A novel excipient composition for maintaining the long-term storage stability of bacteriophages

Abstract

The present invention relates to a novel composition for improving bacteriophage stability for maintaining the long-term distribution storage stability of bacteriophage, and the composition has excellent storage stability of bacteriophage by containing anhydrous sodium sulfate.

Description

[Technical field]

[0001] Cross-reference to related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0047176 dated April 15, 2022, and all contents disclosed in the documents of the Korean Patent Application are incorporated herein by reference.

[0002] The present invention relates to a novel excipient composition for maintaining the long-term storage stability of bacteriophages and its use. [Background technology]

[0003] Foodborne illness caused by foodborne bacteria is a worldwide problem and has been recognized as one of the serious public health issues. Therefore, it is important for the food industry not only to provide consumers with tasty and nutritious food, but also to supply safe food that is free of food poisoning bacteria that may cause illness.

[0004] Bacteriophages are a type of virus that use bacteria as host cells and are non-toxic to humans because they specifically bind only to bacteria. Due to these characteristics, bacteriophages have been widely used in the clinical treatment of infections caused by bacteria, and are particularly used as feed additives to prevent or suppress livestock diseases (Korea Patent Publication No. 10-2021-0145038).

[0005] Meanwhile, during the production and distribution of feed additive bacteriophage products, there is a problem with the conventional excipients, which cause a decrease in productivity and product quality due to a decrease in bacteriophage titer during long-term storage. Therefore, research is needed on excipients that can maintain the quality of feed additive bacteriophage products during long-term distribution. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] KR10-2021-0145038A Summary of the Invention [Problem to be solved by the invention]

[0007] An example of the present application provides a composition for improving bacteriophage stability, comprising anhydrous sodium sulfate, which may be for maintaining and / or increasing the stability (e.g., storage stability) of a bacteriophage for an extended period of time.

[0008] Another example provides a composition comprising a bacteriophage and anhydrous sodium sulfate. The composition may be a feed composition, a food composition, a pharmaceutical composition, or an antimicrobial composition.

[0009] Another example provides a method for improving bacteriophage stability, comprising mixing a bacteriophage with a bacteriophage stability improving composition comprising anhydrous sodium sulfate.

[0010] Another example provides the use of anhydrous sodium sulfate for use in improving bacteriophage stability or in preparing a composition for improving bacteriophage stability.

[0011] Other examples provide uses of bacteriophages and anhydrous sodium sulfate for use in the manufacture of a feed composition, a food composition, a pharmaceutical composition, or an antimicrobial composition.

[0012] Other examples provide uses of bacteriophages and bacteriophage stability improving compositions for use in the manufacture of feed compositions, food compositions, pharmaceutical compositions, or antibacterial compositions. [Means for solving the problem]

[0013] One aspect provides a composition for improving bacteriophage stability comprising anhydrous sodium sulfate.

[0014] Sodium sulfate anhydrous (Na 2 SO 4 Sodium sulfate (Na 2 SO 4 , 142.04 g / mol, CAS Number: 7757-82-6) is a colorless crystal of sodium sulfate. The anhydrous sodium sulfate is bound to 10 water molecules on the outside. 2 SO 4 10H 2 O;322.20 g / mol), so it can absorb moisture in moist environments.

[0015] The composition for improving bacteriophage stability may additionally comprise an excipient component or may be used as an excipient.

[0016] The excipient can refer to a substance added to feed, food, or medicine to make it easier to eat or to create a certain shape. Types of excipients include binders that give binding power to powder raw materials to facilitate molding, disintegrants that promote disintegration, lubricants that are added to facilitate the compression operation of granular products, improve fluidity, and prevent adhesion, and adsorbents that absorb liquid raw materials to make them tablet-shaped. Examples of excipient components that are mainly used include, but are not limited to, one or more selected from the group consisting of hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), lactose (hydrated, anhydrous, monohydroxide, spray-dried form, etc.), microcrystalline cellulose, titanium dioxide, silicon dioxide (colloidal, colloidal silicon dioxide, etc.), magnesium stearate, polyethylene glycol, croscarmellose sodium, pregelatinized starch, sodium starch glycolate, crospovidone, talc, wheat powder, calcium carbonate, silica, etc. The composition for improving bacteriophage stability provided in the present application may further include one or more of the excipient components in addition to anhydrous sodium sulfate.

[0017] The ratio of anhydrous sodium sulfate contained in the composition for improving bacteriophage stability is, based on weight, 1 to 50% (w / w), 1 to 45% (w / w), 1 to 40% (w / w), 1 to 35% (w / w), 1 to 30% (w / w), 5 to 50% (w / w), 5 to 45% (w / w), 5 to 40% (w / w), 5 to 35% (w / w), 5 to 30% (w / w), 10 to 50% (w / w), 10 to 45% (w / w), 10 to 40% (w / w), 10 to 35% (w / w), 10 to 30% (w / w), 15 to 50% (w / w), 15 to 45% (w / w), The amount of the cellulose acetate solution may be, but is not limited to, 15-40% (w / w), 15-35% (w / w), 15-30% (w / w), 20-50% (w / w), 20-45% (w / w), 20-40% (w / w), 20-35% (w / w), 20-30% (w / w), 25-50% (w / w), 25-45% (w / w), 25-40% (w / w), 25-35% (w / w), 25-30% (w / w), 30-50% (w / w), 30-45% (w / w), 30-40% (w / w), or 30-35% (w / w).

