KR20240066487A - Novel bacteriophage LSE1 and uses thereof - Google Patents

Abstract

The present invention provides a bacteriophage having a specific killing ability against Salmonella bacteria, an antibiotic comprising the bacteriophage as an active ingredient; disinfectant; cleaning solution; food additive composition; food composition; feed additive composition; feed composition; It relates to a pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella, and a bacterial sterilization method including the step of treating a subject with bacteriophage.

Description

Novel bacteriophage LSE1 and uses thereof {Novel bacteriophage LSE1 and uses thereof}

The present invention relates to a novel bacteriophage LSE1, and specifically to the bacteriophage LSE1 and its use.

Bacteriophage is a bacteria-specific virus that infects only specific bacteria and controls the growth of bacteria. It cannot self-proliferate without a bacterial host and is often called a phage. Bacteriophage is a simple structure in which single or double strands of DNA or RNA constitute nucleic acid as genetic material, and this nucleic acid is wrapped in a protein envelope. It has an icosahedral head with a tail, an icosahedral head with no tail, etc. It is divided into three basic filament-type structures. The most common type of icosahedral head-tailed bacteriophage is classified into Myoviridae with a contractile tail, Siphoviridae with a long, non-contractile tail, and Podoviridae with a short tail. (Podoviridae), bacteriophages with an icosahedral head and no tail are subdivided according to the shape of the head, the components of the head, and the presence or absence of an envelope.

When infecting a bacterium, the bacteriophage attaches to the surface of the bacterium, injects its genetic material into the cell, and then becomes lytic or lysogenic. In the lytic case, the bacteriophage uses cellular machinery to create its own structures and then destroys or lyses the cell by releasing new bacteriophage particles. In the case of lysogeny, the nucleic acid is incorporated into the chromosome of the bacterial host cell and replicates with the cell without destroying the bacterium, but under certain conditions, it converts to lytic.

After the discovery of bacteriophages, research was conducted to use them as a treatment for infectious diseases, but compared to the characteristics of antibiotics with a broad target spectrum, bacteriophages with a specific target spectrum were outcompeted and did not receive attention. Antibiotics or antibacterial agents are widely used in the treatment of infectious diseases caused by bacterial infections, but the problem of antibiotic-resistant bacteria is becoming more serious due to misuse of antibiotics, and as concerns about the impact of antibiotic residues in food on the human body are increasing, host specificity is lower than that of antibiotics. There is increasing interest in highly bacteriophages.

Meanwhile, Salmonella is a Gram-negative facultative anaerobic bacterium belonging to the Enterobacteriaceae family and a non-spore-forming bacillus that is a pathogenic microorganism widely distributed not only in the intestines of humans, animals, and birds, but also in the natural world. Because Salmonella is a zoonotic infectious agent, it can infect not only humans but also various livestock, including pigs, cows, and chickens, and cause disease. When infected in humans, it causes food poisoning, causing chronic diseases such as diarrhea, vomiting, abdominal pain, fever, and even arthritis, and in livestock such as pigs, cows, and chickens, it causes salmonellosis, causing acute and chronic infections. It is infected by factors such as feed, water, etc.

Salmonella (Salmonella spp.) is broadly classified into two species, Salmonella enterica (S. enterica) or Salmonella bongori (S. bongori), most of which are Salmonella enterica, which is characterized by biochemical characteristics. Accordingly, it is classified into 6 subspecies. After Salmonella species are identified through biochemical tests, the serovar is confirmed according to the Kauffmann-White antigen table, and the strain name is finally written. To date, about 2,659 serotypes have been reported.

Looking at Salmonella infection patterns according to serotype, in the past, outbreaks were concentrated in major serotypes such as Salmonella Enteritidis (S. Enteritidis), Salmonella S. Typhimurium (S. Typhimurium), and Salmonella Typhi (S. Typhi), but recently The frequency of infections with new serotypes of Salmonella is increasing, showing a diversification trend.

Currently, antibiotics are mainly used to prevent and treat Salmonella infections. However, when Salmonella infects animals, it often penetrates into cells, proliferates, and infects, making it difficult for antibiotics, drugs, or other probiotics to penetrate and act. Recently, it has been used in many fields. As the problem of antibiotic resistance is occurring, the cause of this has been identified as misuse of antibiotics, which has led to insufficient use of antibiotics. Accordingly, antibiotics used as growth promoters in mixed feed for industrial animals began to be banned, and the European Union began regulating antibiotic use in 2006 and Korea began regulating antibiotic use in the second half of 2011. In addition, as consumer demands for safe food and food safety are increasing, the use of antibiotics is gradually decreasing. As the use of antibiotics to control bacteria decreases, the occurrence of bacterial diseases is increasing, and the demand for methods to control bacterial diseases without using antibiotics is increasing.

Under this background, the present inventors developed a new bacteriophage specific for Salmonella bacteria and confirmed that an antibacterial composition containing it can be used in pharmaceutical compositions, antibiotics, disinfectants, etc. for preventing or treating infectious diseases caused by Salmonella bacteria. The invention was completed.

