in step (g) on the collector surface by means of electrospinning or electrospraying.

2. The method, according to claim 1, wherein establishing the culture in step (a) is performed using a culture medium selected from the group consisting of DMEM, DMEM-Ham's F-12, IMDM.

3. The method, according to claim 1, wherein the mesenchymal cells used in step a) are mesenchymal stromal cells derived from adipose tissue, bone marrow or Wharton's jelly.

4. The method, according to claim 3, wherein the mesenchymal cells are mesenchymal cells of species selected from the group consisting of dogs, cats, horses and sheep.

5. An antibacterial composition, comprising: an active protein; a polymer; 5% ethyl alcohol, wherein 47.5% is the active protein and 47.5% is the polymer, wherein the active protein is a fibrous, fully water-soluble material containing proteins released by mesenchymal cells from a primary culture, obtained directly from frozen tissue isolate or form direct inoculation of the tissue isolate, and not subjected to further culture passages; and cathelicidin at an amount from 990 to 1230 pg/100 mg of dry weight of the composition, wherein the polymer is an aqueous solution of polyvinyl alcohol, and wherein the composition is produced by electrodeposition of the active protein admixed with the polymer and ethyl alcohol.

6. The antibacterial composition, according to claim 5, wherein the polymer is a 30% aqueous solution (300 mg/ml) of polyvinyl alcohol.

7. The antibacterial composition, according to claim 5, wherein the mesenchymal cells are mesenchymal stromal cells derived from adipose tissue, bone marrow or Wharton's jelly.

8. The antibacterial composition, according to claim 7, wherein the mesenchymal cells are mesenchymal cells of species selected from the group consisting of dogs, cats, horses and sheep.

9. A method of using an antibacterial composition, the method comprising the step of: preparing the composition, according to claim 5 as a medication; and treating humans with the medication.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0045] The invention is shown in the Drawings.

[0046] FIG. 1 is a series of electron microscope images, representing the micro- and ultrastructure of the product obtained by the method according to the invention visualised using a scanning electron microscope under the following magnifications: A) 250×, B) 500×, C) 6500×, D) 35000×.

[0047] FIG. 2 is a graph illustration, representing the cytotoxicity assay for the compositions of the invention against three cell populations—or lines D-17, HAP1 and MSC.

[0048] FIG. 3 is a photographic image, presenting the zone of inhibition of growth (indicated by a circle) of bacteria of the Staphylococcus aureus species (MRSA) on a solid medium under the influence of a composition according to the invention.

[0049] FIG. 4 are photographic images, presenting the zone of growth inhibition (indicated by a circle) of bacteria of the Pseudomonas aeruginosa species on a solid medium under the influence of a composition according to the invention: A) in a view of the entire Petri dish, B) in a close-up detailing the zone of inhibition of bacterial growth, respectively

[0050] FIG. 5 is a photographic image, presenting the effect of an unmodified cellulose disc (negative control) on the growth of Staphylococcus aureus.

[0051] FIG. 6 is a photographic image, presenting the effect of octenidine dihydrochloride (positive control) on the growth of Staphylococcus aureus.

[0052] FIG. 7 is a graph illustration, presenting the concentration of cathelicidin (Canine Cathelicidin 1, CATHL1) in dry weight of the compositions of the invention over 31 days.

[0053] FIG. 8 is a graph illustration, presenting the change in concentration of cathelicidin (Canine Cathelicidin 1, CATHL1) over time in the material obtained after step e′ of the method of the invention (or before electrodeposition).

[0054] FIG. 9 is a photographic image, presenting the effect of the post-culture medium, which is the starting material for the preparation of the composition according to the invention, on the growth of Staphylococcus aureus MRSA.

[0055] FIG. 10 is a graph illustration, showing a comparison of the zones of growth inhibition of Staphylococcus aureus MRSA treated with different formulations.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The invention is presented in details in the following embodiments, wherein all the tests and experimental procedures described below were performed using commercially available test kits, reagents and equipment, following the instructions of the manufacturers of the applied kits, reagents and equipment, unless expressly stated otherwise. All test parameters were measured using standard, commonly known methods used in the art to which this invention belongs.

