Novel excipient compositions for maintaining the long-term distribution and 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] Mutual citation with related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0047176 filed on April 15, 2022, and all contents disclosed in the documents of the said Korean patent application are included as part of the present specification.

[0002] The present invention relates to a novel excipient composition for maintaining long-term distribution and storage stability of bacteriophages and use thereof. BACKGROUND ART

[0003] Foodborne diseases caused by food poisoning bacteria are a problem occurring worldwide and have been recognized as one of the serious public health issues. Therefore, in the food industry, it is not only important to provide consumers with delicious and nutritious foods, but also to supply safe foods free of food poisoning bacteria that may cause diseases.

[0004] Bacteriophages are a type of virus that use bacteria as host cells, and specifically bind only to bacteria, thus exhibiting no human body toxicity. Due to such characteristics, bacteriophages have been widely used in the clinical treatment of infections caused by bacteria, and in particular, are used as feed additives for preventing or suppressing livestock diseases (Korean Patent Publication No. 10-2021-0145038).

[0005] Meanwhile, during long-term storage through production and distribution of bacteriophage products for feed addition, conventionally used excipients have the problem of reduced productivity and product quality due to the decrease in bacteriophage titer. Accordingly, there is a need for research on excipients for quality maintenance during long-term distribution of bacteriophage products for feed addition. PRIOR ART DOCUMENTS PATENT DOCUMENTS

[0006] PATENT DOCUMENT 1 KR10-2021-0145038A [Overview of the project] [Problems that the invention aims to solve]

[0007] One example of this application is the provision of a bacteriophage stability improving composition comprising anhydrous sodium sulfate. The composition may be used to maintain and / or increase the stability of bacteriophages (e.g., storage stability) over a long 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, which includes the step of mixing a bacteriophage with a bacteriophage stability-improving composition containing anhydrous sodium sulfate.

[0010] Other examples include the use of anhydrous sodium sulfate for improving bacteriophage stability or for use in the manufacture of compositions for improving bacteriophage stability.

[0011] Other examples include applications of bacteriophages and anhydrous sodium sulfate for use in the manufacture of feed compositions, food compositions, pharmaceutical compositions, or antimicrobial compositions.

[0012] Other examples include applications for bacteriophages and bacteriophage stability-improving compositions for use in the manufacture of feed compositions, food compositions, pharmaceutical compositions, or antimicrobial compositions. [Means for solving the problem]

[0013] One embodiment provides a bacteriophage stability improving composition containing anhydrous sodium sulfate.

[0014] Anhydrous sodium sulfate (Na2SO4, 142.04 g / mol, CAS Number: 7757-82-6) is a sodium sulfate and is a colorless crystalline substance. This anhydrous sodium sulfate can combine with 10 external water molecules to form sodium sulfate (Na2SO4·10H2O; 322.20 g / mol), and can therefore perform a hygroscopic role in environments with moisture.

[0015] The bacteriophage stability improving composition may additionally contain excipient components or can be used as excipients.

[0016] The aforementioned excipients can refer to substances added to make feed, food, or pharmaceuticals easier to eat or to give them a specific shape. Types of excipients include binders that provide binding force to powdered raw materials to facilitate molding, disintegrants that promote disintegration, lubricants added to facilitate the compression of granular products, improve fluidity, and prevent stickiness, and adsorbents that absorb liquid raw materials to make them into tablets. Examples of commonly used excipient components include, but are not limited to, one or more selected from the group consisting of hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), lactose (hydrated, anhydrous, monohydrate, spray-dried form, etc.), microcrystalline cellulose, titanium dioxide, silicon dioxide (colloidal, etc.), magnesium stearate, polyethylene glycol, croscarmellose sodium, alpha-starch (starch pregelatinized), sodium starch glycolate, crospovidone, talc, wheat flour (grain flour), calcium carbonate, silica, etc. The bacteriophage stability improving composition provided in this application may additionally contain one or more of the above excipient components in addition to anhydrous sodium sulfate.

