Recombinant peptidoglycan hydrolase protein with antibacterial properties, compositions comprising it, their uses and methods utilizing thereof

Abstract

The object of the invention is a recombinant protein with antibacterial properties having an amino acid sequence derived from the sequence of the M23 domain from Streptococcus thermophilus of a peptidoglycan hydrolase character, preferably in the form of a recombinant protein chimera, as well as compositions comprising it, their uses and methods utilizing thereof. The invention is based on recombinant proteins being peptidoglycan hydrolases with peptidase properties - derived from the M23 domain originating from Streptococcus thermophilus, showing a particularly high specificity to Gram (+) bacteria with a direct cross-linking bridges in peptidoglycan, and discloses the proteins of a recombinant protein chimera such as LB24, additionally comprising a bacteria cell wall binding CWT domain from Staphylococcus pettenkoferi.

Description

[0001] Recombinant peptidoglycan hydrolase protein with antibacterial properties, compositions comprising it, their uses and methods utilizing thereof

[0002] DESCRIPTION

[0003] TECHNICAL FIELD

[0004] The object of the invention is a recombinant peptidoglycan hydrolase protein with antibacterial properties, preferably a recombinant protein chimera, compositions comprising it, their uses and methods utilizing thereof. Recombinant proteins are disclosed, which are peptidoglycan hydrolases with peptidase properties - derived from the M23 domain originating from Streptococcus thermophilus, showing a particularly high specificity to Gram (+) bacteria having direct cross-linking bridges in the peptidoglycan (the so-called DAP type) and recombinant M23 domain proteins, such as the recombinant protein chimera LB24, additionally comprising a bacterial cell wall binding domain, preferably the bacterial cell wall binding domain is the CWT domain from Staphylococcus pettenkoferi, what significantly extends the range of the environment in which the recombinant protein chimera operates.

[0005] The invention is used as an antibacterial and bactericidal agent, e.g. in the food industry for disinfecting surfaces, preventing the growth of pathogenic bacteria, in particular in preventing the growth of bacteria Listeria monocytogenes and Bacillus cereus.

[0006] STATE OF ART

[0007] Listeriosis is a disease caused by Gram (+) bacterium Listeria monocytogenes. It is a type of food poisoning that can occur after consuming food contaminated with this bacterium. In most healthy individuals, L. monocytogenes infection can be mild or even asymptomatic. However, in individuals with a weakened immune system, such as the elderly, infants, pregnant women, and those with chronic illnesses, listeriosis can lead to serious complications, including meningitis, sepsis, miscarriages, or neonatal infection. Despite effective antibiotic therapy, listeriosis constitutes a public health problem, causing death in up to 30% of cases [1], A potential source of infection are the so-called "RTE" (Ready-to-Eat) products. These include smoked meats, cold cuts, cheeses, and salads. Secondary infections resulting from insufficient hygiene standards in production facilities have a significant impact on the occurrence of Listeria monocytogenes in these food products. These bacteria are characterized by a wide range of tolerance to environmental conditions, L. monocytogenes survives at temperatures ranging from -2 °C to 45 °C and pH from 4.4 to 9.4. It can survive short-term pasteurization and freezing. Additionally, it easily becomes resistant to preservatives and cleaning agents, forming bacterial biofilms [2], Listeria monocytogenes infections are traditionally treated with penicillins or carbapenems, however, in recent years, an increasing number of Listeria strains exhibiting multiple resistance to antibiotics, including those used to treat infections, are identified. Therefore, new substances capable of eliminating bacteria from the environment are sought. Bacteriocins, which also include peptidoglycan hydrolases, constitute a promising alternative to antibiotics. They are called natural preservatives and are considered safe food additives. In the state of the art such substances are known, including, e.g., nisin A (E234), used in food preservation under the trade name Nisaplin®. Nisin is a polycyclic peptide consisting of 34 amino acids, generated during the fermentation process by Lactococcus lactis lactic acid bacteria. Its activity is based on inhibition of cell wall biosynthesis in bacteria. This substance is active over a wide pH range from 3.5 to 8.0 and is used in a variety of food products.

[0008] In the literature, endolysins, which are peptidoglycan hydrolases capable of eliminating Listeria monocytogenes bacteria in food products are known [3, 4], Due to their specific structure, consisting of a catalytic domain (CD) at the N-terminus, a linker, and a binding domain (CWT) at the C-terminus of the protein, they can act on specific bacteria, specifically on a designated bond in the peptidoglycan (PG). For example, endolysin Ply500 demonstrated the ability to significantly reduce the number of L. monocytogenes on the surface of stored iceberg lettuce, and LysZ5 showed high listericidal activity in soymilk. In turn, PlyLM endolysin demonstrated the ability to remove Listeria monocytogenes biofilms, which are difficult to eliminate with traditional cleaning and disinfection methods. These biofilms can form on production equipment surfaces and in food processing environments, thus posing a serious threat to food microbiological safety. PlyLM effectively destroys biofilm structures, what helps to reduce the risk of food contamination by L. monocytogenes [5],

[0009] Document EP1285069A1 discloses the bacteriocin sakacin G derived from Lactobacillus sake! 2512 and exhibiting activity against Listeria monocytogenes bacteria in food products. This invention prevents product spoilage, however its use for disinfecting surfaces in the food industry has not been disclosed.

[0010] In turn, document EP0705843A1 discloses a bacteriocin obtained from Enterococcus faecium CTC492, which prevents the growth of Listeria monocytogenes in meat and dairy products.

[0011] In the food and biotechnology industries, contamination of work surfaces and products with bacteria such as Listeria monocytogenes and Bacillus cereus is a common problem. These bacteria are responsible for food spoilage and also pose a serious threat to public health, causing food poisoning and other infections. Current methods for combating these pathogens, such as the use of antibiotics and disinfectants, have their limitations. Firstly, bacteria can develop resistance to antibiotics, making it difficult to effectively treat infections. Secondly, chemical agents used for disinfection can be harmful to human health and the environment, and their effectiveness can be limited by the conditions of the production environment — especially often this effectiveness is limited by pH and salinity. Therefore, there exists a need to provide new proteins of peptidoglycan hydrolase character, in particular in the form of recombinant proteins enabling effective reduction of the number of a wide range of bacteria with different peptidoglycan structures in a broad pH range, preferably capable of functioning also in a wide range of salinities, including both low and high salinity. Moreover, there is a need to provide recombinant proteins that are safe for plants, humans, and animals, and do not affect the development cycle of insects— pollinators, in particular. In the state of the art, the issue of increasing bacterial resistance to antibiotics is frequently raised, thus recombinant polypeptides that do not induce such resistance are highly desirable.

[0012] DISCLOSURE OF THE INVENTION

[0013] The invention relates to a recombinant protein with antibacterial properties, which recombinant protein is a recombinant protein chimera comprising

[0014] - a domain of peptidoglycan hydrolase character having an amino acid sequence with at least 90% identity with the sequence of the M23 domain from Streptococcus thermophilus, as shown in SEQ ID No. 1; more preferably having at least 95% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1, more preferably it is an amino acid sequence having the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1;

[0015] - a CWT bacterial cell wall binding domain attached at one of its termini, preferably at the C-terminus, having an amino acid sequence with at least 90% identity with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi; more preferably having at least 95% identity with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi, more preferably it is an amino acid sequence with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi; wherein the recombinant protein has antibacterial properties against Gram (+) bacteria.