[0018] As used herein, the term "improved bacteriophage stability" refers to maintaining, improving, or enhancing the storage stability of a bacteriophage for a long period of time. That is, the effect of improving bacteriophage stability may refer to the effect of maintaining the titer of a bacteriophage or reducing the amount of titer loss.

[0019] Bacteriophage is a common term for viruses that use bacteria and archaea as hosts, and can specifically infect bacteria. Therefore, bacteriophages are used in products such as feed (including feed additives), foods, medicines, and disinfectants due to their antibiotic effect, but there is a problem of reduced productivity and quality of the products because the titer of the bacteriophage decreases during storage in the production or distribution of the products. In other words, the stability improvement effect of the bacteriophage can mean maintaining the titer of the bacteriophage or reducing the amount of decrease in the titer of the bacteriophage during storage of the product containing the bacteriophage.

[0020] In one example, when the composition for improving bacteriophage stability is applied to a bacteriophage (e.g., mixed, etc.), the bacteriophage is incubated at a temperature of 45 to 60°C, 45 to 58°C, 45 to 55°C, 45 to 52°C, 45 to 50°C, 45 to 48°C, 48 to 60°C, 48 to 58°C, 48 to 55°C, 48 to 52°C, 48 to 50°C, 50 to 60°C, 50 to 58°C, 50 to 55°C, or 50 to 52°C; and / or When stored at a relative humidity of 80~100%, 80~99%, 80~98%, 80~97%, 80~96%, 80~95%, 80~92%, 80~90%, 80~88%, 80~85%, 85~100%, 85~99%, 85~98%, 85~97%, 85~96%, 85~95%, 85~92%, 85~90%, 85~88%, 90~100%, 90~99%, 90~98%, 90~97%, 90~96%, 90~95%, 90~92%, 95~100%, 95~99%, 95~98%, 95~97%, or 95~96%. or the bacteriophage titer measured after 1-7 days, 1-14 days, 7-14 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days is reduced by 2 log(pfu / g) or less, 1.9 log(pfu / g) or less, 1.8 log(pfu / g) or less, 1.7 log(pfu / g) or less, 1.6 log(pfu / g) or less, 1.5 log(pfu / g) or less, or 1.4 log(pfu / g) or less, e.g., 1.4 log(pfu / g), as compared to the bacteriophage titer measured on day 0; or It may be reduced to 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, or 15% or less.

[0021] In another example, when the bacteriophage stability improving composition is applied to the bacteriophage (e.g., mixed), compared to a control group (e.g., when anhydrous sodium sulfate is not applied to the bacteriophage), the bacteriophage is exposed to a temperature condition of 45-60°C, 45-58°C, 45-55°C, 45-52°C, 45-50°C, 45-48°C, 48-60°C, 48-58°C, 48-55°C, 48-52°C, 48-50°C, 50-60°C, 50-58°C, 50-55°C, or 50-52°C; and / or When stored at a relative humidity of 80~100%, 80~99%, 80~98%, 80~97%, 80~96%, 80~95%, 80~92%, 80~90%, 80~88%, 80~85%, 85~100%, 85~99%, 85~98%, 85~97%, 85~96%, 85~95%, 85~92%, 85~90%, 85~88%, 90~100%, 90~99%, 90~98%, 90~97%, 90~96%, 90~95%, 90~92%, 95~100%, 95~99%, 95~98%, 95~97%, or 95~96%. The decrease in bacteriophage titer (log(pfu / g)) measured after 1-7 days, 1-14 days, 7-14 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days compared to the titer measured on day 0 was 0.5-3, 0.5-2.5, 0.5-2, 0.5-1.5, 0.6-3, 0.6-2.5 , 0.6~2, 0.6~1.5, 0.7~3, 0.7~2.5, 0.7~2, 0.7~1.5, 0.8~3, 0.8~2.5, 0.8~2, 0.8~1.5, 0.9~3, 0.9~2.5, 0.9~2, 0.9~1.5, 1~3, 1~2.5, 1~2, 1~1.5, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1.0 or more, or 1.1 or more, e.g., even lower by 1.1 or It may be 5-20%p, 5-15%p, 6-20%p, 6-15%p, 7-20%p, 7-15%p, 8-20%p, 8-15%p, 9-20%p, 9-15%p, 10-20%p, 10-15%p, 5%p or more, 6%p or more, 7%p or more, 8%p or more, 9%p or more, 10%p or more, or even lower by 11%p, for example.