[0017] The proportion of anhydrous sodium sulfate contained in the aforementioned bacteriophage stability improving composition is, on a weight basis, 1-50% (w / w), 1-45% (w / w), 1-40% (w / w), 1-35% (w / w), 1-30% (w / w), 5-50% (w / w), 5-45% (w / w), 5-40% (w / w), 5-35% (w / w), 5-30% (w / w), 10-50% (w / w), 10-45% (w / w), 10-40% (w / w), 10-35% (w / w), 10-30% (w / w), 15-50% (w / w), 15-45% ( It may also be, but is not limited to, w / w, 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] In this specification, the term “bacteriophage stability improvement” may mean maintaining, improving, or enhancing the storage stability of bacteriophages over a long period of time. That is, the bacteriophage stability improvement effect may mean the effect of maintaining the potency of bacteriophages or reducing the rate of potency loss.

[0019] Bacteriophages are a general term for viruses that use bacteria and archaea as hosts, and they can specifically infect bacteria. Therefore, their antibiotic effects are used in products such as animal feed (including feed additives), food, pharmaceuticals, and disinfectants. However, during storage in the production or distribution of these products, the potency of the bacteriophages decreases, leading to problems with reduced productivity and quality. In other words, the effect of improving the stability of bacteriophages can be said to mean maintaining the potency of bacteriophages or reducing the amount of decrease in bacteriophage potency during storage of products containing such bacteriophages.

[0020] In one example, when the composition for improving bacteriophage stability is applied to a bacteriophage (e.g., by mixing or the like), the bacteriophage is subjected to a temperature condition 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 under a relative humidity condition of 80 to 100%, 80 to 99%, 80 to 98%, 80 to 97%, 80 to 96%, 80 to 95%, 80 to 92%, 80 to 90%, 80 to 88%, 80 to 85%, 85 to 100%, 85 to 99%, 85 to 98%, 85 to 97%, 85 to 96%, 85 to 95%, 85 to 92%, 85 to 90%, 85 to 88%, 90 to 100%, 90 to 99%, 90 to 98%, 90 to 97%, 90 to 96%, 90 to 95%, 90 to 92%, 95 to 100%, 95 to 99%, 95 to 98%, 95 to 97%, or 95 to 96%, Compared with the bacteriophage titer measured on day 0, the bacteriophage titer measured after 1 to 7 days, 1 to 14 days, 7 to 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 decreases 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, for example 1.4 log(pfu / g), or may decrease 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 composition for improving bacteriophage stability is applied to a bacteriophage (e.g., by mixing or the like), as compared with a control group (e.g., a case where anhydrous sodium sulfate is not applied to the bacteriophage), said bacteriophage under a temperature condition 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 under a relative humidity condition of 80 to 100%, 80 to 99%, 80 to 98%, 80 to 97%, 80 to 96%, 80 to 95%, 80 to 92%, 80 to 90%, 80 to 88%, 80 to 85%, 85 to 100%, 85 to 99%, 85 to 98%, 85 to 97%, 85 to 96%, 85 to 95%, 85 to 92%, 85 to 90%, 85 to 88%, 90 to 100%, 90 to 99%, 90 to 98%, 90 to 97%, 90 to 96%, 90 to 95%, 90 to 92%, 95 to 100%, 95 to 99%, 95 to 98%, 95 to 97%, or 95 to 96%, compared with the titer of the bacteriophage measured on day 0, the reduction amount of the bacteriophage titer (log(pfu / g)) measured after 1 day to 7 days, 1 day to 14 days, 7 days to 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 0.5 to 3, 0.5 to 2.5, 0.5 to 2, 0.5 to 1.5, 0.6 to 3, 0.6 to 2.5, 0.6 to 2, 0.6 to 1.5, 0.7 to 3, 0.7 to 2.5, 0.7 to 2, 0.7 to 1.5, 0.8 to 3, 0.8 to 2.5, 0.8 to 2, 0.8 to 1.5, 0.9 to 3, 0.9 to 2.5, 0.9 to 2, 0.9 to 1.5, 1 to 3, 1 to 2.5, 1 to 2, 1 to 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, for example, further lower by 1.1, or it may be further lower by 5 to 20%p, 5 to 15%p, 6 to 20%p, 6 to 15%p, 7 to 20%p, 7 to 15%p, 8 to 20%p, 8 to 15%p, 9 to 20%p, 9 to 15%p, 10 to 20%p, 10 to 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 11%p or more, for example, 11%p.