[0016] In the preferred recombinant protein with antibacterial properties, the domain with peptidoglycan hydrolase character is linked to the CWT bacterial cell wall binding domain by a linker, preferably having the amino acid sequence SEQ ID No. 5.

[0017] The recombinant protein with antibacterial properties is preferably a recombinant protein chimera LB24 having the amino acid sequence SEQ ID No. 3.

[0018] The invention also relates to a genetic construct that encodes a recombinant protein with antibacterial properties according to the invention.

[0019] The invention also relates to a host cell that comprises a genetic construct according to the invention. The invention also relates to a composition that comprises a recombinant protein with antibacterial properties according to the invention or a mixture thereof and a carrier.

[0020] The composition is preferably in the form of a liquid, solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, ointment, cream, cosmetic or medical powder.

[0021] The composition is preferably intended for use as an antibacterial and / or bactericidal agent.

[0022] The composition is preferably intended for use as an antibacterial and bactericidal agent against Gram (+) bacteria, preferably against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso- DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide.

[0023] The composition is preferably intended for use as an antibacterial and bactericidal agent against bacteria of the genus Listeria spp., Bacillus spp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably against the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0024] The invention also relates to the non-medical use of a recombinant protein according to the invention and / or a composition according to the invention as an antibacterial agent and / or a bactericidal agent.

[0025] In the preferred non-medical use, the antibacterial and / or bactericidal agent is used against Gram(+) bacteria, preferably against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso- DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably against bacteria of the genus Listeria spp., Bacillus spp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably against bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0026] The invention also relates to the use of a recombinant protein with antibacterial properties according to the invention and / or a composition according to the invention as an antiseptic and disinfecting agent.

[0027] The invention also relates to the use of a recombinant protein as an antiseptic and disinfecting agent used in the food and biotechnological industry for decontamination and disinfection of surfaces, devices and products, preferably for decontamination and disinfection from Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2- 4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus spp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0028] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an antibacterial agent for decontamination of surfaces and rooms in food processing, preferably for decontamination of surfaces and / or tools that come into contact with food or semi-finished food products, preferably as an antibacterial agent for decontamination of surfaces and rooms on production lines in the processing of meat, milk and dairy products.

[0029] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an agent preventing and reducing the formation of bacterial biofilms, preferably preventing and reducing the formation of bacterial biofilms produced by Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D- Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus spp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0030] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as a decontaminating agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for decontamination of hospital surfaces, medical and veterinary offices, laboratory surfaces, surfaces of tools, surfaces of devices used in medicine, veterinary medicine, diagnostics.

[0031] The invention also relates to a method for inhibiting or limiting or preventing the growth of bacteria in vitro, which comprises the step of contacting the bacteria with a protein with antibacterial properties according to the invention and / or a composition according to the invention.

[0032] In the preferred method, the bacteria are selected from Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl- 2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus spp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the genus Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0033] The invention also relates to a composition against Gram (+) bacteria, which is intended for use as an antibacterial and bactericidal agent, wherein the composition comprises a carrier, and

[0034] - a recombinant protein with antibacterial properties according to the invention, and / or a recombinant protein consisting of a domain of peptidoglycan hydrolase character having an amino acid sequence with at least 90% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1; more preferably with at least 95% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1, more preferably it is an amino acid sequence having the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1; and / or

[0035] - a recombinant protein in the form of a protein chimera comprising a domain of peptidoglycan hydrolase character having an amino acid sequence with at least 90% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1; more preferably at least 95% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1, more preferably it is an amino acid sequence having the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1 with the bacterial cell wall binding domain CWT attached at one of its termini, preferably at the C-terminus of the chimera, wherein the bacterial cell wall binding domain is preferably a SH3b, LysM, AMIN type domain, preferably the bacterial cell wall binding domain is a CWT domain with an amino acid sequence with at least 90% identity with the amino acid sequence from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi; more preferably at least 95% identity with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi, more preferably it is an amino acid sequence with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi; wherein preferably the peptidoglycan hydrolase domain is linked to the CWT bacterial cell wall binding domain by means of a linker, preferably with the amino acid sequence SEQ ID No. 5.

[0036] The composition against Gram (+) bacteria is preferably in the form of a liquid, solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, ointment, cream, cosmetic or medical powder.

[0037] The composition against Gram (+) bacteria is preferably intended for use as an antibacterial and bactericidal agent against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso- DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide.

[0038] The composition is preferably intended for use as an antibacterial and bactericidal agent against bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; preferably against the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0039] In the preferred composition against Gram (+) bacteria, the carrier is a veterinarily and / or pharmaceutically acceptable and the composition is intended for use in the prevention and treatment of a disease and / or condition caused by the bacteria, preferably wherein the disease is listeriosis.

[0040] The composition against Gram (+) bacteria is preferably used in the prevention and treatment of a disease and / or condition caused by Gram (+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably by bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0041] The invention also relates to the non-medical use of the composition against Gram (+) bacteria according to the invention as an antibacterial agent and / or a bactericidal agent.

[0042] In the preferred non-medical use, the antibacterial and / or bactericidal agent is used against Gram(+) bacteria, preferably against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso- DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably against bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably against bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0043] The invention also relates to the use of a composition against Gram (+) bacteria as an antiseptic and disinfecting agent.

[0044] In the preferred use of the composition, the antiseptic and disinfecting agent is used in the food and biotechnology industry for decontamination and disinfection of surfaces, devices and products, preferably for decontamination and disinfection from Gram(+) bacteria, more preferably bacteria having a crosslinking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0045] The invention also relates to the use of a composition against Gram (+) bacteria as an antibacterial agent inhibiting and preventing microbiological contamination in the food industry, preferably as an additive to food for humans and / or animals, preferably as an additive to milk products, dairy products, meat products, more preferably to milk, yoghurts, cheeses, kefirs, cottage cheeses, milk drinks; cold cuts, sausages, minced meat, frankfurters.

[0046] In the preferred use, the agent is used to inhibit, eliminate and prevent the growth of Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys- D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0047] In the preferred use, the agent is used to prevent infections and diseases caused by microorganisms present in food, preferably listeriosis caused by Listeria monocytogenes. The invention also relates to the use of a composition against Gram (+) bacteria as an antibacterial agent for decontamination of surfaces and rooms in food processing, preferably for decontamination of surfaces and / or tools that come into contact with food or semi-finished food products, preferably as an antibacterial agent for disinfecting surfaces and rooms on production lines in the processing of meat, milk and dairy products.

[0048] The invention also relates to the use of a composition against Gram (+) bacteria as an agent preventing and reducing the formation of bacterial biofilms, preferably preventing and reducing the formation of bacterial biofilms produced by Gram (+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus f aecium, Micrococcus luteus.

[0049] The invention also relates to the use of a composition against Gram (+) bacteria as a decontaminating agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for decontamination of hospital surfaces, medical and veterinary offices, laboratory surfaces, surfaces of tools, surfaces of devices used in medicine, veterinary medicine, diagnostics.