Republic of Korea Patent No. 10-2341100

The purpose of the present invention is to provide a bacteriophage having a specific killing ability against Salmonella bacteria.

Another object of the present invention is to provide an antibiotic containing bacteriophage as an active ingredient.

Another object of the present invention is to provide a disinfectant containing bacteriophage as an active ingredient.

Another object of the present invention is to provide a detergent containing bacteriophage as an active ingredient.

Another object of the present invention is a food additive composition containing bacteriophage as an active ingredient; To provide a food composition.

Another object of the present invention is a feed additive composition containing bacteriophage as an active ingredient; To provide a feed composition.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella bacteria, including bacteriophage as an active ingredient.

Another object of the present invention is to provide a method for sterilizing bacteria, including the step of treating a target with bacteriophage.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

As confirmed in the examples and experimental examples below, the present inventors isolated the new bacteriophage LSE1 (accession number KCTC 15077BP) of the present invention from the natural world, and confirmed the morphological, biochemical and genetic characteristics of the new bacteriophage LSE1. , the present invention was completed by confirming that it is specific for Salmonella and has the ability to inhibit the growth or kill Salmonella.

As an aspect for achieving the above object, the present invention provides a bacteriophage having a specific killing ability against Salmonella bacteria.

As used herein, the term "bacteriophage" is a bacteria-specific virus that infects a specific bacterium and inhibits and inhibits the growth of that bacterium, a virus containing single or double stranded DNA or RNA as genetic material. means.

The term "Salmonella" used in this specification refers to Gram-negative facultative anaerobic bacteria belonging to the Enterobacteriaceae family, including Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Typhi. Typhi ) or Salmonella Paratyphi ( S. Paratyphi), but is not limited thereto.

The bacteriophage may include the base sequence represented by SEQ ID NO: 1 as all or part of the entire gene. In addition, the bacteriophage of the present invention may consist of the base sequence represented by SEQ ID NO: 1 and a functional equivalent of the base sequence. The functional equivalent refers to a sequence that is at least 70% or more, specifically 80% or more, more specifically 90% or more, and even more specifically 95% or more of the base sequence represented by SEQ ID NO. 1 as a result of modification or substitution of the base sequence. Having homology, it means a sequence that exhibits substantially the same physiological activity as the base sequence represented by SEQ ID NO: 1.

As another aspect for achieving the above object, the present invention provides an antibiotic containing bacteriophage as an active ingredient.

As used herein, the term "antibiotic" refers to an agent that can kill bacteria or inhibit the growth of bacteria, and may be a general term for preservatives, disinfectants, and antibacterial agents.

As used herein, the term "active ingredient" refers to an ingredient that exhibits the desired activity alone or can exhibit activity together with a carrier that is not active on its own.

Compared to conventional antibiotics, antibiotics containing the bacteriophage as an active ingredient can kill only specific pathogens without killing beneficial bacteria, and do not induce drug resistance, which can have the effect of extending product lifespan compared to conventional antibiotics.

The antibiotic can be manufactured by a manufacturing method commonly used in the art.

The antibiotic may contain an effective amount of an additive sufficient to reduce quality degradation of the antibiotic, and the additive may be a preservative, stabilizer, excipient, or cryoprotectant. By including the additive, the antibiotic may allow the bacteriophage to survive or maintain activity for a longer period of time within the antibiotic, unlike bacteriophages that exist in nature. The preservatives, stabilizers, excipients, or cryoprotectants may be those commonly used in the art without limitation as long as they can reduce the quality degradation of antibiotics.

As another aspect for achieving the above object, the present invention provides a disinfectant containing bacteriophage as an active ingredient. Another aspect provides a detergent containing the bacteriophage as an active ingredient. The terms bacteriophage, active ingredient, etc. are as described above.

The formulation of the disinfectant or cleaner is not particularly limited, and formulations commonly known in the art can be prepared and used. The disinfectant or cleaner can be manufactured by a manufacturing method commonly used in the art.

The disinfectant may be sprayed to remove salmonella bacteria and may be sprayed on animal activity areas, slaughterhouses, death areas, cooking areas or cooking facilities, but is not limited thereto.

The detergent may be used to clean the skin surface or body parts of animals that have been exposed or may be exposed to salmonella, but is not limited thereto.

The disinfectant or cleaner may contain an effective amount of an additive sufficient to reduce quality degradation, and the additive may be a preservative, stabilizer, excipient, or cryoprotectant. By containing the additive, the disinfectant or detergent may allow the bacteriophage to survive or remain active for a longer period of time in the disinfectant or detergent, unlike bacteriophages that exist in nature. The preservatives, stabilizers, excipients or cryoprotectants can be those commonly used in the art without limitation, as long as they can reduce the quality deterioration of the disinfectant or cleaning agent.