[0022] In another example, when the bacteriophage stability improving composition is applied to the bacteriophage (e.g., mixed), compared to a control group (e.g., when anhydrous sodium sulfate is not applied to the bacteriophage), Temperature conditions of 45-60°C, 45-58°C, 45-55°C, 45-52°C, 45-50°C, 45-48°C, 48-60°C, 48-58°C, 48-55°C, 48-52°C, 48-50°C, 50-60°C, 50-58°C, 50-55°C, or 50-52°C; and / or Relative humidity of 80-100%, 80-99%, 80-98%, 80-97%, 80-96%, 80-95%, 80-92%, 80-90%, 80-88%, 80-85%, 85-100%, 85-99%, 85-98%, 85-97%, 85-96%, 85-95%, 85-92%, 85-90%, 85-88%, 90-100%, 90-99%, 90-98%, 90-97%, 90-96%, 90-95%, 90-92%, 95-100%, 95-99%, 95-98%, 95-97%, or 95-96% When stored for 1-7 days, 1-14 days, 7-14 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days under humidity conditions, The bacteriophage titer may be 2-40%, 2-35%, 2-30%, 2-25%, 2-20%, 2-15%, 2-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, or 15% or more high.

[0023] When the composition for improving bacteriophage stability is applied to a bacteriophage, there is an advantage that the bacteriophage can be stably stored since the titer loss of the bacteriophage is low even when the bacteriophage is stored for a long period of time under extreme conditions such as high temperature and humidity.

[0024] The composition for improving bacteriophage stability may contain anhydrous sodium sulfate, and thus may have a superior effect of improving bacteriophage stability compared to a composition not containing anhydrous sodium sulfate.

[0025] The composition for improving bacteriophage stability may be used for producing a feed composition, a food composition, a pharmaceutical composition, an antibacterial composition, or the like.

[0026] Another embodiment provides a composition comprising a bacteriophage and anhydrous sodium sulfate, such as a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition, or a composition comprising a bacteriophage and the bacteriophage stability improving composition, such as a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition.

[0027] The bacteriophage may be any type or number, and may be one or more (e.g., a combination of two or three) bacteriophages belonging to the Myoviridae, Siphoviridae, and Podoviridae families.

[0028] The bacteriophage may be, but is not limited to, a bacteriophage that specifically infects bacteria belonging to the genus Escherichia (e.g., E. coli, E. albertii, E. fergusonii, E. hermannii, E. vulneris, etc.), the genus Salmonella (e.g., S. bongori, S. enterica, etc.), or the genus Clostridium (e.g., C. botulinum, C. tetani, C. perfringens, C. hisolyticum, C. sordellii, C. butyricum, etc.).

[0029] The initial content (or titer) of the bacteriophage contained in the composition (e.g., feed composition, food composition, pharmaceutical composition, antibacterial composition, etc.) is not particularly limited, but is preferably 1×10 1 ~1×10 15 The amount of bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition) that can be generally used in the pfu / g range or contained in the composition can be appropriately determined by one skilled in the art taking into consideration the intended use of the final composition (e.g., a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition, etc.).

[0030] In the composition (e.g., a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition), the ratio of the bacteriophage and the bacteriophage stability improving composition in the composition is 90 to 99.9% (w / w), 90 to 99.5% (w / w), 90 to 99% (w / w), 90 to 98% (w / w), 90 to 97% (w / w), or the like. (w / w), 90~96%(w / w), 90~95%(w / w), 91~99.9%(w / w), 91~99.5%(w / w), 91~99%(w / w), 91~98%(w / w ), 91~97%(w / w), 91~96%(w / w), 91~95%(w / w), 92~99.9%(w / w), 92~99.5%(w / w), 92~99%(w / w), 92~ 98%(w / w), 92~97%(w / w), 92~96%(w / w), 92~95%(w / w), 93~99.9%(w / w), 93~99.5%(w / w), 93~99%( w / w), 93~98%(w / w), 93~97%(w / w), 93~96%(w / w), 93~95%(w / w), 94~99.9%(w / w), 94~99.5%(w / w), The composition may be, but is not limited to, 94-99% (w / w), 94-98% (w / w), 94-97% (w / w), 94-96% (w / w), 94-95% (w / w), 95-99.9% (w / w), 95-99.5% (w / w), 95-99% (w / w), 95-98% (w / w), 95-97% (w / w), or 95-96% (w / w).

[0031] In the composition (e.g., feed composition, food composition, pharmaceutical composition, or antibacterial composition), the mixing ratio of the bacteriophage to the bacteriophage stability improving composition may be, by weight, 1:8 to 1:999, 1:8 to 5:995, 1:8 to 1:99, 1:8 to 2:98, 1:8 to 3:97, 1:88 to 4:96, 1:8 to 5:95, 1:9 to 1:999, 1:9 to 5:995, 1:9 to 1:99, 1:9 to 2:98, 1:9 to 3:97, 1:9 to 4:96, 1:9 to 5:95, etc. (above, weight of bacteriophage:weight of bacteriophage stability improving composition), but is not limited thereto.