[0050] The invention also relates to the use of a composition against Gram (+) bacteria for in vitro use as an antibacterial agent in the cosmetics industry, as an additive to cosmetics improving their microbiological quality, preferably as a biopreservative, preferably as an additive to liquids, creams, milks, lotions, mists, gels.

[0051] The invention also relates to a cosmetic or care composition for cosmetic, care, or hygiene applications in humans or animals, which comprises a composition against Gram (+) bacteria according to the invention, wherein the composition is intended for external use, and wherein the composition against Gram (+) bacteria is used in the cosmetic or care composition to reduce the occurrence of or eliminate Gram (+) bacteria from the cosmetic or care composition.

[0052] The invention also relates to a method for inhibiting or limiting or preventing the growth of bacteria in vitro, which comprises the step of contacting the bacteria with a protein having antibacterial properties such as the composition against Gram (+) bacteria according to the invention.

[0053] In the preferred method, the bacteria are selected from Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl- 2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus. The invention also relates to a recombinant protein with antibacterial properties which has an amino acid sequence with at least 80% identity with the sequence of the M23 domain from Streptococcus thermophilus peptidoglycan hydrolase as shown in SEQ ID No. 1.

[0054] The preferred recombinant protein with antibacterial properties has an amino acid sequence with at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID No. 1; most preferably it is the M23 domain from Streptococcus thermophilus having the amino acid sequence SEQ ID No. 1.

[0055] Moreover, the preferred recombinant protein with antibacterial properties has a bacterial cell wall binding domain attached to one of its termini, preferably to the C-terminus, creating a recombinant protein chimera, wherein, preferably, the bacterial cell wall binding domain is a SH3b, LysM, AMIN type domain, preferably the bacterial cell wall binding domain is a CWT domain, being the SH3b domain.

[0056] The preferred recombinant protein with antibacterial properties is a recombinant protein chimera with a CWT cell wall binding domain having an amino acid sequence with at least 80% identity with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi, more preferably a recombinant protein chimera with a CWT domain having an amino acid sequence with at least 80% identity with the amino acid sequence SEQ ID No. 2 from Staphylococcus pettenkoferi.

[0057] The preferred recombinant protein with antibacterial properties is a recombinant protein chimera wherein the CWT cell wall binding domain has an amino acid sequence that is at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical to the sequence shown in SEQ ID No. 2; most preferably the CWT cell wall binding domain is the amino acid sequence SEQ ID No. 2 from Staphylococcus pettenkoferi.

[0058] The preferred recombinant protein with antibacterial properties is a recombinant protein chimera having an amino acid sequence with at least 80% identity with the sequence of the recombinant protein chimera LB24 of SEQ ID No. 3, more preferably having an amino acid sequence with at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID No. 3, more preferably it is a recombinant protein chimera LB24 with the amino acid sequence SEQ ID No. 3.

[0059] The invention also relates to a genetic construct that encodes a recombinant protein with antibacterial properties according to the invention.

[0060] The invention also relates to a host cell that comprises a genetic construct according to the invention.

[0061] The invention also relates to a composition, which comprises a recombinant protein with antibacterial properties according to the invention or a mixture thereof and a carrier.

[0062] The composition is preferably in the form of a liquid, solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, ointment, cream, cosmetic or medical powder. The composition is preferably intended for use as an antibacterial and / or bactericidal agent.

[0063] The composition is preferably intended for use as an antibacterial and bactericidal agent against Gram (+) bacteria, preferably against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso- DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide.

[0064] The composition is preferably intended for use as an antibacterial and bactericidal agent against bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; preferably against the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0065] The invention also relates to the non-medical use of the recombinant protein according to the invention and / or the composition according to the invention as an antibacterial agent and / or a bactericidal agent.

[0066] In non-medical use, the antibacterial and / or bactericidal agent is preferably used against Gram(+) bacteria, preferably against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso- DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably against bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably against bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0067] The invention also relates to the use of the recombinant protein with antibacterial properties according to the invention and / or the composition according to the invention as an antiseptic and disinfecting agent.

[0068] In the preferred use of the recombinant protein, the antiseptic and disinfecting agent is used in the food and biotechnology industry for the decontamination and disinfection of surfaces, devices and products, preferably for the decontamination and disinfection from Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl- 2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0069] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an antibacterial agent inhibiting and preventing microbiological contamination in the food industry, preferably as an additive to food for humans and / or animals, preferably as an additive to milk products, dairy products, meat products, more preferably to milk, yoghurts, cheeses, kefirs, cottage cheeses, milk drinks; cold cuts, sausages, minced meat, frankfurters.

[0070] In the preferred use of the recombinant protein, the agent is used to inhibit, eliminate and prevent the growth of Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0071] In the preferred use of the recombinant protein, the agent is used to prevent infections and diseases caused by microorganisms present in food, preferably listeriosis caused by Listeria monocytogenes.

[0072] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an antibacterial agent for decontamination of surfaces and rooms in food processing, preferably for decontamination of surfaces and / or tools that come into contact with food or semi-finished food products, preferably as an antibacterial agent for decontamination of surfaces and rooms on production lines in the processing of meat, milk and dairy products.

[0073] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an agent preventing and reducing the formation of bacterial biofilms, preferably preventing and reducing the formation of bacterial biofilms produced by Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D- Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtlis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0074] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as a decontaminating agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for decontamination of hospital surfaces, medical and veterinary offices, laboratory surfaces, surfaces of tools, surfaces of devices used in medicine, veterinary medicine, diagnostics.

[0075] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an in vitro antibacterial agent in the cosmetics industry, as an additive to cosmetics improving their microbiological quality, preferably as a biopreservative, preferably as an additive to liquids, creams, milks, lotions, mists, gels.

[0076] The invention also relates to a veterinary and / or pharmaceutical composition, which comprises the recombinant protein according to the invention and / or the composition according to the invention and a veterinarily and / or pharmaceutically acceptable carrier for use in the prevention and treatment of a disease and / or condition caused by bacteria, wherein, preferably, the disease is listeriosis.

[0077] The veterinary and / or pharmaceutical composition for use is preferably used in the prophylaxis and treatment of a disease and / or state caused by Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys- murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus spp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0078] The invention also relates to a cosmetic or care composition for cosmetic, care or hygiene applications in humans or animals, which comprises the recombinant protein according to the invention and / or the composition according to the invention, wherein the composition is intended for external use, and wherein the recombinant protein with antibacterial properties is used in the cosmetic or care composition to reduce the occurrence of or eliminate bacteria from the composition, preferably wherein the bacteria are Gram (+) bacteria.

[0079] The invention also relates to a method for inhibiting or limiting or preventing the growth of bacteria in vitro, which comprises the step of contacting the bacteria with a protein having antibacterial properties according to the invention and / or a composition according to the invention.

[0080] In the preferred method, the bacteria are selected from Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl- 2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

[0081] The invention describes recombinant proteins being peptidoglycan hydrolases with the properties of M23 peptidase derived from Streptococcus thermophilus, which exhibits a particular specificity for Gram(+) bacteria having direct cross-links in the peptidoglycan (the so-called m-DAP type) and recombinant proteins of this M23 domain, such as the recombinant protein chimera LB24, additionally comprising a bacterial cell wall binding domain, preferably the CWT domain from Staphylococcus pettenkoferi.

