Phage-containing biocontrol product and use thereof

Abstract

The invention relates to a strategy for using phage products containing one or more host bacteria-specific phages and bacteria, probiotics, antimicrobial agents, bacteriocides, additives or adjuvants for fighting one or more bacteria, with the aim of manipulating the microbiota in an environment or reducing unwanted bacterial load.

Description

[0001] PHAGE-CONTAINING BIOCONTROL PRODUCT AND USE THEREOF

[0002] Related Field

[0003] The present invention relates to a biocontrol product comprising one or more host bacteria- specific phages and adjuvants, such as probiotics or antimicrobial agents, for manipulating the microbiota in an environment or reducing the bacterial load or for fighting multiple bacteria, and to the use of said product.

[0004] State of the Art Related to the Invention (Prior Art)

[0005] Bacteriophages are the most abundant and diverse living organisms on Earth and are generally abundant in environments with bacteria, including soil, oceans, lakes, plants and animals [1, 2].

[0006] Bacteriophage therapy is one of the biocontrol methods that has been used for nearly a century in the fight against bacteria, studied for its effectiveness in fields such as health, agriculture, food, and environment, and still remains relevant today [3, 4], Its specificity to the host bacterium makes it promising for use in many areas threatened by the development of antibiotic-resistant bacteria [1, 5], Although the use of antibiotics has continued extensively to date, the inability to discover new types of antibiotics to prevent antibiotic-resistant bacteria is emerging as a potential threat [4, 6, 7],

[0007] Bacteriophages that lyse the specific bacteria: lyse only the host bacteria, do not harm the natural microflora, are widely present in nature, people are exposed to phages every day without even realizing it, phages are also widely present in the human digestive system and intestinal microflora, and for these reasons, they are natural antibacterials and can be used as biocontrol agents [8], Phages are also used in a variety of biotechnological applications, including phage display, bacterial identification, biofilm degradation and biocontrol of pathogenic bacteria, and in recent years their advantages and potential in various fields such as antibacterial-based cancer therapy, drug delivery and novel vaccine production have enabled their use as biomaterialsfl, 9],

[0008] Numerous studies conclude that phage therapy is not harmful to human health and is an environmentally friendly practice. Apart from health-related application areas, phage application in foods has recently been extensively studied and there are many patents in this field.

[0009] Patent no. US7674467B2, entitled “Salmonella bacteriophage and uses thereof’, includes methods for using bacteriophage specific to Salmonella bacteria or polynucleotides and polypeptides derived therefrom to control contamination of food products by Salmonella. This invention also covers the use of bacteriophage effective against Salmonella bacteria and polynucleotides and polypeptides derived therefrom for the treatment of host infections or environmental contamination by Salmonella

[0010] ,

[0010] Patent no. US6461608B1, entitled “Bacteriophage composition useful in treating food products to prevent bacterial contamination”, is directed to novel phage compositions useful in processing food products to minimize or eliminate bacterial contamination caused by Escherichia coli (E. coli) bacteria, especially toxin-producing E. coli. Methods for processing food products such as meat, fruit juices and spices are also within the scope of the invention [H].

[0011] The phage product, ListShield™, which consists of phages of the bacterium Listeria monocytogenes, is used directly in food production. The company Intralytix has developed a phage preparation for the control of food contamination by Salmonella, E. coli and Listeria bacteria. Similarly, the product Listex®, which contains phages specific to Listeria bacteria, is used to ensure food safety. Omnilytics (Utah, USA) produces phage preparations for use in the food industry and agriculture. The company PhageGuard has developed a phage product to fight Salmonella, E. coli and Listeria bacteria in different types of foods.

[0012] The use of phage products in the design of Theologically and sensory novel products, as well as in manipulating fermentation for the desired end product, has been limited to applications for a single group of bacteria. Patent no. US20090104157A1, entitled “Utilization of bacteriophage to control bacterial contamination in fermentation processes”, involves the addition of a cocktail containing one or more lytic bacteriophages together with a fermentable sugar and an inoculant to the fermentation medium in a fermentation process for the production of ethanol from natural sources. It is based on selecting bacteriophages that infect and lyse bacteria contaminating fermentable sugars. For this purpose, we focused on the selection of lactic acid bacteria from the group consisting of Lactobacillus, Lactococcus, Enterococcus, Weissella, Leuconostoc, Pediococcus, Streptococcus, Oenococcus and combinations of two or more thereof

[0012] ,

[0013] Patent no. W02020205736A1, entitled “Bacteriophage animal feed preservative”, covers the feeding of livestock, such as young poultry or calves, with a feed product containing bacteriophage. The bacteriophage composition is designed to protect the feed product and may exhibit lytic activity specific to one or more pathogenic bacterial species such as Salmonella, Escherichia, Campylobacter and / or Clostridium that may contaminate the feed product

[0013] ,

[0014] Patent no. US20040208853A1, entitled “Method and device for sanitation using bacteriophages” describes methods and devices for sanitation using bacteriophages

[0014] ,

[0015] Summary and Objects of the Invention

[0016] While previous studies have achieved one-dimensional bacterial exchange, the present invention aims to manage more than one bacterium.