[0032] In the composition, for example, a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition, the ratio of the bacteriophage and the anhydrous sodium sulfate in the composition for improving bacteriophage stability is 4 to 45% (w / w), 4 to 40% (w / w), 4 to 30% (w / w), 4 to 20% (w / w), 4 to 18% (w / w), 4 to 15% (w / w), 4 to 10% (w / w), 4 to 5% (w / w), 4 to 4.5% (w / w), 4.5 to 45% (w / w), 4.5 to 40% (w / w), 4.5 to 30% (w / w), 4.5 to 20% (w / w), 4.5 to 18% (w / w), based on weight. (w / w), 4.5-15% (w / w), 4.5-10% (w / w), 4.5-5% (w / w), 15-45% (w / w), 15-40% (w / w), 15-30% (w / w), 15-20% (w / w), 15-18% (w / w), 18-45% (w / w), 18-40% (w / w), 18-30% (w / w), 18-20% (w / w), 20-45% (w / w), 20-40% (w / w), 20-30% (w / w), 30-45% (w / w), 30-40% (w / w), or 40-45% (w / w).

[0033] In one example, a composition comprising the bacteriophage and anhydrous sodium sulfate or a composition for improving bacteriophage stability, such as a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition, Temperature conditions of 45-60°C, 45-58°C, 45-55°C, 45-52°C, 45-50°C, 45-48°C, 48-60°C, 48-58°C, 48-55°C, 48-52°C, 48-50°C, 50-60°C, 50-58°C, 50-55°C, or 50-52°C; and / or When stored at a relative humidity of 80~100%, 80~99%, 80~98%, 80~97%, 80~96%, 80~95%, 80~92%, 80~90%, 80~88%, 80~85%, 85~100%, 85~99%, 85~98%, 85~97%, 85~96%, 85~95%, 85~92%, 85~90%, 85~88%, 90~100%, 90~99%, 90~98%, 90~97%, 90~96%, 90~95%, 90~92%, 95~100%, 95~99%, 95~98%, 95~97%, or 95~96%. the titer of the bacteriophage measured after 1-7 days, 1-14 days, 7-14 days, 1 day later, 2 days later, 3 days later, 4 days later, 5 days later, 6 days later, 7 days later, 8 days later, 9 days later, 10 days later, 11 days later, 12 days later, 13 days later, or 14 days later is reduced by 2 log(pfu / g) or less, 1.9 log(pfu / g) or less, 1.8 log(pfu / g) or less, 1.7 log(pfu / g) or less, 1.6 log(pfu / g) or less, 1.5 log(pfu / g) or less, or 1.4 log(pfu / g) or less, e.g., 1.4 log(pfu / g), as compared to the titer of the bacteriophage measured on day 0; or It may be reduced to 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, or 15% or less.

[0034] In one example, a composition comprising the bacteriophage and anhydrous sodium sulfate or a composition for improving bacteriophage stability, such as a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition, has the following properties compared to a composition not comprising anhydrous sodium sulfate: Temperature conditions of 45-60°C, 45-58°C, 45-55°C, 45-52°C, 45-50°C, 45-48°C, 48-60°C, 48-58°C, 48-55°C, 48-52°C, 48-50°C, 50-60°C, 50-58°C, 50-55°C, or 50-52°C; and / or When stored at a relative humidity of 80~100%, 80~99%, 80~98%, 80~97%, 80~96%, 80~95%, 80~92%, 80~90%, 80~88%, 80~85%, 85~100%, 85~99%, 85~98%, 85~97%, 85~96%, 85~95%, 85~92%, 85~90%, 85~88%, 90~100%, 90~99%, 90~98%, 90~97%, 90~96%, 90~95%, 90~92%, 95~100%, 95~99%, 95~98%, 95~97%, or 95~96%. The decrease in bacteriophage titer (log(pfu / g)) measured after 1-7 days, 1-14 days, 7-14 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days compared to the bacteriophage titer measured on day 0 was 0.5-3, 0.5-2.5, 0.5-2, 0.5-1.5, 0.6-3, or 0.6-2.5. , 0.6~2, 0.6~1.5, 0.7~3, 0.7~2.5, 0.7~2, 0.7~1.5, 0.8~3, 0.8~2.5, 0.8~2, 0.8~1.5, 0.9~3, 0.9~2.5, 0.9~2, 0.9~1.5, 1~3, 1~2.5, 1~2, 1~1.5, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1.0 or more, or 1.1 or more, e.g., even lower by 1.1 or It may be 5-20%p, 5-15%p, 6-20%p, 6-15%p, 7-20%p, 7-15%p, 8-20%p, 8-15%p, 9-20%p, 9-15%p, 10-20%p, 10-15%p, 5%p or more, 6%p or more, 7%p or more, 8%p or more, 9%p or more, 10%p or more, or even lower by 11%p, for example.

[0035] In one example, a composition comprising the bacteriophage and anhydrous sodium sulfate or a composition for improving bacteriophage stability, such as a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition, has a higher stability than a composition not comprising anhydrous sodium sulfate, Temperature conditions of 45-60°C, 45-58°C, 45-55°C, 45-52°C, 45-50°C, 45-48°C, 48-60°C, 48-58°C, 48-55°C, 48-52°C, 48-50°C, 50-60°C, 50-58°C, 50-55°C, or 50-52°C; and / or Relative humidity of 80-100%, 80-99%, 80-98%, 80-97%, 80-96%, 80-95%, 80-92%, 80-90%, 80-88%, 80-85%, 85-100%, 85-99%, 85-98%, 85-97%, 85-96%, 85-95%, 85-92%, 85-90%, 85-88%, 90-100%, 90-99%, 90-98%, 90-97%, 90-96%, 90-95%, 90-92%, 95-100%, 95-99%, 95-98%, 95-97%, or 95-96% When stored for 1-7 days, 1-14 days, 7-14 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days under humidity conditions, The bacteriophage titer may be 2-40%, 2-35%, 2-30%, 2-25%, 2-20%, 2-15%, 2-10%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, or 15% or more high.

