Microcapsules comprising bacterial bacteriophages

Abstract

The present invention relates to the field of protection of bacterial infectious disorders by compositions comprising bacterial bacteriophages. In particular to an antibacterial composition comprising: at least one bacterial bacteriophage selected from the group consisting of brachycaudal bacteriophage, muscular-tail bacteriophage and long-tail bacteriophage as an active agent in a liquid nucleus; and a gel shell encapsulating the liquid core comprising the bacterial bacteriophage and comprising colloidal particles of a dispersed ultraviolet screening agent wherein the ultraviolet screening agent is not a soluble antioxidant compound.

Description

Technical Field

[0001] The present invention relates to the field of protection against bacterial infectious diseases by compositions comprising bacteriophages. Background Art

[0002] Pathogenic bacteria can infect a variety of hosts: humans, animals, plants. Antimicrobial agents can treat bacterial infections by eliminating bacteria (bactericidal compounds) or by preventing the reproduction of bacteria (bacteriostatic compounds). Schematically, antimicrobial agents can be antibiotics of plant or fungal origin, antibiotics of bacterial origin or antibiotics of fossil origin (by extraction or chemical synthesis). Antimicrobial agents can also be of animal origin or even of viral origin. In the latter case, the above-mentioned viruses are bacteriophages (phage) or bacteriophages (bacteriophage) that infect bacteria.

[0003] Bacteria are able to mutate and thus adapt to their environment and antimicrobial agents. It is for this reason that resistance to antibiotics has become a major problem, and awareness of this resistance has led to the consideration of alternative antimicrobial treatments, such as bacteriophages. Furthermore, bacteriophages rapidly adapt to bacterial mutations but do not negatively affect commensal flora. Finally, bacteriophages do not infect eukaryotic cells and are therefore harmless for use in humans, animals, fish or plants.

[0004] In the field of human health, bacteriophages (e.g. coli), which indicates that they are safe to use and have particular applications in the emergence of antibiotic resistance or to supplement antibiotic therapy. Without being limited thereto, ear, nose and throat related infections (such as otitis media) caused by Pseudomonas aeruginosa, and infections of the intestinal system caused by Escherichia coli can be mentioned.

[0005] In the animal health and nutrition sector, where the trend is towards reducing the use of antibiotics, there are real advantages in the use of bacteriophages. For example, in fish farming, the control of pathogens such as Flavobacterium psychrophilum in rainbow trout or salmon, or against pathogens such as Yersinia ruckeri that infect salmon. In poultry farming, phage cocktails are used against Salmonella or Clostridium perfringens, such as those developed by Intralytix.

[0006] In the field of plant health, bacterial infections are the cause of significant economic losses. Most existing biological control methods are based on the use of copper, but the toxicity of copper and its potential side effects have prompted people to look for other biological control options, including the use of bacteriophages.

[0007] Finally, in the agri-food industry, bacteriophages are able to ensure food safety by limiting the ability of pathogens to develop during food processing steps or on surfaces in contact with food. Without being limited thereto, Listeria, Shigella, Salmonella, Escherichia coli or Campylobacter jejuni are priority targets in the agri-food industry.

[0008] One of the difficulties when using bacteriophages is that they are fragile and sensitive to their environment. They have walls composed only of protein, which are thinner and more fragile than those of bacteria. More specifically, the extreme pH of some soils or even ultraviolet radiation (e.g. sunlight) are factors to consider.

[0009] Improving the stability of phages over time and maintaining their activity are major challenges for envisioning large-scale use of phages. There are spray drying techniques or freeze drying techniques (Matinkhoo et al., 2011, JPharm Sci; 100(12): 5197-205; Jones et al., 2012, Bacteriophage 2: 208-214; Vandenheuvel et al., 2013, Eur J PharmBiopharm; 84(3): 578-82; Leung et al., 2016, Pharmaceutical research, 33(6), 1486-1496; Leungd et al., 2017, International journal of Pharmaceutics, 521(1-2), 141-149). Encapsulation techniques designed to improve the stability of phages during storage, transportation and application have been tested.

[0010] In the search for a solution aimed at protecting bacteriophages from external stresses such as pH or UV radiation during storage or application, the Applicant experimented with the co-drying of bacteriophages and yeasts as described in application FR2110984.