[0082] The invention describes recombinant proteins having domain variants with a specified amino acid sequence identity to a given sequence. A person skilled in the art understands that an amino acid sequence identity at the level of at least 80%, more preferably at the level of least 90%, most preferably at the level of least 95%, will result in domain variants with analogous properties. It will be obvious and readily possible for a person skilled in the art to make substitutions of amino acid from one to another without changing the properties of the domain in question, and it is known in the art that an identity at the level of least 90% ensures similar biological properties of a given domain.

[0083] It was shown that the recombinant M23 peptidase protein is capable of highly efficient elimination of bacteria Listeria monocytogenes, Bacillus subtilis and Bacillus cereus but within a narrow range of pH and salinity, which may be very useful in applications requiring such environmental conditions. Due to the fact that the recombinant M23 protein was active in a narrow range of pH and salinity, a method was sought to extend the range of activity of the recombinant protein based on the M23 domain so that its activity was high in a wide range of pH and salinity while maintaining the antibacterial properties resulting from the properties of the M23 domain.

[0084] Unexpectedly, it was found that adding a domain that binds the bacterial cell wall provides such an effect. Unexpectedly, it was found that the produced recombinant proteins comprising the M23 catalytic domain and bacterial cell wall binding domains, preferably recombinant protein chimeras being a combination of the M23 domain from Streptococcus thermophilus with the CWT bacterial cell wall binding domain from Staphylococcus pettenkoferi, produce proteins with peptidoglycan hydrolase activity resulting from the activity of the M23 domain, which are active in a wide range of pH and salinity.

[0085] Another demonstrated advantage is the broad spectrum of activity of the recombinant protein in the form of a recombinant protein chimera according to the invention, particularly advantageous is the high activity of the LB24 protein in conditions close to physiological, due to which the recombinant protein according to the invention effectively eliminates various types of bacteria, including Listeria monocytogenes, Bacillus subtilis and Bacillus cereus, but also Staphylococcus simulans, Enterococcus faecium and Micrococcus luteus. This makes the peptidoglycan hydrolase in the form of a recombinant protein in the form of a recombinant protein chimera according to the invention a universal agent against microbiological contamination, what is particularly important in the food industry, where the diversity of pathogens can be high.

[0086] In order to produce a recombinant protein in the form of a recombinant protein chimera with a higher tolerance to different environmental conditions, a bacterial cell wall binding domain can be added to the active domain M23 derived from Streptococcus thermophilus or an active derivative thereof (having at least 80% amino acid sequence identity within the M23 domain).

[0087] Examples of bacterial cell wall binding domains that can be used to produce a recombinant protein in the form of a recombinant protein chimera according to the invention include

[0088] - SH3b (Src Homology 3 domain superfamily, bacterial) domains that are part of multidomain proteins such as, e.g., lysostaphin, a bacteriocin secreted by Staphylococcus simulans bacteria (GenBank sequence: AAB53783.1, amino acids 402-475); ALE-1, an endopeptidase produced by Staphylococcus capitis (PDB sequence: 1R77_A); endolysin from the Staphylococcus aureus phage StB20 (GenBank sequence: YP_007236686.1);

[0089] - CBD (cell wall binding) domains, which recognize peptidoglycan in the cell walls of Listeria monocytogenes bacteria, found in bacteriophage endolysins, e.g., in the Ply endolysin from the PSA phage genome (PDB database, no. 1XOV); - AMIN domains (Amidase N-terminal domain), which direct periplasmic proteins or proteins from the outside of the cell to the bacterial cell wall, most often to the site of cell division, the so-called septum, found, e.g., in AmiC proteins in E. coli (PDB 4BIN), AMIN~Amidase_3 (YP_001336853^ from Klebsiella pneumoniae, AMIN (NP_938979) from Corynebacterium diphteriae;

[0090] - LysM domains, binding peptidoglycans, for example the domain of transglycosylase D (MltD) in E. coli (PDB 1E0G), or of N-acetylglucosaminidase AltA in Enterococcus faecalis.

[0091] Using a recombinant peptidoglycan hydrolase protein (peptidoglycan hydrolase) based on the M23 domain from Streptococcus thermophilus, or recombinant protein chimeras comprising it, as an antibacterial agent minimizes the development of resistance, which is a significant advantage over the traditional antibiotic methods. In this way, the solutions according to the invention help to combat the growing problem of antibiotic resistance, which is crucial for public health.

[0092] Additionally, the recombinant peptidoglycan hydrolase protein based on the M23 domain from Streptococcus thermophilus as well as recombinant protein chimeras containing an additional bacterial cell wall binding domain are safe for human and animal health and are environmentally friendly. They do not produce toxic byproducts, which is particularly important in the context of food production. The use of such enzymes in the form of recombinant proteins and recombinant protein chimeras according to the invention increases the safety of food products by minimizing the risk of contamination and improving the quality and shelf life of products.

[0093] BRIEF DESCRIPTION OF DRAWINGS

[0094] For a better understanding of the invention, it is illustrated in the embodiments and in the attached drawings, in which:

[0095] Fig. 1. schematically shows the organization of domains in the protein A) LB24; B) VDG62699.1, C) ASE36562.1. The LB24 protein comprises the sequence of the M23 domain (SEQ ID No. 1) and the CWT domain (SEQ ID No. 2). The protein fragment comprising the M23 catalytic domain is derived from the starting protein VDG62699.1 and is located within it at the positions 97-222, and the fragment with the CWT domain is derived from the protein ASE36562.1, hypothetical protein CEP67_04465, in which it is located at the positions 348-446. SP - signal peptide, M23 - catalytic domain, CWT - cell wall binding / recognition domain.

[0096] Fig. 2. shows the separation of the purified, recombinant M23 protein and the recombinant protein chimera LB24 (10 pg per well) on a 15% polyacrylamide gel with a size marker - MW (Prestained Protein Ladder, Thermo Fisher Scientific). Fig. 3. shows the measurement of the lytic activity of the recombinant M23 protein expressed as a % reduction of the initial OD595 of bacterial suspensions of the selected strains after an hour of incubation with 1 pM protein at room temperature in 50 mM glycine pH 8.0 and in 50 mM glycine pH 8.0 with the addition of 100 mM NaCI.

[0097] Fig. 4. shows the measurement of the lytic activity of the recombinant protein chimera LB24 expressed as the % reduction of the initial OD595 of bacterial suspensions of the selected strains after an hour of incubation with lpM LB24 protein at room temperature in 50 mM glycine pH 8.0 with the addition of 100 mM NaCI.

[0098] Fig. 5 shows a comparison of the lytic activity of the recombinant protein - the M23 catalytic domain in comparison to the recombinant protein chimera LB24 under different conditions: (A) in the pH range from 5 to 11 in low conductivity buffers and (B) in conditions with different salt concentration from 0 to 1000 mM in 50 mM glycine pH 8.0 expressed as a % reduction of the initial OD595 of the Listeria monocytogenes DSM 19094 cell suspension.