[0036] A composition (e.g., a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition, etc.) containing the bacteriophage and anhydrous sodium sulfate or a composition for improving bacteriophage stability has the advantage that the titer loss of the bacteriophage is low even when stored for a long period of time in extreme environments such as high temperature and humidity conditions, and the bacteriophage can be stably stored.

[0037] Therefore, the composition containing the bacteriophage and anhydrous sodium sulfate (or a composition for improving bacteriophage stability) can be prepared in various forms according to specific purposes in the fields of household, industry, medicine, and the environment, and for example, can be prepared in the form of a feed composition (including a feed additive composition), a food composition, a pharmaceutical composition, or an antibacterial composition, but is not limited thereto.

[0038] Another embodiment provides a feed composition comprising a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition).

[0039] In this application, "feed" may refer to any natural or artificial diet, meal, or component of said meal that is adapted or suitable for eating, ingesting, and digesting by an animal. An example feed composition additionally includes a concentrate and / or a specialized feed. The concentrated feed may be animal feed such as seeds and fruits including grains such as wheat, oats, and corn; brans including rice bran, wheat bran, and wheat bran as by-products obtained by refining grains; oil cakes which are by-products obtained by extracting oil from beans, rapeseed, sesame, linseed, coconut, and the like; residual starches which are the main components of starch cakes which are the residues left after removing starch from sweet potatoes, potatoes, and the like; fish meal, fish meal, fish solubles which are concentrated fresh liquids obtained from fish; meat meal, blood meal, feather meal, skim milk powder, and dried whey which is the residual liquid when producing cheese from milk and when producing casein from skim milk; yeast, chlorella, and / or seaweed.

[0040] The feed composition according to an example may refer to a feed in a form that is ultimately ingested by an animal, a dietary supplement to be mixed with the feed, and / or a feed additive. The dietary supplement may refer to, for example, a formulation-containing composition that provides a therapeutic agent or digestive agent to an animal, and may refer to a composition that is not a normal calorie intake source, i.e., an energy source, but is ingested in addition to normal animal feed. The feed additive may refer to a substance that is added to the feed for various effects, such as supplementing nutrients and preventing weight loss, increasing digestive availability of fiber in the feed, improving milk quality, preventing reproductive disorders and improving conception rate, and preventing high temperature stress in summer. The feed to which the feed additive can be added may be selected from the group consisting of commercially available feed, or grains, root vegetables, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meal, grain by-products, proteins, inorganic substances, minerals, single-cell proteins, zooplankton, and leftover food and drink, but is not limited thereto.

[0041] In one example, the feed composition may be a feed additive, and when the feed additive according to one example is mixed into a feed (e.g., a compound feed and / or a plain feed that is ultimately ingested by an animal), the feed additive may have a concentration of 0.001% or more, 0.005% or more, 0.01% or more, 0.05% or more, 0.1% or more, 0.5% or more, 1% or less, 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, 0.005% or less, 0.001 to 1%, 0.001 to 0.5%, 0.001 to 0.1%, 0.001 to 0.05%, 0. The compound may be added at a weight ratio of 001-0.01%, 0.001-0.005%, 0.005-1%, 0.005-0.5%, 0.005-0.1%, 0.005-0.05%, 0.005-0.01%, 0.01-1%, 0.01-0.5%, 0.01-0.1%, 0.01-0.05%, 0.05-1%, 0.05-0.5%, 0.05-0.1%, 0.1-1%, 0.1-0.5%, or 0.5-1% and may be blended with feed ingredients, supplementary feed, supplements, and / or other types of additives other than the active ingredients according to the examples.

[0042] By mixing the bacteriophage and anhydrous sodium sulfate (or a composition for improving bacteriophage stability) with feed and feeding it, bacteria in the feed can be reduced or eliminated.

[0043] Another embodiment provides a method for producing a feed composition comprising the step of mixing a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition), said feed composition being as described above.

[0044] Another embodiment provides a food composition comprising a bacteriophage and anhydrous sodium sulfate (or a composition for improving bacteriophage stability). Since the bacteriophage specifically kills bacteria, it can be used to treat processed or non-processed foods or food ingredients to prevent bacterial infection. The food composition according to an example can mean meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, vitamin complexes, health functional foods and health foods, etc., and includes all foods in the usual sense.

[0045] By mixing the bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition) with food and feeding it, bacteria in the food can be reduced or eliminated.

[0046] Another aspect provides a method for producing a food composition comprising the step of mixing a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition), said food composition being as described above.