[0022] In another example, when the bacteriophage is treated with the bacteriophage stability-improving composition (e.g., by mixing), 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 under humidity conditions 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, The potency of the bacteriophage 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 higher.

[0023] When the aforementioned bacteriophage stability improving composition is applied to a bacteriophage, it has the advantage of allowing for stable storage of the bacteriophage, even when stored for a long period of time in extreme environments such as high temperature and high humidity, as the decrease in the potency of the bacteriophage is low.

[0024] The aforementioned bacteriophage stability improving composition may exhibit superior bacteriophage stability improving effects compared to compositions that do not contain anhydrous sodium sulfate, due to the inclusion of anhydrous sodium sulfate.

[0025] The bacteriophage stability improving composition may be used in the manufacture of feed compositions, food compositions, pharmaceutical compositions, or antimicrobial compositions.

[0026] Other embodiments provide compositions comprising a bacteriophage and anhydrous sodium sulfate, for example, a feed composition, a food composition, a pharmaceutical composition, or an antimicrobial composition. Alternatively, they provide compositions comprising a bacteriophage and a composition for improving the stability of the bacteriophage, for example, a feed composition, a food composition, a pharmaceutical composition, or an antimicrobial composition.

[0027] The aforementioned bacteriophages can be used without any particular restrictions on the type or number of bacteriophages, and one or more bacteriophages (for example, a combination of two or three species) belonging to the families Myoviridae, Siphoviridae, and Podoviridae can be selected and used.

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

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

[0030] In the aforementioned compositions (for example, feed compositions, food compositions, pharmaceutical compositions, or antimicrobial compositions), the ratio of bacteriophages to bacteriophage stability improving compositions is, on a weight basis, 90-99.9% (w / w), 90-99.5% (w / w), 90-99% (w / w), 90-98% (w / w), and 90-97%. (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 percentages may be 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), but are not limited to these.

[0031] In the aforementioned compositions (for example, feed compositions, food compositions, pharmaceutical compositions, or antimicrobial compositions), the mixing ratio of the bacteriophage to the bacteriophage stability-improving composition may be 1:8~1:999, 1:8~5:995, 1:8~1:99, 1:8~2:98, 1:8~3:97, 1:88~4:96, 1:8~5:95, 1:9~1:999, 1:9~5:995, 1:9~1:99, 1:9~2:98, 1:9~3:97, 1:9~4:96, 1:9~5:95, etc., based on weight (where the ratio is the weight of the bacteriophage to the weight of the bacteriophage stability-improving composition), but is not limited to these ratios.

[0032] In the aforementioned compositions, such as feed compositions, food compositions, pharmaceutical compositions, or antimicrobial compositions, the ratio of bacteriophages to anhydrous sodium sulfate in the bacteriophage stability improving composition is, by weight, 4-45% (w / w), 4-40% (w / w), 4-30% (w / w), 4-20% (w / w), 4-18% (w / w), 4-15% (w / w), 4-10% (w / w), 4-5% (w / w), 4-4.5% (w / w), 4.5-45% (w / w), 4.5-40% (w / w), 4.5-30% (w / w), 4.5-20% (w / w), and 4.5-18%. (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), etc. are also acceptable, but are not limited to these.

[0033] For example, a composition containing the bacteriophage and anhydrous sodium sulfate or a bacteriophage stability improving composition, such as a feed composition, food composition, pharmaceutical composition, or antimicrobial composition, is: 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 under relative humidity conditions 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%, Compared to the bacteriophage titer measured on day 0, if the bacteriophage titer measured on days 1-7, 1-14, 7-14, 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 decreases 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, for example, by 1.4 log(pfu / g), or It may decrease to 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, or 15% or less.