[0099] Fig. 6. shows the lytic activity of the recombinant protein chimera LB24 on industrial strains of bacteria. (A) various industrial strains of L. monocytogenes (Nofima, MF strains), (B) industrial strains of B. cereus (NMBU strains). Results are expressed as % reduction of the initial OD595 of the bacterial suspension of industrial strains, obtained after 1 hour of incubation with 1 pM of the recombinant protein chimera LB24.

[0100] Fig. 7. shows the ability of the recombinant protein chimera LB24 to eliminate bacterial biofilm biomass obtained on plastic plates under laboratory conditions. Activity was monitored by staining the biofilm with crystal violet and reading absorbance at the wavelength of A=595 nm.

[0101] Fig. 8. shows the bactericidal activity of the recombinant protein chimera LB24 against L. monocytogenes DSM 19094 in a decontamination test of glass, metal and silicone surfaces. The results are presented using a survivability test, monitored by droplet method on a TSB agar plate.

[0102] Fig. 9. shows the activity of the recombinant protein chimera LB24 after 3 months of storage at -80°C and at -20°C after lyophilization process. The results are expressed as a % reduction of the initial OD595 of the bacterial suspension of L. monocytogenes DSM 19094, obtained after an hour of incubation with 1 pM of the recombinant protein chimera LB24.

[0103] Fig. 10. shows the ability of the recombinant protein chimera LB24 to remove bacterial biofilms obtained on metal surfaces incubated for 48 hours in milk. The results are presented using a survivability test of the cells grown from the biofilm, monitored by droplet method on a TSB agar plate.

[0104] EMBODIMENTS OF THE INVENTION The following examples are provided merely for the purpose of illustrating the invention and explaining particular aspects thereof, and are not intended to be limiting and should not be identified with its entire scope, which is defined by the appended claims.

[0105] When the term "comprising" or "including" is used in this description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be the preferred embodiment of the term "comprising" or "including". If a group is defined below that includes at least a number of embodiments, this is also to be understood as disclosing a group that preferably consists of only those embodiments.

[0106] The terms "about" or "approximately" in the context of the present invention mean a range of precision that one skilled in the art would understand to be sufficient to still provide the technical effect of the feature. The term typically means a deviation from the indicated numerical value of ±10%, and preferably ±5%.

[0107] Technical terms are used in accordance with their typical meaning. If a specific meaning is used, the meaning will be described in detail with reference to the context in which a given term is used.

[0108] EXAMPLES

[0109] In the following examples, unless otherwise indicated, standard materials and methods used in the art were used or the manufacturer's recommendations for specific materials and methods were followed.

[0110] Example 1. Production of the recombinant M23 protein and recombinant protein chimera LB24

[0111] Based on BLAST sequence alignment with the M23 domain of the LytM protein from Staphylococcus aureus, the VDG62699.1 protein was identified in the genome of Streptococcus thermophilus NCTC10353 with 31% amino acid sequence identity. This protein consists of 225 amino acids, the first 22 of which constitute the signal peptide, and amino acids 118-214 constitute the M23 catalytic domain (CD). The full- length ORF gene VDG62699 from Streptococcus thermophilus NCTC10353 was codon-optimized for overexpression in Escherichia coli and then chemically synthesized by BioCat (Germany). The fragment encoding the M23 catalytic domain and the adjacent amino acid residues corresponding to the aa 97-222 fragment of the sequence of the protein were amplified by PCR and then cloned by a ligation-independent method into the pET His 6 TEV LIC vector (IB), according to the protocol posted on the Addgene plasmid repository website (Addgene plasmid # 29653; http: / / n2t.net / addgene:29653; RRID:Addgene_29653). The recombinant protein chimera LB24 was prepared by the incomplete polymerase extension cloning method (PIPE cloning), where the insert with the gene encoding the CWT domain from Staphylococcus pettenkoferi (ORF ASE36562.1) located in the pMCSG-7 vector and the His6 TEV LIC expression vector (IB) with the M23 gene insert were amplified in separate reactions, and then mixed together to form a vector- insert hybrid according to the protocol described in [6], Fig. 1 shows a scheme of the structure of the obtained recombinant LB24 protein along with the starting proteins from which the M23 and CWT domains are derived, connected by a linker consisting of several amino acids resulting from the connection of these two domains. The proteins were produced and purified in the same manner.

[0112] The expression vector comprising the recombinant protein gene was introduced into competent Escherichia coll BL21 (DE3) cells. Subsequently, expression was carried out in the auto-induction medium AIM_Super Broth from Formedium. Initially, the culture was carried out at 37°C for 3 hours, and then the bacteria were cultured at 25°C for 24 h. After this time, the bacteria were centrifuged and the bacterial pellet was frozen. The bacteria were lysed using ultrasounds. The recombinant protein was purified by affinity chromatography on a nickel beads (Ni-Sepharose 6 Fast Flow) and then further purified by gel filtration on a Superdex 75 10 / 300 column. Such purified proteins were frozen with the addition of 10% glycerol and stored at -80°C or lyophilized and stored at -20°C. Fig. 2 shows the SDS-PAGE analysis of the obtained protein preparations.

[0113] The recombinant protein M23 and the recombinant protein chimera LB24 produced in Example 1 were used in further analyses and tests.

[0114] Example 2 Activity and specificity of the recombinant M23 protein against different Gram (-) and Gram (+) bacterial species under low and high ionic strength conditions in 50 mM glycine pH 8.0

[0115] In order to investigate the specificity of the M23 protein, a cell wall lysis assay was performed by measuring changes in the optical density of a cell suspension (turbidity reduction assay, TRA). For this purpose, 20 reference strains were selected (Fig. 3).

[0116] Bacterial cells were grown in TSB medium at 37°C with shaking at 80 rpm to OD595=0.6. After this time, the bacteria were centrifuged and suspended in a buffer consisting of 50 mM glycine pH 8.0 or 50 mM glycine pH 8.0 with the addition of 100 mM NaCI to OD595=1. 1 pM of the M23 protein (produced as described for LB24 in Example 1) placed in a 96-well plate was added to the bacterial suspension. The plate was placed in a Bio-Rad plate reader. The OD of the suspension was measured for 60 minutes, with the OD595 reading taken every 2 minutes and shaking for 5 seconds before each reading. The results confirm the activity against Listeria monocytogenes and Bacillus subtilis strains, as a % reduction of the initial OD595 in 50 mM glycine pH 8.0, as well as a strongly limited lytic activity in the presence of 100 mM NaCI (Fig. 3).

[0117] Example 3 Activity and specificity of the recombinant protein chimera LB24 against different species of Gram (-) and Gram (+) bacteria in 50 mM glycine pH 8.0 with 100 mM NaCI

[0118] In order to verify the lytic properties of the recombinant protein chimera LB24 in the presence of 100 mM salt, the TRA assay was performed analogously as in Example 2. The results of this analysis are presented in Tab. 1 along with the level of lytic activity (% reduction of the initial OD) for a given strain with a given type of cross-linking bridge in the peptidoglycan.

[0119] Tab. 1. Specificity of the activity of the recombinant protein chimera LB24 against various bacterial species.