[0017] The invention aims to eliminate the predominant microorganisms in the primary microbiota in the most effective way, while at the same time aiming to develop the secondary microflora as desired.

[0018] In addition, the targeted bacterial species are not only contaminants, but all bacteria that affect the critical control points for maintaining quality criteria from raw materials to the end product are targeted to increase, reduce or protect their numbers. For this purpose, the present invention relates to a biocontrol product comprising one or more phages and at least one of a probiotic, bacteriocide, additive or antimicrobial agent for use in controlling and manipulating microbiota.

[0019] Furthermore, the invention relates to a biocontrol product comprising one or more host bacteria-specific phages and at least of bacteria, probiotics, antimicrobial agents, bacteriocides, additives or adjuvants for manipulating the microbiota or reducing the bacterial load in an environment or for fighting multiple bacteria.

[0020] The invention relates to the use of phage products comprising a combination of bacteriophage and components having synergistic effects for reducing target bacterium or bacteria in a microbiota in order to regulate or manipulate the microbiota in various environments, and for maintaining, or even improving, the dynamics in the microbial load due to competition while reducing such bacterium or bacteria.

[0021] The invention also relates to the use of phage-containing biocontrol products for controlling microbiota in environments such as food, environment and agriculture, and for the prevention and treatment of bacterial infections in living organisms such as animals and humans.

[0022] The present invention relates to the use of phage mixtures targeting the entire ambient microbiota, without being limited to targeting only a specific host bacterium or group of host bacteria, and antimicrobial compounds that exhibit a synergistic effect therewith.

[0023] The invention relates to the use thereof in the reduction or elimination of one or more bacteria, and for managing the microflora that will replace the inhibited bacteria, by being incorporated into or used directly as food additives, food processing aids, disinfectants, antimicrobials, biocontrol agents, or bacteriocides in fields such as production, processing, packaging, and storage in sectors including food, beverage, feed, and health.

[0024] Descriptions of the Drawings Illustrating the Invention

[0025] The figures and related descriptions necessary for a better understanding of the invention are as follows. Fig- 1- Antimicrobial effect of the bacteriophages described in the invention on E. coli bacteria.

[0026] Fig- 2. Antimicrobial effect of the bacteriophages described in invention on P. fluorescens bacteria.

[0027] Fig- 3. Antimicrobial effect of the bacteriophages described in the invention on L. plantarum bacteria.

[0028] Fig. 4. Antimicrobial effect of the bacteriophages described in the invention on host bacteria.

[0029] Fig. 5. Antimicrobial effect of the bacteriophages described in the invention in dual cultures.

[0030] Fig. 6. Antimicrobial effect of bacteriophages described in the invention in a consortium of three bacteria.

[0031] Fig. 7. Antimicrobial activity of the bacteriophages described in the invention in a dual culture of E. coli and I., plantarum bacteria.

[0032] Detailed Description of the Invention

[0033] The invention relates to a biocontrol product comprising one or more host bacteria-specific phages and at least one of bacteria, probiotics, antimicrobial agents, bacteriocides, additives or adjuvants for use in directing, i.e. manipulating, the microbiota.

[0034] This biocontrol product can be liquid, solid, aerosol, gel or cream form. The product in liquid form can be a phage cocktail, while the solid form can be a lyophilized phage cocktail.

[0035] The biocontrol product according to the invention can be used directly as or incorporated in biocontrol agents, antimicrobial agents, food additives, disinfectants and / or bacteriocides in foods, food processes, feed production, health, and generally in all sectors where bacterial contamination occurs or where a microbial community is involved. In alternative embodiments of the invention, said biocontrol product may also contain adjuvants to ensure the efficacy, shelf life and stability during the application period of the bioactive components together with the bacteriophage therein. These can be sucrose, lactose, glycerol, lecithin, trehalose, agar, alginate, pullulan, polyethylene glycol, polyacrylamide or dextran. It may contain sucrose or trehalose in the range 0.1-1 M. It may contain 0-90% w / v lactose, 0-50% w / v glycerol, 0-25% w / v lecithin, 0-2% w / v alginate, 0-5% w / v polyethylene glycol, 0-5% w / v polyacrylamide, 0-4% w / v dextran. It may contain 0-3% w / w agar or 0-10% w / w pullulan.