[0047] Another embodiment provides a pharmaceutical composition for preventing or treating infection with Escherichia, Salmonella, or Clostridium bacteria or a disease induced by Escherichia, Salmonella, or Clostridium bacteria, comprising a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition). Another example provides a method for preventing or treating infection with Escherichia, Salmonella, or Clostridium bacteria or a disease induced by Escherichia, Salmonella, or Clostridium bacteria, comprising administering a pharma- ceutical effective amount of the bacteriophage and bacteriophage stability improving composition to a subject in need of such prevention or treatment. Bacteriophages specifically kill bacteria (eg, Escherichia, Salmonella, or Clostridium bacteria) and can therefore be useful in preventing or treating diseases caused by bacteria.

[0048] The Escherichia bacteria may be Escherichia coli, E. albertii, E. fergusonii, E. hermannii, or E. vulneris, the Salmonella bacteria may be S. bongori or S. enterica, and the Clostridium bacteria may be, but are not limited to, C. botulinum, C. tetani, C. perfringens, C. hisolyticum, C. sordellii, or C. butyricum.

[0049] The infection with or the disease induced by the bacteria of the genus Escherichia may be enteritis, Crohn's disease, ulcerative colitis, bacterial dysentery, urinary tract infection, skin infection, bacteremia, or sepsis, etc., the infection with or the disease induced by the bacteria of the genus Salmonella may be Salmonella infection (Salmonellosis), Salmonella food poisoning, etc., and the infection with or the disease induced by the bacteria of the genus Clostridium may be botulinum food poisoning, Clostridium perfringens food poisoning, Clostridium difficile-induced colitis, Clostridium necrotizing enteritis, neutropenic enterocolitis, or neonatal necrotizing fore-enteritis, etc., but is not limited thereto.

[0050] The pharmaceutical composition according to an embodiment may further include a suitable carrier, excipient, or diluent that is generally used in the manufacture of pharmaceutical compositions. Specifically, the pharmaceutical composition may be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile water for injection by a conventional method.

[0051] The pharmaceutical composition according to the embodiment can be administered in a pharmaceutically effective amount, and in the present application, "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level can be determined by factors including the severity of the disease, the activity of the drug, the age, weight, health, sex, and sensitivity of the patient to the drug, the administration time, route of administration and excretion rate of the composition of the present application used, the duration of treatment, drugs used in combination with or co-used with the composition of the present application, and other factors well known in the medical art. The pharmaceutical composition according to the embodiment can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And, it can be administered singly or multiple times. It is important to administer an amount that can obtain the maximum effect at the minimum amount without side effects, taking into account all of the above factors.

[0052] The dosage of the pharmaceutical composition according to an embodiment may be, for example, 0.0001-10,000 mg / g, 0.0001-5,000 mg / g, 0.0001-1,000 mg / g, 0.0001-500 mg / g, 0.0001-100 mg / g, 0.0001-50 mg / g, or 0.0001-10 mg / g per day, but is not limited thereto. The frequency of administration of the pharmaceutical composition of the present application is not particularly limited thereto, but may be administered once a day or in divided doses for several times administration. The dosage is not intended to limit the scope of the present application in any respect.

[0053] The subject to which the pharmaceutical composition provided herein is administered may be a mammal, including humans, dogs, cats, horses, cows, pigs, goats, rabbits, mice, rats, etc., or a cell, tissue, or culture thereof isolated therefrom, and in one example, the subject may be an individual (a mammal, such as a human) in need of prevention, amelioration, and / or treatment of or having a disease as described above, or a cell, tissue, or culture thereof isolated therefrom. The disease may be infection with E. coli, Salmonella, or Clostridium bacteria, or a disease induced by E. coli, Salmonella, or Clostridium bacteria.

[0054] Another embodiment provides a method for producing a pharmaceutical composition comprising the step of mixing a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition), the pharmaceutical composition being as described above.

[0055] Another embodiment provides an antimicrobial composition comprising a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition).

[0056] In the present specification, antibacterial activity may mean activity such as inhibiting the growth of bacteria and / or killing bacteria, and antibacterial composition may mean a composition having the antibacterial activity. The antibacterial activity may be used interchangeably with antibiotic activity, and the antibacterial composition includes all forms of preparations having the ability to inhibit the growth of bacteria and / or kill bacteria, and unless otherwise specified, may be used interchangeably with antibacterial agents, antibiotics, preservatives, germicides, disinfectants, etc.

[0057] In one example, the antibacterial composition is a generic term for preparations for preventing bacterial infection, and can be used as a disinfectant for everyday life, a disinfectant for food and cooking areas and facilities, and a disinfectant for buildings such as poultry farms and livestock sheds, livestock bodies, drinking water, bedding, egg beds, transport vehicles, tableware, and various other growing items. The bacteria may be, but are not limited to, Escherichia coli, Salmonella, or Clostridium.

[0058] Another aspect provides a method for preparing an antimicrobial composition, comprising mixing a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition), the antimicrobial composition being as described above.