[0120] The recombinant LB24 protein showed the highest bacteriolytic activity against Listeria monocytogenes strains (over 80%), Bacillus cereus (over 80%), as well as Bacillus subtilis strains (over 60%). Activity against Staphylococcus simulans, Enterococcus faecium and Micrococcus luteus was also observed (Fig. 4). The bactericidal activity was confirmed in a droplet test after a series of dilutions of the bacterial suspension of L. monocytogenes DSM 19094 (107CFU / ml) after incubation with different concentrations of the recombinant protein chimera LB24.

[0121] Example 4 The influence of pH on the lytic efficacy of the recombinant M23 protein and the recombinant protein chimera LB24

[0122] L. monocytogenes DSM19094 strain was cultured in TSB medium at 37°C to OD595=0.6, then centrifuged and the bacterial pellet was washed in milli-Q water. Subsequently, the suspension was adjusted to OD595— 1 in milli-Q water and incubated with recombinant M23 proteins or LB24 chimera at a concentration of 1 pM suspended in buffers of different pH. For this purpose, low conductivity buffers were used: 10 mM sodium acetate pH 5.0, 2 mM sodium citrate pH 6.0, 5 mM Tris-HCI pH 7.0, 50 mM glycine pH 8.0, and 50 mM Tris-HCI pH 9.0, 20 mM CAPS pH 10.0 and 10 mM CAPS pH 11.0. The OD reduction test of the L. monocytogenes DSM 19094 bacterial suspension was performed as in Example 2, and the results are presented in Fig. 5A. The recombinant M23 protein showed low pH tolerance. It was active at pH from 6.0 to 8.0. In turn, the recombinant protein chimera LB24 showed high activity over a wide pH range from 6 to 11. The recombinant LB24 protein was almost inactive at pH 5.0. The recombinant LB24 protein showed optimal activity in pH 8.0 buffer (Fig. 5A).

[0123] Example 5 The effect of salt concentration on the lytic efficacy of the recombinant M23 protein and the recombinant protein chimera LB24

[0124] The OD reduction test of L. monocytogenes DSM19094 bacterial suspension was performed analogously as in Example 2 in order to investigate the dependence of the lytic activity on salt concentration. 50 mM glycine buffer pH 8.0 with different concentrations of salt (0 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, and 500 mM NaCI) was used in the turbidity reduction test. The recombinant M23 protein showed high activity only in low-salt conditions, even 10 mM NaCI caused a two-fold decrease in the activity of this protein, and 50 mM NaCI completely inhibited the lytic activity. The recombinant protein chimera LB24 showed high activity in the presence of 0 to 100 mM NaCI. At 250 mM NaCI concentration, the activity was approximately 30%, and at 500 mM, approximately 10% (Fig. 5B).

[0125] Example 6 The effect of the recombinant protein chimera LB24 on industrial strains

[0126] L. monocytogens strains isolated from the food processing environment by the Nofima - food research institute in Norway (Fig. 6. A, MF strains) and Bacillus cereus strains from the collection of the NMBU university in Norway isolated from the dairy processing environment (Fig. 6B) were exposed to the recombinant protein chimera LB24 in an OD reduction test analogously as in Example 2. The results were presented as the % reduction of the initial OD595 of the tested bacterial suspensions after 60-minutes incubation with 1 pM of the recombinant protein chimera LB24 compared to the reference strain DSM19094. The tested L. monocytogenes and Bacillus cereus strains were very susceptible to the activity of the recombinant protein chimera LB24 according to the invention.

[0127] Example 7 The ability to eliminate L monocytogenes biofilm by the recombinant protein chimera LB24 according to the invention

[0128] To assess the biofilm removal efficiency of the recombinant protein chimera LB24 according to the invention, 20 pl of an overnight culture of Listeria monocytogenes MF6331 was added to 180 pl of BHI medium supplemented with 0.6% yeast extract and incubated for another 24 hours in a 96-well plate. After this time, the wells were emptied and washed, and 200 pl of 2 pM recombinant protein chimera LB24 was added. After 2 hours of incubation at 37°C, the cells were washed and then fixed in 96% ethanol for 20 minutes. The ethanol was then removed and the wells were completely air-dried, after which 200 pl of 0.1% crystal violet solution was added. After 30 minutes of staining, the bacterial biofilms were washed 3 x 100 pl with MQ. water and completely air-dried again. After drying, 220 pl of 30% acetic acid was added to each well, incubated for 30 min, and then 200 pl of the solution was transferred to a fresh 96-well plate for absorbance measurement at 595 nm using a Bio-Rad microplate reader. Each experiment was independently repeated twice and was performed in 20 technical replicates. The recombinant protein chimera LB24 was able to eliminate more than 30% of the bacterial biofilm biomass (69% of the obtained biofilm biomass remained) (for results see Fig. 7).

[0129] Example 8 The ability of the recombinant protein chimera LB24 according to the invention to decontaminate surfaces

[0130] Listeria monocytogenes strain DSM 19094 was grown in TSB overnight at 37°C with shaking at 80 rpm. Subsequently, the cells were centrifuged and suspended in water to a density corresponding to 1.8 McFarland, what corresponds to approximately 108CFU / ml. Sterile glass beads, stainless steel cylinders (length 14 mm, inner diameter 6 mm, outer diameter 9 mm) and silicone tubes were immersed in the bacterial suspension and air-dried for 30 min. The prepared materials were incubated for 60 minutes in a 1 pM LB24 solution, and then serially diluted in water and plated on TSB agar. As shown in Fig. 8, LB24 was able to completely eliminate bacterial contamination from every surface used in the experiment.

[0131] Example 9 Storage of the recombinant protein chimera LB24

[0132] The recombinant protein chimera LB24 at a concentration of 100 pM as a glycerol stock (10% glycerol) was frozen in liquid nitrogen and stored at -80°C. Another storage method was lyophilization of the protein (1 mg per ampoule) and storage in the form of a powder (lyophilisate) in glass ampoules at -20°C. After 3 months of storage under such conditions, the activity of such preparations was verified in the OD reduction test. As shown in Fig. 9, there are no significant differences in activity between fresh, frozen, and lyophilized protein preparations of the recombinant protein chimera LB24. The recombinant protein according to the invention retains its activity even after prolonged storage in various forms and under various storage conditions. Its stability makes it suitable for use in the production of preparations for the industrial production of enzyme preparations comprising it.

[0133] Example 10 An example of elimination of bacterial biofilms from stainless steel cylinders incubated in milk

[0134] Stainless steel cylinders (10 mm long, internal diameter of 6 mm) were incubated in milk for 48 hours at room temperature, what allowed the bacteria present in the milk to multiply. The cylinders with bacterial biofilms were then rinsed three times in 50 mM glycine pH 8.0 and placed in a 2 ml Eppendorf tubes. 1 ml of 50 mM glycine pH 8.0 (control) or 1 ml of glycine solution with 2 pM recombinant protein chimera (sample) were added to the Eppendorf tubes. After 2 hours of incubation, the cylinders were rinsed once with 50 mM glycine pH 8.0, and then the bacterial biofilms from the milk were physically removed by scraping them with a swab into 0.5 ml of 50 mM glycine pH 8.0. The bacterial suspensions obtained in this way were serially diluted and plated on a BHI agar plate. As can be seen in Fig. 10, the cylinders that were incubated with the protein of the recombinant protein chimera LB24 were no longer contaminated with bacterial biofilm, while in the control, survival of 105CFU / ml of biofilm-derived bacterial cells is visible.