Example 1

[0057] Method of Encapsulating an Active Protein

[0058] The method of encapsulating an active protein of the invention comprises the following steps:

[0059] Step a: Establishing a Primary Mesenchymal Cell Culture

[0060] The first step of the method according to the invention comprises establishing a primary mesenchymal cell culture containing (at the initial step) from 2,000 to 5,000 source tissue cells (in a culture vessel) and a serum-supplemented culture medium (preferably 10% bovine serum). The culture is then maintained without the use of antibiotics. In the present embodiment, 5,000 cells were used to establish the culture. Cells were counted in the Bürker chamber,

[0061] wherein, the term “primary culture” is understood to mean a non-passaged culture, or a so-called “passage 0” obtained directly from a frozen tissue isolate constituting “stock culture”, or from direct inoculation of the tissue isolate and not subject to further culture passages.

[0062] In this non-limiting embodiment, canine bone marrow mesenchymal cells and DMEM (Dulbecco Modified Eagle Medium) supplemented with 10% serum were used. On the other hand, other mesenchymal cells (e.g., mesenchymal stromal cells derived from adipose tissue, bone marrow or Wharton's jelly isolated from species such as dog, cat, horse and sheep during in vitro culture) may also be used in the method of the invention. The same in the case of the medium, to establish a culture medium of the above-mentioned mesenchymal cells, culture media containing ions necessary for the maintenance and growth of cells in culture other than DMEM, e.g. DMEM-Ham's F-12 (Dulbecco Modified Eagle Medium—Ham's F-12) or IMDM (Iscove's Modified Dulbecco Medium), which additionally ensure the appropriate content of ions to obtain sufficient substrate conductivity for the proper performance of the material synthesis process in the electrodeposition process, can also be used,

[0063] wherein the choice of a specific medium depends on the mesenchymal cells selected for the culture. To obtain a complete culture medium, the medium is supplemented with 10% bovine serum. However, no antibiotics are added to the medium prepared in this way.

[0064] Stage b: Maintaining the Cell Culture Established in Step (a) Until the Culture Surface is Fully Covered by the Cultured Cells

[0065] Cell cultures are performed in standard culture vessels under conditions consistent with the guidelines of the American Type Culture Collection, ATTC), or at a temperature of 37° C. and an atmosphere containing 95% of air and 5% of CO2 at a relative humidity of 90%.

[0066] The culture is maintained under the above-mentioned conditions for 280-340 hours until the culture surface is fully covered by the cultured cells, In this non-limiting embodiment, the culture surface was fully covered after 280 hours,

[0067] wherein, during the culture process (or from the moment of establishing to the moment ending the culture), the culture medium is not replaced. This will allow for the collection of all proteins, growth factors, cytokines and other peptides secreted by the mesenchymal cells from the beginning of the culture.

[0068] Step c: Obtaining a Culture Fluid from the Above of the Cultured Cells

[0069] After the mesenchymal cells cover the culture surface adequately, the culture fluid containing proteins, growth factors, cytokines and peptides secreted by the cultured cells is transferred into a new sterile vessel.

[0070] Step d: purifying the culture fluid obtained in step (c) from cell debris and suspended cells

[0071] The obtained culture medium is centrifuged at 1200×g in order to eliminate cell debris and suspended cells.

[0072] Step e: Transferring the Supernatant Liquid Phase to a New Vessel

[0073] After centrifugation, the obtained supernatant liquid phase is transferred to a new sterile vessel.

[0074] In this embodiment, the medium was then subjected to purification of albumin derived from foetal bovine serum, which may be an allergen. Filtration was performed using an Amicon-type molecular filter with a pore size of 50 kDa, thanks to which proteins larger than 50 kDa are removed from the said media, and proteins, growth factors, cytokines and peptides smaller than 50 kDa are preserved.