[0036] In another alternative embodiment, the biocontrol product may also comprise components produced using immobilization and other technologies, either alone or in combination, such as encapsulation (emulsification, electrospinning, extrusion, etc.), entrapment in liposomes (methods such as water / oil / water emulsion, thin film hydration, etc.), spray drying (spray freeze-drying, spray drying), freeze-drying (lyophilization), and nanotechnology (nanoparticles, nanolayers, etc.), in addition to the polymers mentioned as excipients.

[0037] Furthermore, in another alternative embodiment of the invention, the biocontrol products can be integrated into forms such as carrier electrospun fibers (polyethylene oxide, cellulose diacetate, etc.), emulsions, liposomes or hydrogels for ease of application.

[0038] Although phage studies reduce a single species of bacteria, the balance in these environments can be destroyed because various environments such as food, feed and plants, as in the intestine, contain a specific microbial community. This means that reducing a target bacterium can result in an increase in the count of competing bacteria. In this invention, the phage-containing biocontrol product according to the invention is different and novel from previous phage products as the microbiota is subjected to a holistic evaluation. This holistic approach aims to direct the primary microbiota to the desired secondary microbiota and this guidance is not random but can be achieved by phage-containing biocontrol products added to the environment.

[0039] The Jameson effect can be expressed as one of the components that provide the synergistic effect. According to this effect, both bacterial competition and phage inhibition can suppress or promote bacteria belonging to a community more effectively than a one-to-one effect. For example, within the scope of the present invention, the ability of the Pseudomonas bacterium used for managing food microbiota to reach a number of bacteria 50 times higher than the number it could individually access within a cocktail consisting of the L. plantarum bacterium and the effective LPP11 FSG phage is due to the Jameson effect, which occurs through both the phage and the probiotic bacterium.

[0040] In addition, the Hurdle effect explains another mechanism by which synergistic effects are achieved in situations where the aim is to suppress the bacterial community in every respect. For example, in experiments conducted within the scope of this invention, the lowest bacterial counts were observed not only in groups containing bacteria and the effective phage, but also in groups containing multiple bacteria and effective phages, creating a Hurdle effect with both phages and bacteria.

[0041] For example, in food products, production is carried out using various raw materials, and the microbial load of the environment cannot be reduced until these raw materials are processed; it can only be sustained for a certain period through controlled environments (such as cold storage, humidity control, etc.). Although controlled environments provide these advantages, the microbiota differs from its initial composition under changing temperature conditions.

[0042] For example, psychrophilic species such as Pseudomonas increase in raw milk during cold storage. The presence of 3-log of this bacterium in milk can cause as much damage as 5-log of mastitis-causing bacteria

[0015] , In this regard, controlled conditions may lead to bacteria, such as this one, that technologically adversely affect the raw material becoming predominant in the microflora.

[0043] Within the scope of the present invention, for all environments mentioned, it will be possible to manage the entire load from the acquisition of the raw material and to provide continuous systems in which problems in terms of technological and health-related concerns are eliminated.

[0044] Foods generate their primary and secondary loads at various stages, starting with the raw material and including storage, processing, and distribution. Raw milk contains a wide range of microorganisms due to microbial contamination from various sources such as milking, processing, and cooling [16, 17], However, regardless of microbial diversity, certain species become predominant over time and constitute the majority of the microbiota associated with such food. Studies on raw milk microbiota have shown that some bacteria become more predominant than other species even during short-term storage. Psychrophilic bacteria such as Pseudomonas and Acinetobacter , as well as lactic acid bacteria such as Streptococcus and Lactobacillus, make up approximately 65% of the milk microbiota

[0018] , Pseudomonas species are considered to be factors causing greater spoilage in milk than other psychrophilic bacteria due to the protease, lipase, and phosphatase enzymes they produce. The activities of these enzymes continue throughout cold storage, even if they have undergone heat treatments such as pasteurization and UHT. Pseudomonas fluorescens (P. fluorescens is particularly predominant in chilled milk, and enzyme production must be limited

[0019] , Lactic acid bacteria commonly found in milk play an essential role in the production of fermentation products

[0020] , Strains used as starters are particularly important for the quality and acceptability of the final product. E. coli causes health effects due to pathogenic types in raw milk, as well as technological problems. Although E. coli can generally be eliminated through pasteurization processes, it poses a problem in the milk and dairy industry due to post-pasteurization contamination

[0021] ,

[0045] The phage applications in the prior art focus only on microbial elimination in sectors, such as food, where sensory evaluations are prioritized, and studies related to chemical changes are quite limited. For example, managing the microflora of fermented products produced from raw materials containing complex and variable microbiota, such as raw milk, in the desired direction with this invention not only eliminates microbial risks but also enables the production of products with the desired sensory qualities.