As another aspect for achieving the above object, the present invention provides a food additive composition or food composition containing bacteriophage as an active ingredient.

As described above, the bacteriophage included in the food composition of the present invention specifically kills Salmonella bacteria and thus shows an improving effect on infectious diseases caused by Salmonella bacteria. As long as it has this effect, it can be included in various amounts.

The term "improvement" used in this specification refers to the reduction of diseases caused by Salmonella bacteria.

As used herein, the term "infectious disease caused by salmonella" may include salmonella infection, typhoid fever, or food poisoning, but is not limited thereto.

In addition to the bacteriophage, the food composition of the present invention may further include any compound or natural extract whose safety has already been verified in the art and known to have the corresponding activity in order to improve the convenience of taking or ingesting.

These compounds or extracts include compounds, extracts, and medicinal products listed in compendiums such as the Pharmacopoeia of each country ('Korean Pharmacopoeia' in Korea) and the Code of Health Functional Foods of each country ('Standards and Specifications for Health Functional Foods' notified by the Ministry of Food and Drug Safety in Korea). The laws of each country governing the manufacture and sale of compounds, extracts, and health functional foods that have received product approval in accordance with the laws of each country that govern the manufacture and sale ('Pharmaceutical Affairs Act' in Korea) (the 'Act on Health Functional Foods' in Korea) Accordingly, compounds or extracts with recognized functionality may be included.

The term "food" of the present invention refers to dairy products including meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream, various soups, beverages, tea, drinks, and alcohol. It includes beverages, vitamin complexes, nutritional supplements, functional foods, food additives, health functional foods, and health foods, and includes all foods in the conventional sense.

The term "health functional food" of the present invention is the same as food for special health use (FoSHU), which refers to medicine processed to efficiently exhibit bioregulatory functions in addition to nutritional supply, with high medical effectiveness. It means food. Here, "function" means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Additionally, the food formulation can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in a variety of dosage forms, and unlike general drugs, it has the advantage of being made from food as a raw material and has no side effects that may occur when taking the drug for a long period of time, is excellent in portability, and Food can be consumed as a supplement to improve infectious diseases caused by salmonella bacteria.

Specifically, the health functional foods are foods made by adding bacteriophages to food materials such as beverages, teas, spices, gums, and confectionery, or manufactured into pills, tablets, capsules, powders, suspensions, etc., and consuming them may cause certain health problems. It means bringing about an effect, but unlike regular drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food.

The food composition of the present invention is very useful because it can be consumed on a daily basis and can be expected to improve infectious diseases caused by Salmonella bacteria.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and electrolyte drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, It can be manufactured into foods such as snacks and noodles, and preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc. In addition, the food composition of the present invention may have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food according to Korea's 'Act on Health Functional Foods', or confectionery, tea, beverages, and special food according to each food type according to the food code of Korea's 'Food Sanitation Act' (Ministry of Food and Drug Safety Notification 'Food Standards and Specifications'). It may be food for use, etc.

Additionally, the food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In terms of use, these food additives are divided into sweeteners, flavor enhancers, preservatives, emulsifiers, and flavorings.

The sweetener is used to impart an appropriate sweetness to food, and can be either natural or synthetic. For example, sweeteners such as neotame, lactitol, D-ribose, mannitol, D-maltitol, and sodium saccharin can be used.

The flavor enhancer is a food additive that enhances the taste or aroma of food, and both natural and synthetic agents can be used. For example, flavor enhancers may include disodium 5'-guanylate, L-glutamic acid, glycine, betaine, etc.

As the preservative, sodium metabisulfite, sulfurous anhydride, sorbic acid, benzoic acid, grapefruit seed extract, etc. may be used.

The emulsifier is a food additive that homogeneously mixes or maintains two or more immiscible phases such as water and oil, and may include sodium gluconate, glycerin fatty acid ester, lecithin, magnesium stearate, alginic acid, etc.

The flavoring agent is a food additive that gives a unique flavor to food or reinforces the original flavor of food lost during the manufacturing process. Ethyl vanillin, ethyl octanoate, linalyl acetate, allyl caproate, paramethylacetophenone, etc. may be used. .

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition. The bioactive substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine. Minerals include calcium preparations such as calcium citrate and stearic acid. Magnesium preparations such as magnesium, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose depending on the product type, and with respect to other food additives that may be included in the food composition of the present invention, each country's food code or You can refer to the Food Additives Code.

As another aspect for achieving the above object, the present invention provides a feed additive composition or feed composition containing bacteriophage as an active ingredient. The terms bacteriophage, active ingredient, etc. are as described above.

The feed additive may be prepared in the form of a composition containing bacteriophage and mixed into feed, or may be used by directly adding the bacteriophage during feed production.

The bacteriophage contained in the feed additive may be in a liquid state or a dried solid state, and specifically, may be in the form of a dried powder. The drying method of the bacteriophage may be ventilation drying, natural drying, spray drying, or freeze drying, but is not limited thereto.