[0075] The media prepared in this way can be used to obtain the substrate directly on the day of their preparation, or they can be frozen at a temperature <−18° C. and used later after thawing.

[0076] Step f and g: Mixing the Purified Liquid Phase with an Aqueous Solution of Polyvinyl Alcohol and Ethyl Alcohol;

[0077] The next steps include mixing the purified liquid phase with a solution of polyvinyl alcohol and ethyl alcohol. In this embodiment, the electrodeposition material is obtained by mixing the purified aqueous phase of the post-culture medium (component A) with a 30% polyvinyl alcohol solution (component B) and 99.8% ethyl alcohol (component C) at the following volume ratio:

47.5% of component A+47.5% of component B+5% of component C, [0078] wherein, first, component A is mixed with component B in a gentle manner, preventing the formation of foam.

[0079] In this embodiment, a 30% aqueous solution (300 mg/ml) of polyvinyl alcohol with a molecular weight of 20-30 kDa is used as component B,

[0080] wherein, the aqueous solution of polyvinyl alcohol is prepared by mixing 300 mg of dry polymer with 1 ml of water and heating the mixture at 90° C. until the polymer powder is completely dissolved (approximately 1-2 hours). The solution is then cooled to room temperature.

[0081] After the components A and B are mixed, component C (ethyl alcohol) is added at continuous stirring. The material prepared in this way is used for electrodeposition, which should be started immediately in order to reduce the process of degradation of proteins present in the substrate.

[0082] Stage h: Electrodeposition of the Prepared Material on the Collector Surface

[0083] The prepared material (substrate) is loaded into a disposable syringe. The syringe is connected to a hose ending with a head that supplies electric voltage to the substrate, at the end of which a blunt tip steel needle is installed, wherein, in this embodiment, the outer needle diameter was 1 mm and the inner needle diameter was 0.7 mm.

[0084] A cable supplying positive voltage of 11.8 kV is connected to the head. The syringe is placed on an automatic piston connected to an adjustable stepper motor that allows the speed of liquid flow through the system to be set. The material is deposited on the surface of the collector, in particular a steel collector or an aluminium foil connected to the grounding. wherein the liquid flow rate is 60 μl of substrate/hour and the distance from the end of the needle to the collector is 12 cm, wherein, in this embodiment, the deposition of the material on the collector surface was performed by electrospinning.

[0085] The process allows for the production of a minimum of 4 mg of dry product from 60 μl of substrate per hour (28.5 μl of culture medium), wherein the obtained product contains 6.5% of protein in dry matter. The micro- and ultrastructure of the obtained material are shown in FIG. 1.

Example 2

[0086] Method of encapsulating an active protein according to Example 1, except that: human mesenchymal cells of bone marrow or Wharton's jelly and IMDM medium supplemented with 10% serum were used to establish the primary mesenchymal cell culture.

[0087] wherein 2,000 cells were used, and the culture was maintained for 340 hours. The obtained culture medium was centrifuged at 300×g in order to eliminate cell debris and suspended cells.

[0088] An aqueous solution of polyvinyl alcohol was prepared by mixing 300 mg of dry polymer with 1 ml of water and heating the mixture at 95° C. for 1 hour and then cooling to room temperature.

[0089] The electrodeposition substrate was loaded into a disposable syringe with the outer needle diameter of 0.5 mm and the inner needle diameter of 0.2 mm.

[0090] In turn, the electrodeposition was carried out using the following parameters: [0091] positive voltage of 11.7 kV; [0092] liquid flow rate of 60 μl of substrate/hour [0093] distance from the tip of the needle to the collector of 13 cm. [0094] wherein, in this embodiment, the deposition of the material on the collector surface was performed by electrospraying.

[0095] The process allows for a product containing 5.5% of protein in dry matter.

Example 3

[0096] Antibacterial Composition

[0097] The product obtained by the method according to the invention is an antibacterial composition.