[0046] This invention aims to minimize concerns regarding health, quality, and safety by consciously managing secondary microbiota that may develop due to the limitations of elimination- oriented strategies in all areas involving microbiota.

[0047] This invention is described by way of examples of the strategy and mechanism involved in the invention, as it spans multiple areas. These examples are described in two main categories for use in directing food microbiota and non-food microbiota. Example 1 ) Manipulating food microbiota with a phage-containing biocontrol product

[0048] This example relates to a biocontrol product comprising one or more phages specific to the host bacterium and at least one of probiotics, bactericides, additives, or antimicrobial agents for use in regulating food microbiota.

[0049] In a preferred embodiment of the invention, the community formed by these bacteria in raw milk is manipulated using phages effective against Lactiplantibacillus plantarum (L. plantarum), P. fluorescens, and E. coli.

[0050] In a preferred embodiment of the invention, the use of phage products containing probiotics is intended to reduce the target bacterium E. coli in a simplified microbiota composed of P. fluorescens, representing psychrophilic bacteria that make up the majority of raw milk microbiota, and L. plantarum, representing lactic acid bacteria, and to maintain, or even improve, the dynamics in the microbial load due to competition while reducing such bacteria. The L. plantarum bacterium is a probiotic bacterium, and the microbiota has been manipulated to increase the count of these bacteria in the microbiota.

[0051] In accordance with the objectives specified in Example 1, the strategy covered by this invention was implemented in accordance with the following process steps.

[0052] Determination of the lytic effects of K12.2b, L23.2, and LPP11_FSG phages on host bacteria and preparation of phage cocktails

[0053] K12.2b, L23.2, and LPP11 FSG phages, which exhibit lytic activity against E. coli, P. fluorescens and L. plantarum bacteria, respectively, were selected for the preparation of phage cocktails.

[0054] First, the efficacy of each phage against its host bacteria was determined under the same parameters and used for comparison with other groups.

[0055] Then, bacterial mixtures were prepared in pairs and individual phages and a phage cocktail consisting of two phages inoculated into these bacterial mixtures to determine the inhibition activity of the phages. After inoculation, analysis was performed using the same parameters. Finally, a mixture consisting of the three bacteria (E coli, P. fluorescens, and L. plantarum) was prepared; groups with individual phages, 3 cocktails each containing combinations of two phages from the three different phages, and a cocktail containing all three phages were added to samples containing the bacterial mixture. After analyses were performed using the same parameters, incubations were carried out as described below, and bacterial counts and phage titers were determined by counting.

[0056] The experiment procedure was carried out in three different groups consisting of three bacteria and three phages, respectively, under specified conditions. Bacterial mixtures were prepared for all pairwise combinations of the three bacterial groups (C(3,2)= 3), and phages specific to the bacteria were added to these bacterial mixtures individually and as a cocktail containing two phages. Finally, bacteriophages were applied to the media containing the three bacteria individually, as cocktails containing two phages, and as a cocktail containing all three phages.

[0057] For example, one of the dual combinations consists of P. fluorescens and E. coli bacteria, and the groups formed are three in number: the experimental groups to which L23.2 phage was added; to which K12.2b phage was added; and to which a cocktail of L23.2 and K12.2b phages was added.

[0058] First, the lytic activity was determined using the spread plate method at 25°C for 24 hours using King B agar for Pseudomonas, EMB agar for E. coli, and MRS agar for / .. plantarum. P. fluorescens bacteria count was determined by counting the colonies emitting fluorescence under a UV lamp at 270 nm after 48 hours at 30°C; E. coli bacteria count was determined by counting metallic, bull's eye-shaped colonies after 24 hours at 37°C, and L. plantarum count was determined by counting cream-colored colonies after 24 hours at 37°C.

[0059] In determining phage titers, K12.2b and L23.2 phages were determined using CASO agar and soft agar, while LPP11 FSG was determined using MRS agar and soft agar at the incubation temperatures specified above by plate counting after 24 hours.