The feed additive containing the bacteriophage as an active ingredient may additionally include other additives as needed. Non-limiting examples of the additives that can be used include binders, emulsifiers, and preservatives added to prevent deterioration of feed quality; Amino acids, vitamins, enzymes, probiotics, flavoring agents, non-protein nitrogen compounds (NPN), silicate agents, buffers, colorants, extractants, or oligosaccharides added to increase the effectiveness of feed include these. It may be added singly or two or more types may be added together.

Raw materials other than bacteriophages included in the feed are those commonly used in the industry and can be used without limitation. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, fats and oils, single-cell proteins, zooplanktons or food. These may be used alone or in combination of two or more types.

The feed additives and feeds can be manufactured by manufacturing methods commonly used in the industry.

The feed additive or feed may contain an effective amount of additive sufficient to reduce quality degradation, and the additive may be a preservative, stabilizer, excipient, or cryoprotectant. By containing the additive, the feed additive or feed may allow the bacteriophage to survive or remain active for a longer period of time in the feed additive or feed, unlike bacteriophages that exist in nature. The preservatives, stabilizers, excipients or cryoprotectants are feed additives and those commonly used in the art can be used without limitation as long as they can reduce the quality deterioration of the feed.

As another aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella bacteria, including bacteriophage as an active ingredient.

The terms bacteriophage, active ingredient, infectious disease caused by salmonella, etc. are as described above.

The pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier and provided as a food, medicine, feed additive, or drinking water additive. there is.

As used herein, the term "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound.

The type of carrier that can be included in the composition is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, etc. They can be used alone or in a mixture of two or more types, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella can be administered through oral or parenteral administration, and can also be applied or sprayed to the diseased area. In the case of parenteral administration, it may be administered using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration. Suitable application, spraying, and dosage of the composition are determined by factors such as formulation method, administration method, age, body weight, sex, degree of disease symptoms, food, administration time, administration route, excretion rate, and reaction sensitivity of the subject animal and patient. It varies depending on factors, and usually a skilled doctor or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.

Formulations for oral administration of the pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, powders or granules, emulsions, and hard forms. Alternatively, it can be formulated as a soft capsule, syrup, or elixir. For formulation into dosage forms such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, and magnesium stearate. , may contain a lubricant such as calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax, and in the case of a capsule formulation, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Formulations for parenteral administration of the pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella include injectable forms such as subcutaneous injection, intravenous injection, or intramuscular injection, suppository injection, or inhalable through the respiratory tract. It can be formulated for spray use, such as an aerosol agent. In order to formulate a dosage form for injection, the composition of the present invention can be mixed in water with a stabilizer or buffer to prepare a solution or suspension, and the composition can be formulated for unit administration in ampoules or vials. When formulating for spraying, such as an aerosol, a propellant, etc. may be mixed with additives to disperse the water-dispersed concentrate or wet powder.

The pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella may include a preservative, stabilizer, wetting agent, emulsifier, cryoprotectant, or excipient.

The preservative, stabilizer, or excipient may be included in the composition in an effective amount sufficient to reduce deterioration of the composition.

As used herein, the term "deterioration in quality" may include a reduction in the number of bacteria, a reduction in activity, or a combination thereof of bacteriophage LSE1 included in a composition.

For example, the composition may contain an effective amount of a preservative, stabilizer, or excipient sufficient to reduce degradation of the composition, thereby allowing bacteriophage LSE1 to survive or maintain activity for a longer period of time in the composition, unlike bacteriophages that exist in nature. It could be.

The preservatives, stabilizers or excipients may be those commonly used in the art without limitation, as long as they can reduce quality degradation of the composition. For example, the stabilizer includes alginate, sodium alginate, casein, sodium caseinate, cellulose, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, pectin, gelatin, It may be any one or more selected from the group consisting of gum arabic, xanthan gum, dextran, cyclodextrin, and starch.

The cryoprotectant may be included in the composition in an effective amount sufficient to reduce quality degradation of the composition when the composition is freeze-dried. For example, unlike bacteriophages that exist in a natural state where their activity is reduced or cannot survive in a lyophilized composition, the composition is frozen by containing an effective amount of a cryoprotectant sufficient to reduce degradation of the composition in the lyophilized state. Even in the dried composition, bacteriophage activity may be maintained or bacteriophages may be able to survive.

As long as the cryoprotectant is intended to reduce quality degradation of the freeze-dried composition, those commonly used in the art may be used without limitation. For example, the cryoprotectant may be one or more selected from the group consisting of glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), glucose, trehalose, maltodextrin, dextrin, skim milk, and starch. .

As another aspect for achieving the above object, the present invention provides a method of bacterial sterilization, comprising the step of treating the subject with the bacteriophage.

Specifically, the method of sterilizing bacteria may include treating the bacteria with an effective amount of a disinfectant or detergent containing the bacteriophage of the present invention as an active ingredient, but is not limited thereto.