[0098] Said composition comprises an active protein, a polymer, and ethyl alcohol, wherein, the active protein is a fibrous, fully water-soluble material containing proteins released by mesenchymal cells (e.g., canine bone marrow mesenchymal cells), including cathelicidin. In this embodiment, the mesenchymal cells are canine bone marrow mesenchymal cells. On the other hand, other mesenchymal cells e.g., mesenchymal stromal cells derived from adipose tissue, bone marrow or Wharton's jelly of human origin or isolated from species such as dog, cat, horse and sheep during in vitro culture may also be used during in vitro culture.

[0099] In this embodiment, the composition of the invention comprises cathelicidin at an amount of 1230 pg/100 mg of the dry weight of the composition. In turn, the polymer is an aqueous solution of polyvinyl alcohol, preferably a 30% aqueous solution (300 mg/ml) of polyvinyl alcohol with a molecular weight of 20-30 kDa. In this embodiment, the antibacterial composition of the invention comprises 47.5% of the active protein, 47.5% of polyvinyl alcohol aqueous solution and 5% of ethyl alcohol.

Example 4

[0100] Antibacterial Composition

[0101] Antibacterial composition as in Example 3 except that the concentration of cathelicidin is 990 pg/100 mg of dry weight of the composition.

Example 5

[0102] Antibacterial Composition

[0103] Antibacterial composition as in Example 3 except that the concentration of cathelicidin is 270 pg/100 mg of dry weight of the composition.

Example 6

[0104] Antibacterial Composition

[0105] Antibacterial composition as in Example 3 except that the concentration of cathelicidin is 420 pg/100 mg of dry weight of the composition.

Example 7

[0106] Antibacterial Composition

[0107] Antibacterial composition as in Example 3 except that the concentration of cathelicidin is 820 pg/100 mg of dry weight of the composition.

Example 8

[0108] Antibacterial Composition

[0109] Antibacterial composition as in Example 3 except that the concentration of cathelicidin is 550 pg/100 mg of the composition.

Example 9

[0110] Analysis of the Cytotoxicity of the Compositions of the Invention

[0111] The cytotoxicity assay was performed using canine adipose tissue-derived mesenchymal stromal cells (MSC, passage 3), dog osteosarcoma reference line (ATCC® D-17), and human leukaemia haploid line (HAP1, HorizonDiscovery). For the experiment, cells were grown in wells in a 24-well plate.

[0112] The following sample designations were used: [0113] AM-API-1 test sample—a 10% solution of the composition according to the invention in DMEM (Dulbecco Modified Eagle Medium) containing 10% foetal bovine serum; [0114] negative control—10% solution of the negative material in DMEM containing 10% of foetal bovine serum, where the negative material is fresh (or has never come into contact with cells) medium encapsulated according to the method of the invention; [0115] positive control—DMEM containing 10% of foetal bovine serum

[0116] In the first stage of the study, the cultures of the MSC, D-17 and HAP1 cell lines were established and the cultures were maintained until the culture surface was fully covered. After the culture surface was fully covered, a 10% solution of AM-API-1 in DMEM containing 10% of foetal bovine serum was added to the test group cultures. A 10% solution of AM-API-1 analogous material was added to the negative control culture, wherein the culture medium was replaced with fresh medium. Complete medium (DMEM+10% of foetal bovine serum) was added to the positive control. After 24 hours of culture, cells were harvested with the use of trypsin and tested for viability using 0.4% trypan blue (staining dead cells) and a BioRad TC20 automated cell counter. The analysis was performed in ten replicates for each cell line,

[0117] wherein the culture density (number of cells/cm2) differed between individual lines, wherein in the case of: [0118] the MSC line—the culture density at the time of harvest after the experiment was ˜1×105 cells/cm2, while 10ט1×105 cells were counted in the experiment; [0119] the D-17 line—the culture density at the time of harvest after the experiment was ˜1.8×105 cells/cm2, while 10ט1.8×105 cells were counted in the experiment; [0120] the HAP1 line—the culture density at the time of harvest after the experiment was 3.2×105 cells/cm2, while 10ט3.2×105 cells were counted in the experiment.