[0059] Another embodiment provides a method for improving bacteriophage stability, comprising the step of mixing a bacteriophage with anhydrous sodium sulfate (or a bacteriophage stability improving composition), wherein the bacteriophage or bacteriophage stability improvement is as described above.

[0060] Another embodiment provides an antibacterial method comprising applying a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition) to a target in need of antibacterial treatment. The antibacterial method may be, but is not limited to, a method for controlling bacteria (a method for controlling bacteria), a method for reducing bacteria (a method for reducing bacteria), a method for inhibiting bacteria (e.g., a method for inhibiting bacterial growth, etc.), or a method for killing bacteria.

[0061] Another embodiment provides a cleaning method comprising applying a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition) to a locus in need of cleaning.

[0062] Another aspect provides the use of anhydrous sodium sulfate for use in improving bacteriophage stability or in the manufacture of a composition for improving bacteriophage stability.

[0063] Another aspect provides a use of a bacteriophage and anhydrous sodium sulfate for use in the manufacture of a feed composition, a food composition, a pharmaceutical composition, or an antimicrobial composition.

[0064] Another aspect provides the use of the bacteriophages and bacteriophage stability improving compositions for use in the manufacture of a feed composition, a food composition, a pharmaceutical composition, or an antibacterial composition. Effect of the Invention

[0065] The present application relates to a composition for improving bacteriophage stability, comprising anhydrous sodium sulfate, which can be used to improve the storage stability of bacteriophages. [Brief description of the drawings]

[0066] [Figure 1] A bacteriophage sample (4 g of a five-type mixed bacteriophage and excipient not containing anhydrous sodium sulfate) was stored at 50±2°C and 90±5% RH, and the bacteriophage titer was measured on the 0th, 7th, and 14th days, and the results are shown in a graph. [Diagram 2] A bacteriophage sample (4 g of a five-type mixed bacteriophage and a vehicle containing 20% ​​(w / w) anhydrous sodium sulfate) was stored at 50±2°C and 90±5% RH, and the bacteriophage titer was measured on the 0th, 7th, and 14th days, and the results are shown in a graph. [Diagram 3]A bacteriophage sample (4 g of a five-type mixed bacteriophage and a vehicle containing 50% (w / w) anhydrous sodium sulfate) was stored at 50±2°C and 90±5% RH, and the bacteriophage titer was measured on the 0th, 7th, and 14th days, and the results are shown in a graph. [Figure 4] The figure shows a graph of the reduction in bacteriophage titer (Δlog(pfu / g)) measured on days 0 and 14 after storing bacteriophage samples at 50±2°C and 90±5% RH. The excipients used were an excipient containing no anhydrous sodium sulfate, an excipient containing 20% ​​(w / w) anhydrous sodium sulfate, and an excipient containing 50% (w / w) anhydrous sodium sulfate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The present invention will now be described in more detail with reference to the following examples, which are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0068] Example 1. Evaluation of storage stability of anhydrous sodium sulfate 1. Bacteriophage Sample Preparation 4g of the five-kind mixed bacteriophage was mixed with 36g of excipient to prepare two packets per sample, as shown in Table 1 below, of bacteriophage samples 1 to 3. Sample 1 used an excipient prepared by mixing wheat flour (Sazodong Awang, wheat flour (grade 2), gravity grade 2) 90% (w / w), silica (FuJian SanMing Zheng Yuan Chemical, Precipitated Sillica, ZJ-516B) 5% (w / w), and calcium carbonate (Omya Korea, calcium carbonate) 5% (w / w). Samples 2 and 3 used a mixed excipient prepared by mixing the excipient components (existing excipient) prepared by mixing wheat flour, silica, and calcium carbonate as described above with anhydrous sodium sulfate. The compositions of Samples 1 to 3 are summarized in Table 1 below.

[0069] The five-type bacteriophage mixture was prepared with the following composition: CJ7: 6×10 9 PFU / g, CJ11: 4×10 9 PFU / g, CJ23: 1×10 9 PFU / g, CJ24:5×109PFU / g, CJ22:3×10 8 PFU / g.

[0070] [Table 1]

[0071] The species names, target strains, and sources of the bacteriophages are shown in Table 2 below.

[0072] [Table 2]

[0073] 2.Storage conditions 40g of each sample was placed in an aluminum bag and heat sealed three times. Each sample was stored at 50±2℃ and 90±5% RH (relative humidity).

[0074] 3. Phage titer measurement After 7 and 14 days of sample storage, each sample was diluted with SM buffer (5.8 g / L NaCl, 2 g / L MgSO 4 7H 2 The bacteriophage solution was diluted 10-fold in 0.05 M Tris-Cl (pH 7.5) (20 g sample + 180 ml SM buffer). 50 ml of the diluted solution was centrifuged (10,000 rpm, 10 min), and the bacteriophage solution was filtered through a 0.2 μm filter.

[0075] 4. Plaque assay titration The bacteriophage solution was serially diluted to the appropriate concentration, and then 100 μl of the bacteriophage sample diluted in 5 ml of 0.7% soft agar (BD DIFCO, Cat. No. 44164) was added to 50 μl of the phage-specific indicator strain (OD 600nm =2) were mixed.