[0011] Surprisingly, encapsulating bacteriophage used as active agent in a shell comprising dispersed UV-shielding colloidal particles can maintain the activity and antimicrobial effect of the bacteriophage. The encapsulated bacteriophage thus provides effective control of bacterial pathogens, advantageously effective control of bacterial pathogens against humans, animals or plants.

[0012] Therefore, the present invention relates to an antimicrobial composition comprising at least one bacteriophage, selected from short-tailed phage, muscle-tailed phage and long-tailed phage, as an active agent in a liquid core; the liquid core comprises the bacteriophage encapsulated in a gel shell, the gel shell comprising colloidal particles of dispersed UV-screening agent, wherein the UV-screening agent is not a soluble antioxidant compound. Summary of the invention

[0013] The terms "phage" or "bacteriophage" are used indiscriminately. Phages or bacteriophages are viruses that are able to infect bacteria in a highly specific manner without any significant side effects on the human, animal or plant host. Therefore, the antimicrobial composition subject of the present invention can be directed against any potential pathogenic bacteria, whether the host is human, animal or plant. By antimicrobial action, it is understood that bacteria are lysed after infection by bacteriophages.

[0014] In one embodiment, the target pathogen is a bacterium that can infect plants and / or harvested products. Bacterial diseases of plants cause huge losses to agricultural and vegetable crops. Few treatments are available, and many are copper-based. Iriarte et al. (Bacteriophage, 2012; 2(4): 215-224) showed that bacteriophages can penetrate the interior of plants, move from roots to leaves, and remain in the plant for up to 7 days.

[0015] For the aerial parts of the plant, the target pathogens are selected from: Erwinia species, or more specifically Erwinia amylovora; Xanthomonas species, or more specifically Xanthomonas citri (whose host is citrus, where this pathogen can cause the disease commonly known as citrus greening); Xylella species, or more specifically Xylella fastidiosa, which can infect about 600 plant species belonging to more than 80 different plant families: grapevines, citrus fruits, fruit trees, almond trees, olive trees, cherry trees, coffee plants, avocado trees, alfalfa, oleander, oak trees, maple trees, etc. The disease affecting grapevines is called Pierce's disease.

[0016] For soil diseases (needing to be applied through a drip irrigation system or to the seeds), the target pathogens are selected from: Pseudomonas species, or more specifically Pseudomonas syringae pv. tomato (which can cause a disease known as bacterial spot in the host tomato plant); Ralstonia species, or more specifically Ralstonia solanacearum (whose host is also a tomato plant and can cause a disease known as bacterial wilt).

[0017] For plant-harvested products (direct application to the product), pathogens particularly targeted are species of the genera Pectobacterium and Dickieella that infect potato plants.

[0018] Erwinia amylovora is a plant pathogenic bacterium that causes fire blight, an infectious disease that affects apple and pear trees and other plants in the Rosaceae family (Momol MT; Aldwinckle HS, 2000. Genetic diversity and host range of Erwinia amylovora. In: Vanneste JL, ed., Fire blight the disease and its causative agent, Erwinia amylovora. Wallingford, Oxon UK: CABI Press, 55-72; Zwet T van der; Keil HL, 1979. Fire blight, a bacterial disease of Rosaceous plants. Agriculture Handbook, Science and Education Administration USDA. Beltsville, USA: USDA, No. 510: 200 pp). A screen of 33 phages that infect this species of Erwinia was performed, from which, for example, five phages of the family Myoviridae were isolated that were able to infect a variety of bacterial strains of Erwinia amylovora. The bacteriophages were named BPH1, 31Dx, 26C, P2 and 2By, respectively, and were used for the encapsulation test of the antimicrobial composition of the present invention.

[0019] In one embodiment, the target pathogen is a bacterium that can infect humans, including but not limited to bacterial strains of Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens, or Salmonella.

[0020] In one embodiment, the target pathogen is a bacterium that can infect animals, such as Escherichia coli, Salmonella, Campylobacter, Staphylococcus, Flavobacterium psychrophilum, Yersinia ruckeri, Clostridium perfringens. The animals that need to be treated are not only cattle, but also sheep, pigs, poultry, fish, crustaceans, etc.

[0021] In one embodiment, the target pathogen is one that the agri-food industry seeks to protect dairy products, meat, or fish against, such as Listeria, Salmonella, Shigella, E. coli, Campylobacter jejuni, and the like.