[0060] Here, phages and bacteria were taken into CASO broth at different multiplicity of infection (MOI) values and incubated at 25°C for 24 hours. In the group to which L23.2 was added, the bacterial count was approximately 100 CFU / ml at 24 hours, while in the control group containing only bacteria, this count was determined to be approximately 100 million CFU / ml (Fig. 2).

[0061] In the group to which K12.2b phage was added, the bacterial count was approximately 10 thousand CFU / ml at 24 hours, while in the control group containing only bacteria, this count was more than 1 billion CFU / ml (Fig. 1).

[0062] In the group to which LPP11 FSG phage was added, the bacterial count was 1 million CFU / ml at 24 hours, while in the control group containing only bacteria, this count was determined to be 10 times higher than in the experimental group (Fig. 3).

[0063] Furthermore, all these results have been confirmed by ODeoo values (Fig. 4).

[0064] Following the experimental results, products containing phages specific to this simplified microbiota were created and their effect on the microbiota was evaluated under in vitro conditions.

[0065] Phage-containing products were created with different combinations of three phages at the same titer (POB / ml) and added to sterile CASO broth at different MOI values to determine their in vitro efficacy on the bacteria, and then incubated at 25°C for 24 hours. Bacteria and phage counts were performed on samples taken after application in the manner described above.

[0066] Following in vitro analyses, the role and effect of the use of the phage-containing product according to this invention in accordance with the specified strategy in producing the following results has been scientifically determined (Figs. 1-3, Figs. 5-6).

[0067] This invention enabled the simultaneous reduction of competing E. coli and P. fluorescens (Hurdle effect) and even reduced the P. fluorescens count to undetectable levels, based on the strategy described above. The total count of these two bacteria is less than 10 thousand CFU / ml even after 24 hours (Figs. 1-2). Even after 16 hours in the in vitro environment where raw milk is modeled, the total bacteria count is below the legal limit of 100 thousand CFU / ml (Figs. 1-3).

[0068] The Pseudomonas bacteria counts were kept below 100 CFU / ml at room temperature (25°C) for 24 hours (Fig. 2).

[0069] - E. coli counts could not be detected in in vitro tests because it was less than 10 CFU / ml (Fig. 1).

[0070] L. plantarum counts remained below the acceptable limit for milk for 16 hours (Fig. 3).

[0071] As a result, the strategy described in this invention scientifically proves its effectiveness by eliminating unwanted bacteria (a total of 150 CFU / ml) for 16 hours in an environment where milk is modeled, keeping the desired bacteria under control, and ensuring that the total count remains below 100 thousand CFU / ml.

[0072] Until now, phage-containing products have only been able to induce changes in the target bacteria, but they cannot produce the same effect in a microbiota where more complex interactions occur. This invention has made it possible to change a microbiota as desired.

[0073] Until now, even if the target bacteria in a product has been reduced, the number of other bacteria competing therewith has increased significantly. With this invention, a product design approach has been adopted by taking into account the competing bacteria.

[0074] Again, with this invention, the concept of simplified microbiota for food has had a significant impact on the acceptability of raw milk in terms of changes in total bacterial load.

[0075] Furthermore, with this invention, the Pseudomonas bacteria count, which has a negative impact on sustainability due to major technological problems and product losses, have been kept under control for 24 hours under controlled conditions. Additionally, this invention has enabled the bacterial count of E. coli bacteria, which may include pathogenic strains, to be reduced to undetectable levels within 24 hours under the conditions described above.

[0076] Again, with this invention, the counts of a lactic acid bacterium that plays an important role in the production of fermented products have been kept under control for up to 16 hours even at room temperature.

[0077] This invention is particularly important in Turkiye, where the vast majority of milk production is carried out by small businesses. The great potential of the products to be manufactured with this invention in terms of usage and its contribution as a measure against the food crisis predicted to occur by 2050 are significant in terms of preventing the waste of raw materials and products. Continuity in food supply is dependent on sustainable food production; the present invention, which aims to achieve this continuity, targets the biocontrol of critical control points in the process from producer to consumer.

[0078] In alternative embodiments of the invention, the use of the biocontrol product within the scope of the present invention in directing the food microbiota can reduce, control, or completely eliminate pathogenic bacteria and bacteria that cause spoilage in meat and meat products, milk and dairy products, fruits and vegetables, and other foods, as well as in ready- to-eat products. Examples of such pathogenic bacteria include Escherichia species, Campylobacter species, Salmonella species, Clostridium species, Staphylococcus species, Bacillus species, Shigella species, Vibrio species, and Listeria species. Examples of bacteria that cause spoilage include Escherichia species, Pseudomonas species, Enterobacter species, Micrococcus species, Acinetobacter species, Serratia species, Flavobacterium species, Achromobacter species, and Corynebacterium species.