[0135] REFERENCES:

[0136] 1. Pagliano P, Arslan F, Ascione T (2017) Epidemiology and treatment of the commonest form of listeriosis: meningitis and bacteraemia. Infez Med 25:210-216

[0137] 2. Walczycka M Metody inaktywacj i hamowania wzrostu Listeria monocytogenes w przetworach mi^snych. ZYWNOSC Nauka Technologia Jakosc 2:61-72

[0138] 3. Gray JA, Chandry PS, Kaur M, et al (2018) Novel Biocontrol Methods for Listeria monocytogenes Biofilms in Food Production Facilities. Front Microbiol 9:605.

[0139] 4. Bai J, Kim Y-T, Ryu S, Lee J-H (2016) Biocontrol and rapid detection of food-borne pathogens using bacteriophages and endolysins. Frontiers in Microbiology 7:.

[0140] 5. Nazir A, Xu X, Liu Y, Chen Y (2023) Phage Endolysins: Advances in the World of Food Safety. Cells 12:2169.

[0141] 6. Klock HE, Lesley SA (2009) The Polymerase Incomplete Primer Extension (PIPE) Method Applied to High-Throughput Cloning and Site-Directed Mutagenesis. In: Doyle SA (ed) High Throughput Protein Expression and Purification: Methods and Protocols. Humana Press, Totowa, NJ, pp 91-103

[0142] SEQUENCE LISTING

[0143] SEQ ID No. 1 Amino acid sequence containing the recombinant M23 protein constituting residues 97-222 of the "peptidase M23 / M37 family" protein sequence from Streptococcus thermophilus NCTC10353 (VDG62699.1).

[0144] GQKVVLPAEGRFTSGYGPRWGRMHNGIDIANGIGTPIYSVMDGTVINAGPAQGFGKWVRVRHDDGTITVYGHVHSF NVSVGQRVTAGEQIAEMGNEGQSTGPHLHFEVRPGGGDAIDPVPWLKERG

[0145] SEQ ID No. 2 Amino acid sequence containing the CWT domain of the LB24 protein (bacterial cell wall binding domain) constituting residues 348-446 of the hypothetical protein CEP67_04465 from Staphylococcus pettenkoferi (GenBank: ASE36562.1), on a gray background there is a linker sequence, the sequence of the CWT domain of the LB24 protein amino acids 9-99 (91 AA long) KNPSTSNGFKTNKYGTLYKSEKASFTPNTNIITRKTGPFRSMKQAGILKAGKKINYDEVMKQDGYVWLGYGSKKNRKYV PIRTWNRNTNAMGPIWGKIS

[0146] SEQ ID No. 3 Amino acid sequence of the entire protein of the recombinant protein chimera LB24, the linker sequence is highlighted in gray GQKVVLPAEGRFTSGYGPRWGRMHNGIDIANGIGTPIYSVMDGTVINAGPAQGFGKWVRVRHDDGTITVYGHVHSF NVSVGQRVTAGEQIAEMGNEGQSTGPHLHFEVRPGGGDAIDPVPWLKERGKN PSTSNGFKTNKYGTLYKSEKASFTP NTNIITRKTGPFRSMKQAGILKAGKKINYDEVM KQDGYVWLGYGSKKNRKYVPIRTWNRNTNAMGPIWGKIS

[0147] SEQ ID No. 4: Nucleotide sequence encoding the protein of the recombinant protein chimera LB24 Ggccagaaggtcgtccttccggcagagggccgcttcacctcaggctacggcccgcgttggggccgcatgcacaacggcatcgacatcgccaacggc atcggcaccccgatctactccgtcatggacggcacagtcatcaatgccggccccgcacagggcttcggtaagtgggtccgcgtccgccacgacgacg gcaccatcaccgtgtacggccacgtccactccttcaacgtgagcgtgggccagcgtgtgaccgccggtgagcaaattgccgaaatgggcaacgaggg ccagtccaccggcccgcacctccacttcgaggtccgcccgggcggcggcgatgccatcgacccggtgccgtggctcaaggaacgcggcaagaaccc atctacttctaacggattcaaaactaataaatatggaacgctttataaatctgaaaaagcatcatttacaccaaatacaaatattatcactagaaagac gggtccttttagaagtatgaaacaagcaggaattttaaaagctggtaagaaaattaactatgatgaagtaatgaaacaagatggttatgtttggttag gttatggaagtaaaaagaaccgtaaatacgtaccaattagaacttggaatagaaatactaacgctatgggaccaatttgggggaaaattagt

[0148] SEQ ID No. 5 Amino acid sequence of the linker constituting a fragment of SpM23b. KNPSTSNG

Claims

CLAIMS1. A recombinant protein with antibacterial properties, characterized in that the recombinant protein is a recombinant protein chimera comprising- a domain of peptidoglycan hydrolase character having an amino acid sequence with at least 90% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1; more preferably having at least 95% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1, more preferably it is an amino acid sequence having the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1;- a CWT bacterial cell wall binding domain attached at one of its termini, preferably at the C-terminus, having an amino acid sequence with at least 90% identity with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi; more preferably having at least 95% identity with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi, more preferably it is an amino acid sequence with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi; wherein the recombinant protein has antibacterial properties against Gram (+) bacteria.

2. The recombinant protein with antibacterial properties according to claim 1, characterized in that the domain with peptidoglycan hydrolase character is linked to the CWT bacterial cell wall binding domain by a linker, preferably having the amino acid sequence SEQ ID No. 5.

3. The recombinant protein with antibacterial properties according to claims 1-2, characterized in that it is a recombinant protein chimera LB24 having the amino acid sequence SEQ ID No. 3.

4. A genetic construct characterized in that it encodes the recombinant protein with antibacterial properties as defined in claims 1-3.

5. A host cell characterized in that it comprises a genetic construct as defined in claim 4.

6. A composition characterized in that it comprises the recombinant protein with antibacterial properties as defined in claims 1-3 or a mixture thereof and a carrier.

7. The composition according to claim 6, characterized in that it is in the form of a liquid, solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, ointment, cream, cosmetic or medical powder.

8. The composition according to claims 6-7, characterized in that it is intended for use as an antibacterial and / or bactericidal agent.

9. The composition according to claims 6-8, characterized in that it is intended for use as an antibacterial and bactericidal agent against Gram (+) bacteria, preferably against bacteria having a cross-linking bridgein the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide.

10. The composition according to claims 7-9, characterized in that it is intended for use as an antibacterial and bactericidal agent against bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably against the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

11. A non-medical use of the recombinant protein as defined in claims 1-3 and / or the composition as defined in claims 8-10 as an antibacterial agent and / or a bactericidal agent.

12. The non-medical use according to claim 11, characterized in that the antibacterial and / or bactericidal agent is used against Gram(+) bacteria, preferably against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably against bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably against bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

13. Use of the recombinant protein with antibacterial properties as defined in claims 1-3 and / or the composition as defined in claims 8-10 as an antiseptic and disinfecting agent.