The term "sterilization" used in this specification refers to killing bacteria using bacteriophages, and the bacteria refers to Salmonella, but is not limited thereto.

The term "object" used in this specification refers to food exposed or likely to be exposed to Salmonella bacteria, the skin surface of animals, each body part, as well as the animal activity area, slaughterhouse, death area, cooking area or cooking equipment, etc. This may be the target, but is not limited to this.

The new bacteriophage of the present invention is specific for Salmonella and has the ability to inhibit or kill Salmonella growth, so it can be used as an antibiotic, disinfectant, detergent, food additive, food, feed additive, feed, or pharmaceutical for preventing or treating infectious diseases caused by Salmonella. It can be widely used in compositions, etc.

Figure 1 is a photograph showing a plaque formed by bacteriophage LSE1 isolated in the present invention.
Figure 2 is a photograph showing the results of microscopic observation of bacteriophage LSE1 isolated in the present invention.
Figure 3 is a graph showing the host lytic activity over time of the bacteriophage LSE1 isolated in the present invention.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Isolation of bacteriophage LSE1

A sewage sample was collected from a public sewage treatment facility in Gimje-si, Jeollabuk-do, and 20 ml of sewage sample, 20 ml of twice-concentrated TSB medium, 2mM CaCl ₂ , and host Salmonella Enteritidis NCCP 14547 culture were added to a 50 mL tube and incubated at 37°C for 16 minutes. cultured for some time.

The grown culture was centrifuged and filtered (0.45 μm) to remove foreign substances and bacteria from the sample. Salmonella cultured for more than 12 hours was mixed with Top Agar (0.4%) and poured into 1.5% Agar medium to solidify. Then, 10 μl of the growth sample was added and cultured at 37°C for 8 hours.

When the desired bacteriophage appeared on the plate, independent and transparent plaques were observed (Figure 1). This plaque area was separated using a tip and then placed in a new bacterial culture, and it was observed that the culture fluid became clear.

Afterwards, the culture medium was centrifuged, and only the supernatant was taken to recover a filtrate containing only bacteriophages. Pure bacteriophages were obtained from the bacteriophage filtrate recovered through ultra-high-speed centrifugation using cesium chloride (CsCl) density difference.

The bacteriophage obtained in this way has an excellent killing effect on Salmonella bacteria, and in particular, the rate of resistant bacteria appearing even after a long period of time is low, so the killing effect is excellent, so it was named LSE1 and deposited at the Korea Research Institute of Bioscience and Biotechnology, with accession number KCTC. Received 15077 BP.

Example 2: Analysis of morphological characteristics of obtained phages

Electron micrographs of the bacteriophage obtained in Example 1 were analyzed (Figure 2). The electron microscope used was a transmission electron microscope. The bacteriophage sample to be observed was placed on a carbon-coated copper grid and negatively stained with 2% liquid uranyl acetate. The stained sample was observed using a transmission electron microscope (Hitach 7650) at a voltage of 100 kV.

The bacteriophage LSE1 observed in this way has the form of a siphovirus consisting of a head of about 80 nm and a non-contractile tail of about 200 nm, based on the standard for classification by shape.

Example 3: Base sequence analysis of the obtained phage

To analyze the base sequence of the isolated phage, the phage genes were isolated. 0.5 M EDTA (pH 8.0), proteinase K, and SDS were added to 1 mL of pure isolated bacteriophage, and the concentrations after addition of the reagents were 20 mM, 50 μg/mL, and 0.5%, respectively. The mixed solution was kept at 56°C for 2 hours and then cooled to room temperature. The same amount of phenol was added and centrifuged at 3000g for 5 minutes. Only the supernatant was obtained, treated again with PCI (phenol-chloroform-isoamylalcohol), and centrifuged under the same conditions. An equal amount of chloroform was added to the supernatant obtained above and centrifuged. Repeat this process, add 1 mL of 95% ethanol and 100 μL of 3 M sodium acetate (pH 5.2) to precipitate DNA at -80°C, centrifuge for 15 minutes, and add 70% ethanol once. Further centrifugation was performed. After removing the ethanol and drying the DNA, the DNA was dissolved in distilled water.

The nucleotide sequence of the total pure isolated gene was determined, and the nucleotide sequence of bacteriophage LSE1 has a total size of 112,766 bp (SEQ ID NO. 1), and the nucleotide sequence is shown in SEQ ID NO. 1. The determined base sequence was analyzed using the 'RAST (Rapid Annotation using Subsystem Technology)' program. As a result of the analysis, 146 open reading frames (ORFs) and 26 tRNAs were predicted in the LSE1 genome, of which 94 ORFs were hypothetical proteins whose functions have not yet been discovered. It is judged.

The 52 ORFs can be broadly divided into four groups: metabolism, packaging, structure, and lysis. Since the integrase or repressor that maintains the bacteriophage in a lysogenic state was not predicted, it was confirmed again that LSE1 is a lytic phage. The putative functions of the LSE1 gene are shown in Table 1 below.