[0079] Example 2) Manipulating the microbiota of non-food environments

[0080] This example illustrates the use of biocontrol products according to the invention in the microbiota of non-food environments. Accordingly, in another preferred embodiment of the invention, the use of a phage specific to Erwinia amylovora (E. amylovora bacteria controls the bacteria that cause fire blight and Salmonella bacteria that are predominant in poultry houses.

[0081] The use of local phages in the phage-containing biocontrol product described by the present invention contributes to the production of region-specific solutions.

[0082] The use of local phage or bacteria is important in reducing unwanted predominant bacteria in the microbiota described by the present invention. i. Use of phage-containing products in plants

[0083] The predominance of disease-causing microorganisms in the environment can limit the growth of other microorganisms. Within the scope of this analysis, a cocktail consisting of EAP2 and EAP4 phages was applied at different MOI values to plant parts such as leaves, branches, and apples for the treatment of fire blight caused by E. amylovora in the preferred embodiment of the invention.

[0084] The following results were obtained in the experiments conducted:

[0085] The use of the relevant phages has resulted in a significant reduction in the count of disease-causing bacteria.

[0086] The use of local phage products for biocontrol purposes is one of the important parameters in manipulating the microbiota in the application area. However, the importance of the strategy described by this invention is to prevent other bacteria commonly present in the environment from becoming predominant after the elimination of this bacterium in the environment, thereby causing economic damage to the treated products.

[0087] The concept of simplified microbiota specific to the area, as described in Example 7, and the production of phage products targeting microbiota, also serve as examples of the significant potential for increasing bacterial reduction in such studies. This preferred embodiment of the invention has led to the development of a phage product for treating fire blight, for which there is no effective phage product available in Turkiye. The effects of using local phages, which are an important element in microbiota management, offer a solution to the disadvantages of the prior art by producing phage products as described by this invention. ii. Use of the phage-containing product in an ambient microbiota

[0088] Another study, conducted in a poultry house, addressed why the strategy described by the invention should be used in the application of phages in abiotic environments.

[0089] A cocktail consisting of SK-E1, SK-Til, and SK-T2 phages was applied at different MOI values to sawdust used in poultry houses. Samples taken after application were mixed with peptone water to ensure that the bacteria passed into the liquid. Liquid samples were spread on XLD agar and incubated at 37°C for 18-24 hours. Typical black colonies were counted after incubation. The following results were obtained in the experiments conducted:

[0090] Although the count of Salmonella bacteria was kept under control, typical E. coli bacterial growth was observed in these samples. This situation serves as an example of why microbiota should be taken into consideration in biocontrol applications involving phages, as described in this invention. iii. Use of phage-containing products related to human health

[0091] In recent years, bacteriophages have been used in biotic environments to prevent various health disadvantages caused by antibiotic use, such as the development of resistant colonies and the elimination of beneficial bacteria in the microbiota. However, the effect of bacteriophages on a narrow range of bacteria results in the growth of unwanted bacteria in the microbiota to replace the eliminated bacteria. This invention describes a strategy based on directing the microbiota to overcome problems encountered in health applications of bacteriophages to date.

[0092] Accordingly, in another preferred embodiment of the invention, the elimination of bacteria such as E. coli O157:H7, E. faecalis. and S. aureus, which can cause pathogenic effects in human and animal health and become predominant in the microbiota in such cases, has been investigated in in vitro conditions using local phages (DI for E. coli O157:H7; EFl for E. faecalis SAI for S. aureus) and / or bacteria.

[0093] First, host bacteria and local phage isolates were added to CASO broth media at MOI values of 1, 10, and 100, and samples were taken at 37°C for 4-6 hours to determine the ODeoo values.

[0094] In the control group without phage addition, ODeoo values exceeded 2 after 4 hours, whereas ODeoo values for Enterococcus faecalis (E. faecalis) and Staphylococcus aureus (S. aureus) remained below 0.5. In fact, at MOI values of 10 and 100, the ODeoo value yielded an absorbance close to 0.

[0095] With regard to the elimination of E. coli O157:H7 bacteria using bacteriophages, due to resistance mechanisms, the maximum reduction in bacterial counts was 1-log CFU / ml, and within 24 hours, this value reached 100 million CFU / ml bacteria.

[0096] Although the use of local isolates produces effective results for E. faecalis and S. aureus, the same effectiveness cannot be claimed for A. coli O157:H7.