[0034] For example, a composition containing the bacteriophage and anhydrous sodium sulfate, or a bacteriophage stability improving composition, such as a feed composition, food composition, pharmaceutical composition, or antimicrobial composition, is superior to a composition that does not contain 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 under relative humidity conditions 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%, Compared to the bacteriophage titer measured on day 0, the decrease in bacteriophage titer (log(pfu / g)) measured on days 1-7, 1-14, 7-14, 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 was 0.5-3, 0.5-2.5, 0.5-2, 0.5-1.5, 0.6-3, and 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 higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.9 or higher, 1.0 or higher, or 1.1 or higher, for example, 1.1 is even lower, or 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 11%p or more, which could be as low as, for example, 11%p.

[0035] For example, a composition containing the bacteriophage and anhydrous sodium sulfate or a bacteriophage stability improving composition, such as a feed composition, food composition, pharmaceutical composition, or antimicrobial composition, is superior to a composition that does not contain 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 under humidity conditions 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, The potency of the bacteriophage 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 higher.

[0036] The aforementioned composition containing the bacteriophage and anhydrous sodium sulfate or a bacteriophage stability improving composition (for example, a feed composition, a food composition, a pharmaceutical composition, or an antimicrobial composition) has the advantage of allowing for stable storage of the bacteriophage with minimal decrease in potency even when stored for long periods in extreme environments such as high temperature and high humidity.

[0037] Therefore, compositions containing the bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition) can be manufactured in a variety of forms to suit specific purposes in the household, industrial, medical, and environmental fields, and can be manufactured in the form of, for example, feed compositions (including feed additive compositions), food compositions, pharmaceutical compositions, or antimicrobial compositions, but are 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” can mean any natural or artificial prescribed diet, single serving, or components of such single serving that an animal eats, ingests, and digests. An example of a feed composition may further include concentrated feed and / or specialty feed. The concentrated feed may also include seed fruits and grains such as wheat, oats, and corn; brans such as rice bran, gluten, and wheat bran, which are by-products obtained from refining grains; residues such as oilseed meal, which is a by-product obtained from extracting oil from beans, rapeseed, sesame, flaxseed, and coconut palm; residual starches, which are the main components of starch residue left after removing starch from sweet potatoes, potatoes, etc.; fish soluble, which is concentrated fish meal, fish meal, and fresh liquid obtained from fish; meat meal, blood meal, feather meal, skim milk powder, dried whey, which is the residue left when making cheese from milk or when making casein from skim milk; yeast, chlorella, and / or seaweed.

[0040] An example of a feed composition can mean feed in the form that is ultimately ingested by an animal, a dietary supplement added to the feed, and / or a feed additive. The dietary supplement can mean, for example, a formulation-containing composition that provides an animal with a therapeutic or digestive agent, which is not normally a source of caloric intake, i.e., an energy source, but is ingested in addition to a normal animal feed. The feed additive can mean a substance added to feed for various purposes, such as nutrient supplementation and prevention of weight loss, improvement of the digestibility of fiber in the feed, improvement of milk quality, prevention of reproductive disorders and improvement of conception rates, and prevention of summer heat stress. The feed to which the feed additive can be added can be selected from, but is not limited to, commercially available feed, or from the group consisting of grains, root vegetables, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meal, grain by-products, proteins, inorganic substances, mineral substances, single-cell proteins, zooplankton, leftover food and drink, etc.

[0041] For example, the feed composition may be a feed additive, and when the feed additive according to this example is added to feed (e.g., compound feed and / or single-ingredient feed that is ultimately ingested by animals), the amount is 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, 0.001~1%, 0.001~0.5%, 0.001~0.1%, 0.001~0.05%, 0. It can be added by weight in amounts of 0.01-0.01%, 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 blended with feed ingredients, supplemental feeds, additives, and / or other types of additives other than active ingredients, as an example.

[0042] By mixing the aforementioned bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition) with feed and supplying it, the 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). The feed composition is as described above.

[0044] Another embodiment provides a food composition comprising a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition). Since the bacteriophage specifically kills bacteria, it can be used in the treatment of processed foods or unprocessed foods or food ingredients to prevent bacterial infection. An example food composition may include meats, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, beverages, alcoholic beverages, vitamin complexes, health functional foods and health foods, and encompasses all foods in the ordinary sense.