Locus tag start closing function LSE1_1 1697 33 Probable A1 protein LSE1_2 2503 2087 A2 protein LSE1_3 2853 2602 Hypothetical protein LSE1_4 4340 5341 Hypothetical protein LSE1_5 5731 6225 Hypothetical protein LSE1_6 6485 6697 Hypothetical protein LSE1_7 7154 7366 hypothetical protein LSE1_8 10283 9399 Hypothetical protein LSE1_9 10710 10360 Hypothetical protein LSE1_10 11321 10710 Hypothetical protein LSE1_11 11452 11321 Hypothetical protein LSE1_12 11673 11506 Hypothetical protein LSE1_13 12935 12513 Hypothetical protein LSE1_14 13287 12946 Phage capsid and scaffold LSE1_15 13738 13271 Hypothetical protein LSE1_16 14362 14033 Hypothetical protein LSE1_17 14597 14352 Hypothetical protein LSE1_18 14875 14594 Hypothetical protein LSE1_19 15291 14872 Hypothetical protein LSE1_20 15542 15291 Hypothetical protein LSE1_21 16048 15617 Hypothetical protein LSE1_22 17094 16675 Phosphoesterase LSE1_23 17561 17193 Hypothetical protein LSE1_24 18424 17561 Serine/threonine protein phosphatase (EC 3.1.3.16) LSE1_25 18576 18427 hypothetical protein LSE1_26 19100 18810 Thioredoxin, phage-associated LSE1_27 19503 19093 Hypothetical protein LSE1_28 20486 20073 Phage endolysin LSE1_29 21139 20483 Phage holin LSE1_30 21895 21296 ATP-dependent Clp protease proteolytic subunit (EC 3.4.21.92) LSE1_31 22660 21908 Deoxynucleotide monophosphate kinase (EC 2.7.4.13) LSE1_32 23393 22944 Hypothetical protein LSE1_33 24048 23350 Hypothetical protein LSE1_34 24542 24195 Hypothetical protein LSE1_35 24943 24659 Hypothetical proteins LSE1_36 25236 24940 Hypothetical protein LSE1_37 25609 25190 Hypothetical proteins LSE1_38 25901 25602 Hypothetical protein LSE1_39 26175 25894 Hypothetical proteins LSE1_40 26606 26253 Hypothetical protein LSE1_41 26851 26666 Hypothetical proteins LSE1_42 27071 26916 Hypothetical proteins LSE1_43 27659 27291 Pyruvate formate-lyase (EC 2.3.1.54) LSE1_44 28040 27846 hypothetical protein LSE1_rna.1 28153 28078 tRNA-Met-CAT LSE1_rna.2 28326 28250 tRNA-Ile-GAT LSE1_45 28534 28328 Hypothetical proteins LSE1_46 28827 28534 Hypothetical protein LSE1_rna.3 28976 28902 tRNA-Thr-TGT LSE1_47 29151 28987 Hypothetical protein LSE1_48 29365 29144 Hypothetical protein LSE1_rna.4 29455 29381 tRNA-Gly-TCC LSE1_rna.5 29538 29463 tRNA-Gln-TTG LSE1_rna.6 29620 29545 tRNA-Gln-CTG LSE1_49 29829 29629 Hypothetical proteins LSE1_rna.7 29928 29854 tRNA-Arg-ACG LSE1_50 30579 30406 Hypothetical proteins LSE1_51 31360 30776 Phage-associated homing endonuclease LSE1_rna.8 31461 31377 tRNA-Leu-TAG LSE1_rna.9 31546 31468 tRNA-Ala-TGC LSE1_52 32256 31903 Hypothetical protein LSE1_rna.10 32355 32282 tRNA-Val-TAC LSE1_rna.11 32437 32360 tRNA-Lys-TTT LSE1_53 33055 32891 Hypothetical protein LSE1_rna.12 33154 33077 tRNA-Met-CAT LSE1_rna.13 33238 33161 tRNA-Pro-TGG LSE1_rna.14 33681 33607 tRNA-Gly-GCC LSE1_rna.15 33961 33885 tRNA-Lys-CTT LSE1_54 34154 33963 hypothetical protein LSE1_rna.16 34245 34169 tRNA-Asp-GTC LSE1_55 34440 34255 hypothetical protein LSE1_rna.17 34621 34544 tRNA-Asn-GTT LSE1_rna.18 35212 35138 tRNA-Cys-GCA LSE1_56 35488 35216 Hypothetical protein LSE1_57 35810 35538 Hypothetical protein LSE1_rna.19 35902 35828 tRNA-Phe-GAA LSE1_rna.20 35985 35909 tRNA-Pseudo-CCA LSE1_rna.21 36070 35994 tRNA-Glu-TTC LSE1_rna.22 36168 36078 tRNA-Pseudo-GTA LSE1_58 36634 36359 Hypothetical proteins LSE1_59 36930 36724 Hypothetical protein LSE1_60 37066 36944 Hypothetical protein LSE1_rna.23 37158 37082 tRNA-Leu-TAA LSE1_61 37505 37167 Hypothetical protein LSE1_62 37692 37507 Phage antitermination protein Q LSE1_rna.24 37787 37710 tRNA-Met-CAT LSE1_rna.25 37881 37793 tRNA-Ser-GCT LSE1_63 38160 37882 Phage-associated homing endonuclease LSE1_64 38758 38462 Hypothetical proteins LSE1_65 39542 38865 Ribosyl nicotinamide transporter, PnuC-like LSE1_66 40599 39544 NadR transcriptional regulator LSE1_67 41815 40871 Hypothetical proteins LSE1_68 42027 41827 Hypothetical protein LSE1_rna.26 42128 42054 tRNA-Arg-TCT LSE1_69 42646 42137 hypothetical protein LSE1_70 44153 43710 Phage recombination related exonuclease (EC 3.1.11.