[0097] The strategy of directing the microbiota as described in this invention has been used to model a situation in which a pathogenic bacterium is predominant in the human intestinal microbiota in vitro, to eliminate this bacterium and ensure that a probiotic bacterium becomes predominant in the microbiota, thereby preventing the growth of unwanted bacteria.

[0098] In this application, E. coli bacteria were selected as the target microorganism for the simulation of pathogenic bacteria, and E. coli counts were reduced using phage application. In addition, the addition of L. plantarum bacteria to the environment has ensured that this bacterium becomes predominant in the microbiota (Fig. 7).

[0099] By maintaining the target bacterial value below 100 CFU / ml at 25°C for 24 hours, the probiotic bacteria completely dominated the microbiota (>6 log CFU / ml). However, under the same conditions, although E. coli bacteria counts can be reduced for a certain period of time, the bacteria count begins to increase rapidly again within 24 hours after resistance develops. In the latter case, the target bacterial count reaches values 250 times higher than those obtained with this invention.

[0100] In addition, in alternative embodiments of the invention, said biocontrol product may be used to direct microbiota for human health purposes to treat diseases such as bacterial diseases caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, or Pseudomonas aeruginosa, Klebsiella pneumoniae, Clostridium difficile, chronic wounds and burns, diabetic foot ulcers, respiratory tract infections, gastrointestinal infections, urinary system infections, orthopedic infections, prosthesis and implant infections, eye infections, bacterial keratitis, and sepsis.

[0101] Although the use of phage preparations in the prior art can reduce target bacteria in many areas, in most sectors characterized by complex microbiota, this can result in random bacteria becoming predominant in place of the reduced bacteria.

[0102] The most important stage of standardization in the use of biocontrol products and the most important deficiency of phage application, namely the isolation of local phages and control of the microbiota that will form after application, can be achieved with the present invention.

[0103] This is because this invention describes the use of phage-containing biocontrol products that allow for the addition of different types of microbial and antimicrobial components to eliminate the microorganisms that are predominant in the primary microbiota in the most effective way and to promote the desired development of the secondary microflora.

[0104] The use of a biocontrol product containing a phage or phage components specific to the target bacterium or bacteria, and at least one of bacteria, probiotics, bactericides, antimicrobial agents, or adjuvants capable of providing the desired effect to control and direct the microbiota by changing the count of at least one of the bacteria that make up the bacterial load of the microbiota reduces the count or completely eliminates one or more target bacterial species or strains by providing one or more different bacteriophages, controls the count of the target bacterial species or strain by using components with a synergistic effect to increase the efficiency of said combination of one or more different bacteriophages, manipulates the microbiota after the elimination of harmful bacterium or bacteria as it contains bacteria suitable for the purpose together with bacteriophage and components with synergistic effect.

[0105] References

[0106] 1. Wu, Q., et al., Phages in Fermented Foods: Interactions and Applications.

[0107] Fermentation, 2023. 9(3): p. 201.

[0108] Strathdee, S.A., et al., Phage therapy: From biological mechanisms to future directions. Cell, 2023. 186(1): p. 17-31.

[0109] Kutateladze, M. and R. Adamia, Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends in Biotechnology, 2010. 28(12): p. 591-595.

[0110] Kortright, K.E., et al., Phage Therapy: A renewed approach to combat antibioticresistant bacteria. Cell Host & Microbe, 2019. 25(2): p. 219-232.

[0111] Aranaga, C., et al., Phage Therapy in the Era of Multidrug Resistance in Bacteria: A Systematic Review. International Journal of Molecular Sciences, 2022. 23(9). tarihi: 1 Mayis 2022). Available from: https: / 7wvtrw.pewtrusts.org / en / research-and- an ly set en ti f~i c-roadmap-for-antibioti c-di scovery ,

[0112] Merabishvili, M., et al., Production of Phage Therapeutics and Formulations:

[0113] Innovative Approaches, in Phage Therapy: A Practical Approach, A. Gorski, R. Mi^dzybrodzki, and J. Borysowski, Editors. 2019., Springer International Publishing: Cham.

[0114] Garvey, M., Bacteriophages and Food Production: Biocontrol and Bio-Preservation Options for Food Safety. Antibiotics, 2022. 11(10): p. 1324.

[0115] Hudson, J. A., et al., Bacteriophages as Biocontrol Agents in Food. Journal of Food Protection, 2005. 68(2): p. 426-437.

[0116] Sulakvelidze, A., S. Sozhamamnnan, and G.R. Pasternack, Salmonella bacteriophage and uses thereof. 2010, Google Patents.