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

[0046] Another embodiment 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). The food composition is as described above.

[0047] Another embodiment provides a pharmaceutical composition for the prevention or treatment of infection by or diseases induced by bacteria of the genera Escherichia, Salmonella, or Clostridium, comprising a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition). Another example provides a method for the prevention or treatment of infection by or diseases induced by bacteria of the genera Escherichia, Salmonella, or Clostridium, comprising the step of administering a pharmaceutically effective amount of the bacteriophage and the bacteriophage stability improving composition to a subject in need of prevention or treatment of infection by or diseases induced by bacteria of the genera Escherichia, Salmonella, or Clostridium, comprising the step of administering to a subject in need of prevention or treatment of infection by or diseases induced by bacteria of the genera Escherichia, Salmonella, or Clostridium, comprising the steps of administering a pharmaceutically effective amount of the bacteriophage and the bacteriophage stability improving composition to a subject in need of prevention or treatment of infection by or diseases induced by bacteria of the genera Escherichia, Salmonella, or Clostridium. Bacteriophages specifically kill bacteria (e.g., bacteria of the genera Escherichia, Salmonella, or Clostridium), and can therefore be usefully used to prevent or treat diseases induced by these bacteria.

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

[0049] The infection with or disease induced by the Escherichia bacteria may include enteritis, Crohn's disease, ulcerative cochlearitis, bacterial dysentery, urinary tract infection, skin infection, bacteremia, or sepsis; the infection with or disease induced by the Salmonella bacteria may include salmonellosis, salmonella food poisoning, or other similar conditions; and the infection with or disease induced by the Clostridium bacteria may include botulism, Clostridium perfringens food poisoning, Clostridium difficile-induced colitis, Clostridium necrotizing enterocolitis, neutropenic enterocolitis, or neonatal necrotizing foreglutitis, but is not limited to these.

[0050] An example of a pharmaceutical composition may additionally include suitable carriers, excipients, or diluents commonly used in the manufacture of pharmaceutical compositions. Specifically, each of these pharmaceutical compositions can be prepared by conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile water for injection.

[0051] An example of a pharmaceutical composition can be administered in a pharmacokinetically effective amount, where, in this application, “pharmacokinetically effective amount” means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level can be determined by factors including the severity of the disease, the activity of the drug, the patient’s age, weight, health, sex, the patient’s sensitivity to the drug, the time of administration, route of administration and elimination ratio of the composition used, the duration of treatment, and other factors well known in the medical field, including drugs compounded or used concurrently with the composition used. An example of a pharmaceutical composition can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or concurrently with conventional therapeutic agents. It can also be administered single or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, taking all of the aforementioned factors into consideration.

[0052] For example, the dosage of a pharmaceutical composition can be administered to mammals at doses of 0.0001 to 10,000 mg / g, 0.0001 to 5,000 mg / g, 0.0001 to 1,000 mg / g, 0.0001 to 500 mg / g, 0.0001 to 100 mg / g, 0.0001 to 50 mg / g, or 0.0001 to 10 mg / g per day, but is not limited thereto. The frequency of administration of the pharmaceutical composition of this application is not particularly limited, but can be administered once a day or in divided doses several times a day. The aforementioned dosages do not limit the scope of this application in any respect.

[0053] The subjects of the pharmaceutical compositions provided herein may be mammals, including humans, dogs, cats, horses, cattle, pigs, goats, rabbits, mice, rats, etc., or cells, tissues, or cultures thereof isolated therefrom, for example, the subjects may be individuals (such as humans or other mammals) or cells, tissues, or cultures thereof isolated therefrom, who require the prevention, improvement, and / or treatment of the aforementioned diseases, or who have the aforementioned diseases. The aforementioned diseases may be infections with Escherichia coli, Salmonella, or Clostridium bacteria, or diseases induced by Escherichia 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 is 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 this specification, antimicrobial activity can mean activity that inhibits the growth of bacteria and / or kills them, and antimicrobial composition can mean a composition having said antimicrobial activity. The said antimicrobial activity can be used interchangeably with anti-vitality and so on, and the said antimicrobial composition encompasses all forms of formulations having the ability to inhibit and / or kill bacteria, and unless otherwise specified, can be used interchangeably with antimicrobial agents, antibiotics, preservatives, fungicides, disinfectants, and so on.