[0022] Advantageously, encapsulation enables the antimicrobial composition of the present invention to maintain its antimicrobial properties, as shown in the experimental results given in the examples below. In order to demonstrate the activity of phages and to enumerate phages, it is essential that the phages remain effective against the pathogens they target.

[0023] The capsule used in the antimicrobial composition of the present invention has a shell made from a liquid solution comprising a biopolymer having gel properties and comprising dispersed colloidal particles of an ultraviolet shielding agent, wherein the ultraviolet shielding agent is not a soluble antioxidant compound.

[0024] The microcapsules used in the antimicrobial composition of the present invention are capable of confining bacteriophages but not immobilizing them.

[0025] Microcapsules are well known to those skilled in the art. They can be formed by different techniques and can have different shell compositions. Typically, the microcapsules used in the present invention are produced according to the manufacturing method described in French patent FR 2939012 and international application WO2010063937 A1.

[0026] They consist of a liquid core comprising at least one bacteriophage forming the active agent of the antimicrobial composition of the invention. The liquid core may comprise a mixture of different bacteriophages.

[0027] The liquid core is encapsulated by a gelatinous, substantially solid outer cover, which is referred to as a shell. In particular, the shell is solid. Preferably, the shell of the microcapsule is mainly composed of a biopolymer with gel properties. In particular, the shell is composed of the biopolymer with gel properties. This biopolymer that accounts for the majority of the shell is the main biopolymer. For example, the biopolymer with gel properties is alginate, gellan gum, xanthan gum, pectin, chitosan, agar or carrageenan. Preferably, the shell of the microcapsule forming the antibacterial composition of the present invention is mainly composed of alginate, and in particular, the shell is composed of alginate.

[0028] When in hydrated form, for example, a suspension in an aqueous solution, the microcapsules of the antibacterial composition of the present invention have an average diameter between 50 microns and 4000 microns, preferably between 100 microns and 2000 microns, more particularly between 200 microns and 1000 microns, advantageously between 200 microns and 600 microns. The average diameter can be measured by a variety of techniques known to those skilled in the art, such as particle size analysis based on laser diffraction, sieving classification or optical microscopy imaging. They can also be in dehydrated form. Preferably, the dehydration is partial dehydration, and after dehydration, the hydrated microcapsules preferably have a water content (measured by a moisture analyzer) less than 10%.

[0029] Preferably, the microcapsules of the present invention are freestanding, ie they are not contained within another structure (eg a film, bead, gel or secondary encapsulation), and they are in direct contact with their surrounding medium, typically a liquid (eg a suspension) or a gas.

[0030] The colloidal particles of the UV shielding agent refer to particles with an average particle size between 10 nanometers and 10 micrometers, more preferably between 100 nanometers and 5 micrometers. Generally speaking, the smaller the particle, the more effective its UV protection is. These shielding agents can also make the microcapsules opaque.

[0031] In the context of the present invention, UV screeners are not soluble antioxidant compounds. Such compounds degrade faster when exposed to ultraviolet rays. The UV screeners used in the microcapsules forming the antimicrobial composition of the present invention can be organic screeners or inorganic screeners, or a mixture of organic screeners and inorganic screeners. The UV screeners are preferably selected from titanium oxide, carbon black, biochar, charcoal, latex, silicon dioxide, clay and mixtures thereof. Biochar is the product of biomass pyrolysis and is used as a soil conditioner in agriculture. For example, the clay used as UV screeners is talcum powder, kaolin and mixtures thereof. Kaolin is a clay that is also used to protect some crops from the effects of pests in agriculture.

[0032] Preferably, the UV shielding agent used in the microcapsules forming the antimicrobial composition of the present invention is biodegradable and / or edible and / or pharmaceutically acceptable.

[0033] "Dispersed particles of UV screeners" refer to particles preferably dispersed in the shell of the microcapsule to provide a uniform anti-UV effect on the surface of the microcapsule. Preferably, the core of the capsule of the present invention does not include particles of UV screeners. In particular, the concentration of the UV screener is between 0.05% and 30% (volume / volume), more particularly between 0.1% and 20% (volume / volume), relative to the total volume of the shell.

[0034] Preferably, in the methods and uses of the invention, the microcapsules are used in such a way that they are not included in another structure (such as a film, beads, gel or secondary encapsulation) but are used directly, optionally in the form of a suspension in a liquid.