14. The use of a recombinant protein according to claim 13, characterized in that the antiseptic and disinfecting agent is used in the food and biotechnological industry for decontamination and disinfection of surfaces, devices and products, preferably for decontamination and disinfection from Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys- D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

15. Use of the recombinant protein as defined in claims 1-3 and / or the composition as defined in claims 8-10 as an antibacterial agent for decontamination of surfaces and rooms in food processing, preferably for decontamination of surfaces and / or tools that come into contact with food or semi-finished food products, preferably as an antibacterial agent for decontamination of surfaces and rooms on production lines in the processing of meat, milk and dairy products.

16. Use of the recombinant protein as defined in claims 1-3 and / or the composition as defined in claims 8-10 as an agent preventing and reducing the formation of bacterial biofilms, preferably preventing and reducing the formation of bacterial biofilms produced by Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

17. Use of the recombinant protein as defined in claims 1-3 and / or the composition as defined in claims 8-10 as a decontaminating agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for decontamination of hospital surfaces, medical and veterinary offices, laboratory surfaces, surfaces of tools, surfaces of devices used in medicine, veterinary medicine, diagnostics.

18. A method for inhibiting or limiting or preventing the growth of bacteria in vitro, characterized in that it comprises the step of contacting the bacteria with the protein with antibacterial properties as defined in claims 1-3 and / or the composition as defined in claims 8-10.

19. The method according to claim 18, characterized in that the bacteria are selected from Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D- Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the genus Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

20. A composition against Gram (+) bacteria, characterized in that it is intended for use as an antibacterial and bactericidal agent, wherein the composition comprises a carrier, and- the recombinant protein with antibacterial properties as defined in claims 1-3, and / or- a recombinant protein consisting of a domain of peptidoglycan hydrolase character having an amino acid sequence with at least 90% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1; more preferably with at least 95% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1, more preferably it is an amino acid sequence having the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1; and / or- a recombinant protein in the form of a protein chimera comprising a domain of peptidoglycan hydrolase character having an amino acid sequence with at least 90% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1; more preferably at least 95% identity with the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1, more preferably it is an amino acid sequence having the sequence of the M23 domain from Streptococcus thermophilus as shown in SEQ ID No. 1with the bacterial cell wall binding domain CWT attached at one of its termini, preferably at the C- terminus of the chimera, wherein the bacterial cell wall binding domain is preferably a SH3b, LysM, AMIN type domain, preferably the bacterial cell wall binding domain is a CWT domain with an amino acid sequence with at least 90% identity with the amino acid sequence from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi; more preferably at least 95% identity with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi, more preferably is an amino acid sequence with the sequence of amino acids from 9 to 99 of SEQ ID No. 2 from Staphylococcus pettenkoferi; wherein preferably, the peptidoglycan hydrolase domain is linked to the CWT bacterial cell wall binding domain by means of a linker, preferably with the amino acid sequence SEQ ID No. 5.

21. The composition according to claim 20, characterized in that it is in the form of a liquid, solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, ointment, cream, cosmetic or medical powder.

22. The composition according to claims 20-21, characterized in that it is intended for use as an antibacterial and bactericidal agent against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide.

23. The composition according to claims 20-22, characterized in that it is intended for use as an antibacterial and bactericidal agent against bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; preferably against the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

24. The composition according to claims 20-23, characterized in that the carrier is veterinarily and / or pharmaceutically acceptable, and that the composition is intended for use in the prevention and treatment of a disease and / or condition caused by bacteria, preferably wherein the disease is listeriosis.

25. The composition according to claim 24, characterized in that it is used for the prevention and treatment of a disease and / or condition caused by Gram (+) bacteria, preferably bacteria having a crosslinking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably by bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

26. A non-medical use of the composition as defined in claims 20-23 as an antibacterial agent and / or a bactericidal agent.

27. The non-medical use according to claim 26, characterized in that the antibacterial and / or bactericidal agent is used against Gram(+) bacteria, preferably against bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably against bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably against bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

28. Use of the composition as defined in claims 20-23 as an antiseptic and disinfecting agent.

29. The use of the composition according to claim 28, characterized in that the antiseptic and disinfecting agent is used in the food and biotechnology industry for decontamination and disinfection of surfaces, devices and products, preferably for decontamination and disinfection from Gram(+) bacteria, more preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys- D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

30. Use of the composition as defined in claims 20-23, characterized in that it is used as an antibacterial agent inhibiting and preventing microbiological contamination in the food industry, preferably as an additive to food for humans and / or animals, preferably as an additive to milk, dairy products, meat products, more preferably to milk, yoghurts, cheeses, kefirs, cottage cheeses, milk drinks; cold cuts, sausages, minced meat, frankfurters.

31. The use according to claim 30, characterized in that the agent is used to inhibit, eliminate and prevent the growth of Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

32. The use according to claims 30-31, characterized in that the agent is used to prevent infections and diseases caused by microorganisms present in food, preferably listeriosis caused by Listeria monocytogenes.

33. Use of the composition as defined in claims 20-23, characterized in that it is used as an antibacterial agent for decontamination of surfaces and rooms in food processing, preferably for decontamination of surfaces and / or tools that come into contact with food or semi-finished food products, preferably as anT1antibacterial agent for decontamination of surfaces and rooms on production lines in the processing of meat, milk and dairy products.

34. Use of a composition as defined in claims 20-23, characterized in that it is used as an agent preventing and reducing the formation of bacterial biofilms, preferably preventing and reducing the formation of bacterial biofilms produced by Gram (+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D-Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus f aecium, Micrococcus luteus.

35. Use of a composition as defined in claims 20-23, characterized in that it is used as a decontaminating agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for decontamination of hospital surfaces, medical and veterinary offices, laboratory surfaces, surfaces of tools, surfaces of devices used in medicine, veterinary medicine, diagnostics.

36. Use of the composition as defined in claims 20-23, characterized in that it is used for in vitro use as an antibacterial agent in the cosmetics industry, as an additive to cosmetics improving their microbiological quality, preferably as a biopreservative, preferably as an additive to liquids, creams, milks, lotions, mists, gels.

37. A cosmetic or care composition for cosmetic, care and hygiene applications in humans or animals, characterized in that it comprises the composition as defined in claims 20-23, wherein the composition is intended for external use, and wherein composition, against Gram (+) bacteria is used in the cosmetic or care composition to reduce the occurrence of or eliminate Gram (+) bacteria from the cosmetic or care composition.

38. A method for inhibiting or limiting or preventing the growth of bacteria in vitro, characterized in that it comprises the step of contacting the bacteria with a protein having antibacterial properties such as the composition as defined in claims 20-23.

39. The method according to claim 38, characterized in that the bacteria are selected from Gram(+) bacteria, preferably bacteria having a cross-linking bridge in the peptidoglycan of the type: meso-DAP-D- Ala, L-Lys-D-Asp, L-Lys-Gly2-4-L-Serl-2-Gly, L-Lys-murein peptide, more preferably bacteria of the genus Listeria spp., Bacillus ssp., Staphylococcus spp., Enterococcus spp., Micrococcus spp.; more preferably bacteria of the species Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus simulans, Enterococcus faecium, Micrococcus luteus.

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