[0057] For example, the aforementioned antibacterial composition is a general term for preparations for preventing bacterial infections and can be used as a general household disinfectant, a disinfectant for food and cooking areas and equipment, a disinfectant for buildings such as poultry farms and livestock barns, livestock bodies, drinking water, bedding straw, egg trays, transport vehicles, tableware, and various other breeding supplies. The bacteria may be, but are not limited to, Escherichia coli, Salmonella, or Clostridium.

[0058] Another embodiment provides a method for producing an antimicrobial composition, comprising the step of mixing a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition). The antimicrobial composition is 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). The bacteriophage or bacteriophage stability improvement is as described above.

[0060] Other embodiments provide an antimicrobial method comprising the step of applying a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition) to an object requiring antimicrobial action. The antimicrobial method may be, but is not limited to, a method for controlling bacteria, a method for reducing bacteria, a method for inhibiting bacteria (e.g., a method for inhibiting bacterial growth), or a method for killing bacteria.

[0061] Another embodiment provides a cleaning method comprising the step of applying a bacteriophage and anhydrous sodium sulfate (or a bacteriophage stability improving composition) to an object that needs to be cleaned.

[0062] Other embodiments provide uses for anhydrous sodium sulfate for improving bacteriophage stability or for use in the manufacture of compositions for improving bacteriophage stability.

[0063] Other embodiments provide uses for bacteriophages and anhydrous sodium sulfate for use in the manufacture of feed compositions, food compositions, pharmaceutical compositions, or antimicrobial compositions.

[0064] Other embodiments provide uses for bacteriophages and bacteriophage stability improving compositions for use in the manufacture of feed compositions, food compositions, pharmaceutical compositions, or antimicrobial compositions. [Effects of the Invention]

[0065] This application describes how the storage stability of bacteriophages can be improved by using a bacteriophage stability improving composition containing anhydrous sodium sulfate. [Brief explanation of the drawing]

[0066] [Figure 1] The bacteriophage samples (4g of a mixed bacteriophage of 5 types and an excipient without anhydrous sodium sulfate) were stored at 50±2℃ and 90±5%RH, and the titers of the bacteriophages were measured on day 0, day 7, and day 14, and the results are shown as a graph. [Figure 2] This graph shows the results of measuring the potency of bacteriophages on days 0, 7, and 14 after storing a bacteriophage sample (4 g of a mixed bacteriophage of 5 types and an excipient containing 20% ​​(w / w) anhydrous sodium sulfate) at 50±2°C and 90±5% RH. [Figure 3]This graph shows the results of measuring the potency of bacteriophages on days 0, 7, and 14 after storing a bacteriophage sample (4 g of a mixed bacteriophage of 5 types and an excipient containing 50% (w / w) anhydrous sodium sulfate) at 50±2°C and 90±5% RH. [Figure 4] This graph shows the decrease in bacteriophage titer (Δlog(pfu / g)) measured on day 0 and day 14 after storing bacteriophage samples at 50±2°C and 90±5% RH. Excipients used included an excipient without anhydrous sodium sulfate, an excipient containing 20% ​​(w / w) anhydrous sodium sulfate, and an excipient containing 50% (w / w) anhydrous sodium sulfate. [Modes for carrying out the invention]

[0067] The present invention will be described more specifically below with reference to the following embodiments. However, these are merely illustrative examples of the present invention, and the scope of the present invention is not limited by these embodiments. [Examples]

[0068] Example 1. Evaluation of storage stability of anhydrous sodium sulfate 1. Bacteriophage sample preparation Four g of a five-component mixed bacteriophage was mixed with 36 g of excipients to prepare two packets each of bacteriophage samples 1 to 3, as shown in Table 1 below. Sample 1 used an excipient prepared by mixing 90% (w / w) wheat flour (sazodone awang, wheat flour (grade 2), gravity grade 2), 5% (w / w) silica (FuJian SanMing Zheng Yuan Chemical, Precipitated Silica, ZJ-516B), and 5% (w / w) calcium carbonate (Omya Korea, calcium carbonate). Samples 2 and 3 used a mixed excipient prepared by mixing the excipient components (existing excipients) 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-component bacteriophage was prepared with the following composition: CJ7: 6×10 9 PFU / g, CJ11: 4×10 9 PFU / g, CJ23: 1 x 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 aforementioned bacteriophages are as shown in Table 2 below.