[0035] The present invention also relates to a method for preventing and / or treating a plant from resisting at least one pathogenic bacterium, comprising the following steps:

[0036] Providing an antimicrobial composition comprising at least one bacteriophage as an active agent against at least one pathogenic bacterium, wherein the bacteriophage is protected from the effects of ultraviolet light in a microcapsule comprising colloidal particles of an ultraviolet light shielding agent dispersed in a shell, wherein the ultraviolet light shielding agent is not a soluble antioxidant;

[0037] The composition is applied to the above-ground parts of the plants.

[0038] In particular, the at least one bacteriophage is selected from the group consisting of short-tailed phages, muscle-tailed phages and long-tailed phages.

[0039] In one embodiment, the harvested product of the plant is antimicrobial prophylactic and / or treated.

[0040] In one embodiment, the antimicrobial composition can also be applied by ground broadcasting. In another embodiment, the antimicrobial composition can be applied around the seeds, particularly but not limited to by coating or film coating. In another embodiment, the product can be applied after harvest.

[0041] In another embodiment, the present invention relates to the preferred non-therapeutic use of the antimicrobial composition of the present invention in animal and / or human nutrition and / or nourishment. In this embodiment, the bacteriophages used are preferably not therapeutic bacteriophages. In other words, the bacteriophages are interesting from a nutritional or nourishing point of view, but they are not able to prevent or treat the disease of the animal or individual consuming these bacteriophages.

[0042] "Nutrient" refers to normal or frequent food intake. The antimicrobial composition may include a plurality of bacteriophages that can improve the properties of food, including bioavailability or shelf life.

[0043] "Nutrients" refer to food supplements taken occasionally or during treatment, usually to prevent or compensate for a deficiency. The antimicrobial composition of the present invention can include a variety of bacteriophages that can improve nutrient intake by improving the bioavailability of nutrients or can alter intestinal flora.

[0044] The present invention also relates to a pharmaceutical composition comprising the antibacterial composition of the present invention.

[0045] The present invention also relates to the antibacterial composition or pharmaceutical composition of the present invention for use as an antibacterial agent, in particular as a medicinal product, and more particularly for the treatment of conditions caused by bacteria, in a subject in need thereof.

[0046] In this embodiment, the antibacterial composition of the present invention advantageously comprises a bacteriophage selected from bacteria targeting pathogenic bacteria of humans or animals, more specifically a bacteriophage targeting bacteria selected from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or Salmonella strains.

[0047] The term "pharmaceutical composition" as defined herein refers to a mixture or solution comprising at least one therapeutic agent that is administered to a subject to prevent or treat a particular condition affecting the subject.

[0048] Therefore, a pharmaceutical composition as defined herein additionally and preferably comprises a pharmaceutically acceptable excipient.

[0049] As used herein, "pharmaceutically acceptable" refers to compositions and molecular entities that do not produce secondary, allergic or other adverse reactions when administered to a subject.

[0050] The term "subject" herein refers to a living being, preferably a mammal, and more particularly a human being.

[0051] "Therapeutically effective amount" herein refers to the dosage and time period necessary to obtain the desired therapeutic outcome. This amount may vary due to a variety of factors, such as the disease, the extent of the disease, the age, sex and weight of the subject, and the ability of the microcapsules to induce the desired therapeutic outcome. The therapeutically effective amount encompasses an amount in which any toxic or deleterious effects are offset by the therapeutically beneficial effects. The therapeutically effective amount also encompasses an amount sufficient to confer a benefit (e.g., a clinical benefit).

[0052] The pharmaceutical composition is preferably adapted to the route of administration.

[0053] In a specific embodiment, the above-mentioned pharmaceutical composition is suitable for oral, sublingual, buccal, intranasal or topical administration.

[0054] As mentioned above, the present invention also relates to the use of the antibacterial composition or pharmaceutical composition of the present invention as a pharmaceutical product. The present invention also relates to the use of the antibacterial composition of the present invention for preparing a pharmaceutical composition.

[0055] For these uses, the antimicrobial composition of the invention advantageously comprises a bacteriophage selected from bacteria targeting pathogenic bacteria of humans or animals, more particularly bacteria targeting bacteria selected from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or Salmonella strains.

[0056] The use of the antibacterial composition of the present invention can facilitate the storage, packaging or preparation of the above-mentioned pharmaceutical composition while preserving active bacteriophages.