-) LSE1_71 44323 44153 Hypothetical proteins LSE1_72 44841 44392 Hypothetical proteins LSE1_73 45164 44847 Hypothetical protein LSE1_74 46246 45608 Phage tail fiber protein LSE1_75 46483 46301 Hypothetical proteins LSE1_76 47256 46555 Metallopeptidase, phage-associated LSE1_77 47499 47287 Hypothetical protein LSE1_78 47756 47541 Phage tail length tape-measure protein LSE1_79 48333 47818 Hypothetical protein LSE1_80 48656 48417 Hypothetical proteins LSE1_81 49248 48772 Ribonuclease HI (EC 3.1.26.4) LSE1_82 49517 49248 Hypothetical protein LSE1_83 50718 49864 Thymidylate synthase (EC 2.1.1.45) LSE1_84 51245 50715 Dihydrofolate reductase, phage-associated LSE1_85 52390 51245 Ribonucleotide reductase of class Ia (aerobic), beta subunit (EC 1.17.4.1) LSE1_86 54888 52498 Ribonucleotide reductase of class Ia (aerobic), alpha subunit (EC 1.17.4.1) LSE1_87 55397 54909 Phage-associated homing endonuclease LSE1_88 55703 55461 Hypothetical protein LSE1_89 56457 55705 Phosphate starvation-inducible protein PhoH, predicted ATPase LSE1_90 56810 58684 Ribonucleotide reductase of class III (anaerobic), large subunit (EC 1.17.4.2) LSE1_91 58783 59064 Hypothetical protein LSE1_92 59074 59277 Hypothetical protein LSE1_93 59440 60291 NAD-dependent protein deacetylase of SIR2 family LSE1_94 60269 60427 Hypothetical protein LSE1_95 60424 60609 Hypothetical protein LSE1_96 60824 60940 Hypothetical proteins LSE1_97 60968 61102 Hypothetical protein LSE1_98 61105 61533 Hypothetical protein LSE1_99 61543 61938 Hypothetical protein LSE1_100 62555 65344 Phage DNA primase C LSE1_101 65328 65561 Hypothetical protein LSE1_102 65631 66335 Hypothetical protein LSE1_103 66328 66573 hypothetical protein LSE1_104 66681 67091 Hypothetical protein LSE1_105 67128 67424 Hypothetical protein LSE1_106 67595 67783 Hypothetical protein LSE1_107 67968 68141 hypothetical protein LSE1_108 68142 68336 hypothetical protein LSE1_109 68329 69300 DNA ligase, phage-associated LSE1_110 69503 70273 DNA ligase, phage-associated LSE1_111 70276 71043 Hypothetical proteins LSE1_112 71132 72598 Hypothetical protein LSE1_113 72595 73485 DNA primase (EC 2.7.7.-) / DNA helicase (EC 3.6.1.-), phage-associated LSE1_114 73548 76115 DNA polymerase I (EC 2.7.7.7), phage-associated LSE1_115 76602 77954 DNA helicase, phage-associated LSE1_116 78450 79223 Hypothetical protein LSE1_117 79263 80240 Phage-associated recombinase LSE1_118 80263 82059 Phage recombination related exonuclease (EC 3.1.11.-) LSE1_119 82063 82545 Hypothetical protein LSE1_120 82545 83420 Phage ribonuclease H (EC 3.1.26.4) LSE1_121 83417 83863 Deoxyuridine 5'-triphosphate nucleotidohydrolase (EC 3.6.1.23) LSE1_122 83841 84098 Hypothetical protein LSE1_123 87315 84364 Phage tail fibers LSE1_124 87737 87315 Hypothetical protein LSE1_125 89740 87743 Phage tail fibers LSE1_126 92650 89801 Phage tail length tape-measure protein LSE1_127 93261 92647 Hypothetical protein LSE1_128 97080 93370 Phage tail length tape-measure protein LSE1_129 97508 97161 Hypothetical protein LSE1_130 97995 97591 Hypothetical protein LSE1_131 98891 97992 Phage tail fibers LSE1_132 100305 98896 Phage major tail protein LSE1_133 101588 100821 Hypothetical protein LSE1_134 102100 101588 Hypothetical protein LSE1_135 103536 102160 Phage major capsid protein LSE1_136 104186 103554 Phage capsid and scaffold LSE1_137 104513 104190 Phage tail fibers LSE1_138 105727 104510 Phage portal (connector) protein LSE1_139 105867 105727 Hypothetical protein LSE1_140 107595 106279 Phage terminase, large subunit LSE1_141 108077 107595 Hypothetical proteins LSE1_142 109869 108088 Phage-associated receptor-binding protein LSE1_143 109953 110219 Hypothetical protein LSE1_144 110842 110979 hypothetical protein LSE1_145 112116 111382 degrades 5'-deoxyribonucleotides to their corresponding deoxyribonucleosides LSE1_146 112596 112204 Hypothetical proteins