[0121] The conducted analysis revealed that the 10% AM-API-1 solution showed a slight cytotoxicity in relation to the tested cells of the D-17, HAP1 and MSC lines (FIG. 2). No statistically significant difference between the experimental group and the negative control was recorded, which indicates that polyvinyl alcohol is probably responsible for the slight cytotoxic effect, which may affect the properties of the medium (osmotic concentration, density).

Example 10

[0122] Analysis of the Antibacterial Properties of the Compositions of the Invention

[0123] The test of antibacterial properties was performed on two reference strains—Staphylococcus aureus MRSA and Pseudomonas aeruginosa using the A.D.A.M. method previously described by the team of Adam F. Junka (Junka A. F., ?ywicka A., Szymczyk P., Dziadas M., Bartoszewicz M., Fija?kowski K. A.D.A.M. test (Antibiofilm Dressing's Activity Measurement)—Simple method for evaluating anti-biofilm activity of drug-saturated dressings against wound pathogens. J Microbiol Methods 2017; 143:6-12).

[0124] In the experiment, 2 ml of a suspension of 105 colony-forming units (CFU) of Komagataeibacter xylinus/1 ml of dedicated Herstin-Schramm medium, which was introduced into the well of a 24-well plate and incubated at 28° C. for 7 days until bacterial cellulose (BC) mats were produced, were used. Subsequently, the BC mats were cleaned by alkaline lysis to remove cells, and then rinsed with water until pH=7. On the discs obtained in the said way, 5 mg of AM-API-1 (composition according to the invention—10% solution of AM-API-1 in water) or the appropriate control was applied and spread over the surface of the BC discs. BC discs modified in this way were applied to agar plates on which Staphylococcus aureus or Pseudomonas aeruginosa were inoculated in a manner analogous to the commonly used disc diffusion method used in the determination of antibiotic resistance, and incubated at 37° C. for 24 hours. After the incubation time had elapsed, the zones of growth inhibition around the BC discs were read out. As a control of the experiment, an unmodified BC disc (negative control) and a disc soaked with a substance with confirmed antimicrobial activity—octenidine dihydrochloride (positive control) were used. The test with octenidine dihydrochloride was performed for Staphylococcus aureus. The test was performed in triplicate.

[0125] The conducted microbiological tests showed that the composition according to the invention, dissolved in a physiological buffer (water) at a concentration of 10%, shows a strong antibacterial effect against bacteria of the species Staphylococcus aureus MRSA (FIG. 3), as well as a moderate effect against bacteria of the species Pseudomonas aeruginosa (FIGS. 4A and 4B).

[0126] For the negative control, no antimicrobial activity was observed (FIG. 5). In the case of the positive control—octenidine dihydrochloride—the expected inhibition of bacterial growth was recorded (FIG. 6). The obtained results confirm the antimicrobial properties of the compositions according to the invention, which, in combination with the lack of cytotoxicity (confirmed by the analysis according to example 9), make the composition according to the invention suitable to be used as an active ingredient in medicinal products for human use, in particular for the treatment of bacterial infections.

Example 11

[0127] Analysis of the Cathelicidin Content in the Composition According to the Invention

[0128] The basis for obtaining reproducible amounts of cathelicidin in the composition according to the invention is obtaining conditioned media from cultures conducted according to a specific, reproducible and validated method, or the method according to the invention.

[0129] In this embodiment, the composition of the invention (designated AM-API-1) was obtained using mesenchymal cells derived from a dog.

[0130] Antimicrobial properties of canine cathelicidin (Canine Cathelicidin 1, hereinafter CATHL1) are well known (Santoro D, Marsella R, Bunick D, Graves T K, Campbell K L Expression and distribution of canine antimicrobial peptides in the skin of healthy and atopic beagles. Vet Immunol Immunopathol. 2011 Dec. 15; 144(3-4):382-8. doi: 10.1016/j.vetimm.2011.08.004).