[0057] "Treatment" or "treat" herein refers to partially or substantially achieving one or more of the following results: partially or completely alleviating the extent of the disease, improving clinical symptoms or signs associated with the disease, delaying, inhibiting or preventing the progression of the disease. In particular, in the present invention, the disease to be treated is a disease caused by bacteria, especially those diseases mentioned above.

[0058] The following examples are given for illustrative purposes and are not to be construed as limiting the scope of the present invention. DETAILED DESCRIPTION

[0059] Phage counting method

[0060] Methods for demonstrating the activity and enumeration of bacteriophages must use the bacteria targeted by the bacteriophage being tested. Generally, the method for enumerating bacteriophages of unknown concentration is the semi-solid agar overlay method known to those skilled in the art.

[0061] A preculture of the target bacteria is obtained in liquid LB medium (Luria Bertani broth). In some cases, the preculture step in liquid medium can be performed twice to achieve sufficient volume and concentration, which is assessed by OD (optical density) measurement at 600 nm.

[0062] Serial dilutions of the phages to be counted were made in liquid LB medium.

[0063] Serial dilutions of the phage were brought into contact with the bacterial culture on LBA medium (Luria Bertani Agar) pre-supplemented with semi-solid Luria Bertani agar medium containing 0.75% agar.

[0064] After incubation at 30°C for 16 to 24 hours, the cleavage areas were read and counted.

[0065] The Newport Salmonella strain was used to isolate the Salmonella phage from Intralytix. TM ) to count.

[0066] Anti-Listeria phages provided by Intralytix were enumerated using Listeria monocytogenes strains.

[0067] The Erwinia amylovora strain Ea4 provided by the French National Institute for Agri-Food and Environment (INRAe) was used for enumeration of anti-Erwinia phages (phage BPH1, phage 31Dx, phage 26C, phage P2 and phage 2By).

[0068] Example 1: Phage encapsulation test and post-encapsulation stability assessment

[0069] The above bacteriophage BPH1 was encapsulated in capsules without UV shielding agents. Drying tests were performed using three different drying matrices. The activity over time and the stability with temperature were evaluated.

[0070] Among glycerol, SM-sucrose and trehalose as drying excipients, SM-sucrose was the excipient that provided the most acceptable drying yield.

[0071] The stability of the encapsulated phage was tested at +4°C, +25°C and +40°C for one month. At +4°C or +25°C, the titer of the encapsulated phage in liquid form dropped very little. At +40°C, the titer dropped by about 2 logs. In solid form (i.e., after drying with SM-sucrose as an excipient), the titer remained essentially the same regardless of the storage temperature, dropping by less than 1 log at +40°C.

[0072] Example 2: Protection against Erwinia phage in capsules containing UV-screening agents

[0073] The above-mentioned phages BPH1, phage 31Dx, phage 26C, phage P2 and phage 2By were encapsulated in shells containing biochar as an anti-UV shielding agent. Phages alone, capsules containing phages with or without biochar were exposed to UV radiation in liquid form.

[0074] The above-mentioned bacteriophages are deposited in accordance with the Budapest Treaty with a microbial culture collection: National Collection of Microorganisms, Institut Pasteur, 25, rue Dr. Roux, 75015 Paris, France:

[0075] BPH1 was deposited on December 17, 2021, with accession number I-5803;

[0076] 31Dx was deposited on May 6, 2022, with accession number I-5845;

[0077] 26C was deposited on May 6, 2022, with registration number I-5846;

[0078] P2 was deposited on May 6, 2022, with registration number I-5847; and

[0079] 2By deposited on May 6, 2022, registration number I-5848.

[0080] The experimental method for exposure to ultraviolet radiation is as follows:

[0081] Use a sterile cell strainer to obtain a uniform capsule sample of 100 μm in size;

[0082] Weigh 3 g of wet capsules or 1 g of dry capsules respectively and sieve them into a sterile petri dish;

[0083] Add 0.1 M sodium chloride buffer and dilute 1:10 with sterile saline;

[0084] Control samples were taken before exposure;

[0085] Exposure to UV light was performed at an intensity of 105 mW / cm2 using preheated lamps as required.

[0086] Place the Petri dish in the center of the oven.