Experimental Example 1: Analysis of Salmonella lytic activity of bacteriophage LSE1

Host S. Enteritidis NCCP 14547 The host lytic ability of LSE1 was confirmed by mixing a certain concentration of bacteriophage LSE1 into the culture medium, measuring the absorbance at regular intervals, and comparing it with the control group.

Compared to the control group in which Salmonella alone was cultured, when Salmonella was mixed with bacteriophage LSE1, the absorbance was significantly reduced and no resistant bacteria appeared even 5 hours after phage infection, showing that bacteriophage LSE1 has an excellent lytic effect on Salmonella hosts. Confirmed (Figure 3).

Experimental Example 2: Analysis of host specificity of bacteriophage LSE1

To confirm the host specificity of bacteriophage LSE1, its lytic activity against various strains of Salmonella was analyzed.

The host specificity of bacteriophage LSE1 was determined through the dropping method. After mixing the bacterial culture cultured for 16 hours with 0.4% agar medium, it was poured into 1.5% agar medium and dried at room temperature for 15 minutes. The phage culture medium was dropped on top of it and incubated for 8 hours at 37°C. If the strain is susceptible to phage, a transparent area, or plaque, appears on the plate, which proves that it is the result of complete lysis of the bacterial cells. If sensitivity is weak, hazy areas may appear or independent bold spots may appear.

As a result of the experiment, the bacteriophage LSE1 of the present invention was isolated from S. In addition to Enteritidis, it showed lytic activity on other serotypes such as Salmonella Paratyphi, Salmonella Typhi, and Salmonella Typhimurium, confirming its broad lytic activity within Salmonella bacteria. The lytic activity was divided into three levels according to the transparency of the plaque, and the lytic activity of LSE1 against Salmonella is shown in Table 2 [+++: clear lysis, ++: slightly turbid, +: very very turbid, -: no lysis].

host bacteria Bacteriophage LSE1 Salmonella Enteritidis ATCC 13076 ++ Enteritidis KCCM 12021 +++ Enteritidis NCCP 14547 +++ Enteritidis NCCP 14771 ++ Enteritidis NCCP 16206 +++ Paratyphi NCCP 14759 +++ Typhi NCCP 13699 + Typhi NCCP 14641 ++ Typhimurium ATCC 14028 + Typhimurium ATCC 43971 + Typhimurium ATCC 6994 +++ Typhimurium DT104 + Typhimurium NCCP 12219 + Typhimurium NCCP 12241 + Typhimurium NCCP 12245 + Typhimurium NCCP 16207 +

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15077BP 20220919

Sequence list electronic file attached

Claims (15)

Bacteriophage with specific killing ability against Salmonella bacteria. In paragraph 1
The bacteriophage is a bacteriophage deposited as KCTC 15077BP. According to paragraph 1,
The salmonella is a bacteriophage that is at least one selected from Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Typhi, and Salmonella Paratyphi. An antibiotic comprising the bacteriophage of claim 1 as an active ingredient. The antibiotic according to claim 4, wherein the antibiotic has the ability to inhibit the growth of Salmonella bacteria or to lyse them. A disinfectant containing the bacteriophage of paragraph 1 as an active ingredient. A detergent containing the bacteriophage of claim 1 as an active ingredient. A food additive composition comprising the bacteriophage of claim 1 as an active ingredient. A food composition comprising the bacteriophage of claim 1 as an active ingredient. A feed additive composition comprising the bacteriophage of claim 1 as an active ingredient. A feed composition comprising the bacteriophage of claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating infectious diseases caused by Salmonella bacteria, comprising the bacteriophage of claim 1 as an active ingredient. According to clause 12,
The composition, wherein the infectious disease caused by Salmonella is Salmonella infection, typhoid fever, or food poisoning. Comprising the step of processing the bacteriophage of paragraph 1 on the target,
How to sterilize bacteria. According to clause 14,
A sterilization method wherein the bacteria are salmonella.