[0117] Averback, P. and J. Gemmell, Bacteriophage composition useful in treating food products to prevent bacterial contamination. 2002, Google Patents.

[0118] Baruch, E. and S.D. Okull, Utilization of bacteriophage to control bacterial contamination in fermentation processes. 2009, Google Patents.

[0119] Kamezos, T. and K. Russo, Bacteriophage animal feed preservative . 2020, Google Patents.

[0120] Sulakvelidze, A., et al., Method and device for sanitation using bacteriophages. 2004, Google Patents.

[0121] Walstra, P., Dairy Technology: Principles of Milk Properties and Processes. 1999: CRC Press.

[0122] Robinson, R. and P. Itsaranuwat, Dairy Microbiology Handbook: The Microbiology of Milk and Milk Products. 2005, John Wiley & Sons: New York, NY. p. 175-211.

[0123] Robinson, R.K., Dairy microbiology handbook: the microbiology of milk and milk products. 2005: John Wiley & Sons.

[0124] Yuan, H., et al., Microbial Properties of Raw Milk throughout the Year and Their Relationships to Quality Parameters. Foods, 2022. 11(19): p. 3077.

[0125] Samarzija, D., S. Zamberlin, and T. Pogacic, Psychrotrophic bacteria and milk and dairy products quality. Mljekarstvo / Dairy, 2012. 62: p. 77-95.

[0126] Quigley, L., et al., The complex microbiota of raw milk. FEMS Microbiol Rev, 2013. 37(5): p. 664-98.

[0127] Frank, J.F. and E.H. Marth, Survey of Soft and Semisoft Cheese for Presence of Fecal Coliforms and Serotypes of Enteropathogenic Escherichia coli. Journal of Food Protection, 1978. 41(3): p. 198-200.

Claims

CLAIMS1. A biocontrol product containing one or more host bacteria-specific phage and at least one of bacteria, probiotics, antimicrobial agents, bactericides, additives, or adjuvants, for use in controlling and manipulating the microbiota by changing the count of at least one of the bacteria that make up the bacterial load of the microbiota.

2. The biocontrol product according to claim 1, characterized in that it is in liquid, solid, aerosol, gel, or cream form.

3. The biocontrol product according to claim 1, characterized in that it comprises sucrose, lactose, glycerol, lecithin, trehalose, agar, alginate, pullulan, polyethylene glycol, polyacrylamide or dextran.

4. The biocontrol product according to any one of claims 1-3, for use in regulating food microbiota by changing the count of at least one of the bacteria that make up the bacterial load.

5. The biocontrol product according to any one of claims 1-3, for use in regulating raw milk microbiota by changing the count of at least one of the bacteria that make up the bacterial load.

6. The biocontrol product according to any one of claims 1-3, comprising at least one phage specific to Lactobacillus plantarum, Pseudomonas fluor escens or E. coli K12 bacteria, for use in regulating raw milk microbiota by changing the count of at least one of the bacteria that make up the bacterial load.

7. The biocontrol product according to any one of claims 1-3, comprising at least one of the K12.2b, L23.2 or LPP11 FSG phages, for use in regulating raw milk microbiota by changing the count of at least one of the bacteria that make up the bacterial load.

8. The biocontrol product according to any one of claims 1-3, for use in inhibiting Erwinia amylovora bacteria, which causes fire blight.

9. The biocontrol product according to any one of claims 1-3, comprising at least one phage specific to Erwinia amylovora bacteria, for use in inhibiting Erwinia amylovora bacteria, which causes fire blight.

10. The biocontrol product according to any one of claims 1-3, comprising EAP2 and EAP4 phages for use in inhibiting Erwinia amylovora bacteria, which causes fire blight.

11. The biocontrol product according to any one of claims 1-3, for use in the treatment of bacterial infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Clostridium difficile.

12. The biocontrol product according to any one of claims 1-3, for use in the treatment of chronic wounds and burns.

13. The biocontrol product according to any one of claims 1-3, for use in the treatment of respiratory tract infections, gastrointestinal infections, or urinary system infections.

14. The biocontrol product according to any one of claims 1-3, for use in the treatment of diabetic foot ulcers.

15. The biocontrol product according to any one of claims 1-3, for use in the treatment of orthopedic infections, prosthetic and implant infections.

16. The biocontrol product according to any one of claims 1-3, for use in the treatment of eye infections or bacterial keratitis.

17. The biocontrol product according to any one of claims 1-3, for use in the treatment of sepsis.

Images