[0076] The bacteriophages were quantified by pouring and plating onto general LB (Luria-Bertani, aerobic) or BHI (Brain Heart Infusion, anaerobic) media and culturing in an incubator (aerobic; 30°C, 18 h, anaerobic; 37°C, 18 h).

[0077] 5. Experimental Results The bacteriophage quantification results for each sample after 7 and 14 days of sample storage are shown in Figures 1 to 3 and Tables 2 to 4 (Sample 1: Figure 1, Table 2; Sample 2: Figure 2, Table 3; Sample 3: Figure 3, Table 4). The difference between the titer value on day 0 (log(pfu / g)) and the titer value on day 14 (log(pfu / g)) is shown as Δlog, which was divided by the titer value on day 0 to indicate the amount of titer reduction. Figure 4 shows a graph of the titer reduction (Δlog) of the five bacteriophages (CJ7, CJ11, CJ23, CJ24, CJ22) contained in Samples 1 to 3 after 14 days of sample storage.

[0078] [Table 3]

[0079] [Table 4]

[0080] [Table 5]

[0081] Quantitative results showed that when the existing excipient (Sample 1) was used, the five bacteriophages (CJ7, CJ11, CJ23, CJ24, CJ22) in the excipient showed an average 2.5 log reduction in bacteriophage titer (average 27.2% reduction) when exposed to 50±2°C and 90±5% RH for 14 days (Figure 1, Table 3).

[0082] On the other hand, when a mixed excipient consisting of the excipient and anhydrous sodium sulfate was used, the five bacteriophage species in the excipient showed a reduction in bacteriophage titer of approximately 1.3 log (average reduction of 14.2%) (sample 2, Figure 2, Table 4) and 1.2 log (average reduction of 12.3%) (sample 3, Figure 3, Table 5), respectively.

[0083] That is, compared to the existing excipients, the use of the excipient mixture containing anhydrous sodium sulfate showed an average bacteriophage stability maintaining effect of 1.3 logs.

[0084] From the above description, it should be understood by those skilled in the art to which the present invention pertains that the present invention can be embodied in other specific forms without changing the technical idea or essential features thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below and their equivalent concepts rather than the above detailed description.

Claims

1. A composition for improving bacteriophage stability comprising anhydrous sodium sulfate.

2. 2. The composition for improving bacteriophage stability according to claim 1, which contains 5 to 50% by weight of anhydrous sodium sulfate.

3. 2. The composition for improving bacteriophage stability according to claim 1, which is for improving the stability of one or more bacteriophages selected from the group consisting of Myoviridae, Siphoviridae, and Podoviridae bacteriophages.

4. The composition for improving bacteriophage stability according to any one of claims 1 to 3, which is for use in the production of a feed, food, or antibacterial composition.

5. A feed composition comprising a bacteriophage and the bacteriophage stability improving composition according to any one of claims 1 to 3.

6. The feed composition according to claim 5, wherein a mixing ratio of the bacteriophage and the bacteriophage stability improving composition contained in the feed composition is 1:9 to 1:99 by weight (weight of the bacteriophage:weight of the bacteriophage stability improving composition).

7. The feed composition comprises: When stored for 1 to 14 days at a temperature of 45 to 60°C and a relative humidity of 80 to 100%, 6. The feed composition according to claim 5, wherein the titer of the bacteriophage contained in the feed composition is at least 10% higher than that of a feed composition not containing anhydrous sodium sulfate.

8. A food composition comprising a bacteriophage and the bacteriophage stability improving composition according to any one of claims 1 to 3.

9. The food composition according to claim 8, wherein the mixing ratio of the bacteriophage and the composition for improving bacteriophage stability contained in the food composition is 1:9 to 1:99 by weight (weight of bacteriophage:weight of the composition for improving bacteriophage stability).

10. The food composition comprises: When stored for 1 to 14 days at a temperature of 45 to 60°C and a relative humidity of 80 to 100%, 9. The food composition of claim 8, wherein the titer of the bacteriophage contained in the food composition is at least 10% higher than that of a food composition not containing anhydrous sodium sulfate.

11. An antibacterial composition comprising a bacteriophage and the bacteriophage stability improving composition according to any one of claims 1 to 3.

12. The antibacterial composition according to claim 11, wherein the mixing ratio of the bacteriophage and the bacteriophage stability improving composition contained in the antibacterial composition is 1:9 to 1:99 by weight (weight of bacteriophage:weight of the bacteriophage stability improving composition).

13. The antibacterial composition comprises: When stored for 1 to 14 days at a temperature of 45 to 60°C and a relative humidity of 80 to 100%, 12. The antibacterial composition according to claim 11, wherein the titer of the bacteriophage contained in the antibacterial composition is at least 10% higher than that of an antibacterial composition not containing anhydrous sodium sulfate.

14. A method for improving bacteriophage stability, comprising the step of mixing a bacteriophage and the composition for improving bacteriophage stability according to any one of claims 1 to 3.

15. 4. An antibacterial method comprising the step of applying a bacteriophage and a composition for improving bacteriophage stability according to any one of claims 1 to 3 to a target in need of antibacterial treatment.

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