[0087] The exposure times were 10 min, 20 min, and 30 min for unencapsulated phage, encapsulated phage without UV-screening agent, encapsulated phage with UV-screening agent, and control sodium chloride buffer, respectively.

[0088] From each sample exposed to UV radiation, 200 μl was taken for counting.

[0089] Prior to counting as described above, as much supernatant as possible was removed, the capsules were weighed, opened by the addition of 1 ml of phosphate-buffered saline (PBS) containing ethylenediaminetetraacetic acid (EDTA), stirred on a rotating disk agitator at medium speed for 5 min, and then serially diluted and counted according to the protocol given above.

[0090] Table 1 shows the titration results of BPH1 phage after UV exposure time of 0, 5, 10, 20 and 30 minutes. LDD is the limit of detection.

[0091] Table 1

[0092]

[0093]

[0094] These results indicate that the titer of phages dropped dramatically (more than 4 log values) after 5 minutes of UV exposure at the same rate as that of "free" phages used as controls.

[0095] In contrast, the titer of phages encapsulated with biochar decreased more significantly: from a 1-log drop after 5 min exposure to a 2-log drop after 30 min exposure. These results clearly indicate that the capsules containing biochar effectively protected the phages from inactivation.

[0096] The encapsulated and dried phages were exposed to UV light after rehydration and similar results as shown in Table 2 were obtained after 10 minutes of exposure.

[0097] Table 2

[0098]

[0099] Tables 3 and 4 show the titration results of 31Dx phage after UV exposure time of 0, 5, 10, 20 and 30 minutes, respectively. LDD represents the limit of detection of wet capsules and dry capsules, respectively.

[0100] Table 3

[0101]

[0102]

[0103] Table 4

[0104]

[0105]

[0106] Tables 5 and 6 show the titration results of 26C phage after UV exposure time of 0, 5, 10, 20 and 30 minutes, respectively. LDD represents the detection limit of wet capsules and dry capsules, respectively.

[0107] Table 5

[0108] sample Exposure time Concentration (PFU / mL or PFU / g) Negative control 0 0.00E+00 Free phage 0 1.56E+09 Wet Capsule E1 0 7.94E+07 Wet Capsule E2 0 8.21E+06 Negative control 10 0.00E+00 Free phage 10 2.07E+04 Wet Capsule E1 10 3,27E+07 Wet Capsule E2 10 2.89E+06 Negative control 20 0.00E+00 Free phage 20 Detection limit Wet Capsule E1 20 1.16E+07 Wet Capsule E2 20 2.22E+06 Negative control 30 0.00E+00 Free phage 30 Detection limit Wet Capsule E1 30 1.78E+07 Wet Capsule E2 30 1.72E+06

[0109] Table 6

[0110] sample Exposure time Concentration (PFU / mL or PFU / g) Negative control 0 0.00E+00 Free phage 0 1.50E+09 Dry capsule E1 0 1.18E+08 Dry capsule E2 0 9.47E+08 Negative control 10 0.00E+00 Free phage 10 9.07E+04 Dry capsule E1 10 1.94E+07 Dry capsule E2 10 1.13E+08 Negative control 20 0.00E+00 Free phage 20 Detection limit Dry capsule E1 20 4.45E+08 Dry capsule E2 20 2.50E+08 Negative control 30 0.00E+00 Free phage 30 Detection limit Dry capsule E1 30 4.68E+07 Dry capsule E2 30 4.69E+07

[0111] Tables 7 and 8 show the titration results of P2 phage after UV exposure time of 0, 5, 10, 20 and 30 minutes, respectively. LDD is the detection limit for wet capsules and dry capsules, respectively. NA means not applicable (data not available due to technical reasons).

[0112] Table 7

[0113]

[0114]

[0115] Table 8

[0116]

[0117]

[0118] Tables 9 and 10 show the titration results of 31Dx phage after UV exposure for 0, 5, 10, 20 and 30 minutes, respectively. LDD is the detection limit of wet capsules and dry capsules, respectively.