[0072] [Table 2]

[0073] 2.Storage conditions Each 40g 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 sample storage, each sample was diluted 10-fold in SM buffer (5.8 g / L NaCl, 2 g / L MgSO4·7H2O, 0.05 M Tris-Cl (pH 7.5)) 7 and 14 days later (20 g sample + 180 ml SM buffer). 50 ml of the diluted solution was centrifuged (10,000 rpm, 10 minutes), and the bacteriophage solution was filtered through a 0.2 μm filter.

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

[0076] After pouring and plating the samples into general LB (Luria-Bertani, aerobic) or BHI (Brain Heart Infusion, anaerobic) medium, the samples were incubated in an incubator (aerobic: 30°C, 18h; anaerobic: 37°C, 18h). The number of plaques was then measured to quantify the bacteriophages.

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

[0078] [Table 3]

[0079] [Table 4]

[0080] [Table 5]

[0081] Quantitative results showed that when using the existing excipient (Sample 1), five bacteriophages (CJ7, CJ11, CJ23, CJ24, CJ22) within the excipient showed an average decrease in bacteriophage titer of 2.5 logs (average decrease of 27.2%) 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 containing the excipient and anhydrous sodium sulfate was used, the five bacteriophages in the excipient showed a decrease in bacteriophage titer of approximately 1.3 logs (average decrease of 14.2%) (Sample 2, Figure 2, Table 4) and 1.2 logs (average decrease of 12.3%) (Sample 3, Figure 3, Table 5), respectively.

[0083] Specifically, compared to existing excipients, the mixed excipient containing anhydrous sodium sulfate showed an average bacteriophage stability maintenance effect of 1.3 log.

[0084] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In connection with this, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts described below rather than from the above detailed description.

Claims

1. A bacteriophage stability improving composition comprising 5 to 50% by weight of anhydrous sodium sulfate, The aforementioned composition is a powdered solid phase composition for improving bacteriophage stability.

2. The bacteriophage stability improving composition according to claim 1, comprising 15 to 50% by weight of anhydrous sodium sulfate.

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

4. A bacteriophage stability improving composition according to any one of claims 1 to 3, for use in the manufacture of feed, food, or antimicrobial compositions.

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

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

7. The aforementioned feed composition is When stored for 1 to 14 days at a temperature of 45-60°C and a relative humidity of 80-100%, The feed composition according to claim 5, wherein the potency of the bacteriophage contained in the feed composition is 10% or more higher than that of a feed composition that does not contain anhydrous sodium sulfate.

8. A food composition comprising a bacteriophage and a 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 bacteriophage stability improving composition contained in the food composition is 1:9 to 1:99 by weight (wherein the weight of the bacteriophage is equal to the weight of the bacteriophage stability improving composition).

10. The aforementioned food composition, When stored for 1 to 14 days at a temperature of 45-60°C and a relative humidity of 80-100%, The food composition according to claim 8, wherein the potency of the bacteriophage contained in the food composition is 10% or more higher than that of a food composition that does not contain anhydrous sodium sulfate.

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

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

13. The aforementioned antibacterial composition is When stored for 1 to 14 days at a temperature of 45-60°C and a relative humidity of 80-100%, The antimicrobial composition according to claim 11, wherein the potency of the bacteriophage contained in the antimicrobial composition is 10% or more higher than that of the antimicrobial composition that does not contain anhydrous sodium sulfate.

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

15. The step includes applying a bacteriophage and a bacteriophage stability improving composition according to any one of claims 1 to 3 to an object requiring antimicrobial action. An antimicrobial method in which the target is an animal other than a human.

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