[0131] A commercially available ELISA (CATHL1 ELISA Kit, MyBioSource) was used to confirm the cathelicidin (Canine Cathelicidin 1, hereinafter CATHL1) content in the composition of the invention (AM-API-1).

[0132] In order to perform a measurement, a 10% solution of AM-API-1 was prepared by dissolving 100 mg of the material (composition according to the invention) in 1 ml of DMEM. As a negative control, the material prepared according to the invention (analogous to AM-API-1) was applied, which was prepared using fresh DMEM containing 10% of bovine serum instead of medium obtained from the culture (or fresh, sterile medium that had never come into contact with tissue culture). The assay was prepared according to the instructions provided by the manufacturer with the ELISA kit (CATHL1 ELISA Kit, MyBioSource). Measurements were performed using a ThermoScientific Multiskan FC spectrophotometric microplate reader.

[0133] The study was carried out in five replicates with the use of five different batches of the culture medium. In each replicate, the control and test sample were prepared using five independent source cell cultures.

[0134] In the study, both the peptide content in the dry weight of AM-API-1 on the first day after isolation, and the stability of the material stored under refrigerated conditions (2-8° C.) over a 31-day period were determined. The results are shown in Table 1 below:

TABLE-US-00001 TABLE 1 Analysis of CATHL1 content in dry matter of AM-API formulation during 31 days Time (days) 1 6 25 31 Content in 990-1230 820-970 400-550 270-420 100 mg of dry matter (pg)

[0135] It has been shown that refrigerated storage allows the stability of the test protein in AM-API-1 to be maintained for a minimum of 31 days (FIG. 7).

[0136] Moreover, the concentration of cathelicidin in the conditioned media constituting the starting material for AM-API-1 (or the material obtained after step e′ of the method according to the invention, but before its mixing with the aqueous solution of polyvinyl alcohol) was tested analogously. The media were stored under refrigerated conditions (or at a temperature in the range of 2-8° C.) for no more than 30 days after being harvested from the culture.

[0137] The media were dissolved and stored in the refrigerator for 10 consecutive days to determine the decrease in peptide concentration over time. Concentration was measured on days 1 and 6 after thawing the media. The test was performed in triplicate. CATHL1 concentration was measured using an ELISA assay (CATHL1 ELISA kit, MyBioSource). As shown in FIG. 8, after the collection of the culture medium, the cathelicidin concentration was above 2000 pg/ml, while after 6 days of refrigerated storage of the medium, the cathelicidin concentration dropped to 0.

[0138] The presented analysis confirms that the encapsulation of the active ingredient using the method according to the invention provides a product with extended stability.

Example 12

[0139] Bioavailability Analysis

[0140] As part of the drug bioavailability study, the antibacterial properties of the post-culture medium constituting a starting material for the composition of the invention were compared with the composition of the invention (designated AM-API-1). The experiment was conducted in the same manner as in the case of the analysis of antimicrobial properties presented in Example 10.

[0141] In this embodiment, the following formulations were analysed: [0142] AM-API-1—the composition according to the invention; [0143] Placebo—a negative control, which were unmodified BC discs; [0144] Octenidine dihydrochloride—a substance with known antibacterial activity, which served as a positive control; [0145] Conditioned medium—or post-culture medium (prepared according to steps a-e′ of the method according to the invention) constituting the starting material for the composition according to the invention.

[0146] The BC discs were soaked with the above-mentioned formulations and placed in Petri dishes according to the methodology described above. The media was tested for growth inhibition of Staphylococcus aureus MRSA. As presented in Fig. the conditioned medium (starting material) showed a similar antibacterial effect as the composition according to the invention. Measurements of the diameters of the zones of growth inhibition and their comparison with the results for AM-API-1 showed similar effectiveness of the starting material and AM-API-1 in inhibiting the growth of S. aureus MRSA (FIG. 10).