[0119] Table 9

[0120]

[0121]

[0122] Table 10

[0123] sample Exposure time Concentration (PFU / mL or PFU / g) Negative control 0 0.00E+00 Free phage 0 5.83E+06 Dry capsule E1 0 5.14E+06 Dry capsule E2 0 4.21E+06 Negative control 10 0.00E+00 Free phage 10 Detection limit Dry capsule E1 10 1.75E+06 Dry capsule E2 10 5.65E+06 Negative control 20 0.00E+00 Free phage 20 Detection limit Dry capsule E1 20 3.39E+06 Dry capsule E2 20 1.58E+06 Negative control 30 0.00E+00 Free phage 30 Detection limit Dry capsule E1 30 1.22E+06 Dry capsule E2 30 6.44E+06

[0124] Example 3: Protection of phage cocktail in capsules containing UV shielding agents

[0125] Intralytix sells Salmofresh, a phage cocktail against the bacterium Salmonella. TM , encapsulated in capsules containing UV shielding agents. In parallel, the two individual phages that make up the cocktail, phage SBA 1781 and phage SPT, were encapsulated in the same type of capsules. All of these phages were exposed to UV light and counted according to the experimental protocol given above, and comparisons were made between the phage cocktail or free phage and negative controls.

[0126] The experiments were performed on wet and dry capsules.

[0127] The results are given in Tables 11 and 12 below.

[0128] Table 11

[0129]

[0130] Table 12

[0131]

[0132]

[0133] These results show that the number of phages in the free phage control (i.e., unencapsulated phages) is greatly reduced after the UV exposure time reaches 10 minutes and thereafter, while the phages in the capsules containing the UV shielding agent remain active. Since the activity is demonstrated by the antibacterial effect of the phages on their targeted bacteria, these results show the stability of the antibacterial composition of the present invention.

Claims

1. An antibacterial composition comprising: at least one bacteriophage selected from the group consisting of short-tailed phage, muscle-tailed phage, and long-tailed phage as an active agent in the liquid core; A gel shell encapsulating the liquid core including the bacteriophage and including colloidal particles of dispersed UV screeners, wherein the UV screeners are not soluble antioxidant compounds.

2. The antibacterial composition according to claim 1, wherein The ultraviolet shielding agent is selected from titanium oxide, carbon black, biochar, charcoal, latex, silica, clay, and mixtures thereof.

3. The composition according to any one of claims 1 or 2, wherein The bacteriophage targets at least one plant pathogenic bacterium.

4. The composition according to claim 3, wherein The at least one plant pathogenic bacterium is selected from the group consisting of Erwinia species, Xanthomonas species, Xylella species, Pseudomonas species, and Ralstonia species.

5. The composition according to claim 4, wherein The plant pathogenic bacteria are of the genus Erwinia.

6. The composition according to any one of claims 1 or 2, wherein The bacteriophage targets at least one pathogenic bacterium capable of infecting humans, and the at least one pathogenic bacterium is preferably selected from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or Salmonella.

7. The composition according to any one of claims 1 or 2, wherein The bacteriophage targets at least one animal pathogenic bacterium, and the at least one animal pathogenic bacterium is preferably selected from Escherichia coli, Salmonella, Campylobacter, Staphylococcus, Flavobacterium, Yersinia, Clostridium.

8. The composition according to any one of claims 1 or 2, wherein The bacteriophage targets at least one pathogenic bacterium capable of contaminating agri-food industry products, preferably selected from Listeria, Salmonella, Shigella, Escherichia coli, Campylobacter jejuni.

9. Use of the antimicrobial composition according to any one of claims 1 to 5 in the treatment of plant crops, their seeds or harvested products.

10. Use of the antimicrobial composition according to any one of claims 1 to 5 in soil treatment.

11. A method for preventing and / or treating a plant against at least one pathogenic bacterium, comprising the following steps: Providing an antimicrobial composition comprising at least one bacteriophage as an active agent against said at least one pathogenic bacterium, said bacteriophage being protected from the effects of ultraviolet light in a core of a microcapsule comprising colloidal particles of an ultraviolet light shielding agent dispersed in a shell encapsulating said core, wherein said ultraviolet light shielding agent is not a soluble antioxidant compound; The composition is applied to the above-ground parts of the plants and / or by ground broadcasting, or around the seeds.

12. Use of the antimicrobial composition according to any one of claims 1, 2 or 6 in human nutrition and / or food.

13. Use of the antimicrobial composition according to any one of claims 1, 2 or 7 in animal nutrition and / or feed.

14. A pharmaceutical composition comprising the antibacterial composition according to any one of claims 1 or 2.

15. Use of a composition according to claim 14 for the treatment of a mammal, preferably a human.

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