AU2020100856A4 - Method for preserving freeze-dried powder capsules of phages - Google Patents
Method for preserving freeze-dried powder capsules of phages Download PDFInfo
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- AU2020100856A4 AU2020100856A4 AU2020100856A AU2020100856A AU2020100856A4 AU 2020100856 A4 AU2020100856 A4 AU 2020100856A4 AU 2020100856 A AU2020100856 A AU 2020100856A AU 2020100856 A AU2020100856 A AU 2020100856A AU 2020100856 A4 AU2020100856 A4 AU 2020100856A4
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
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Abstract
Austracy
The present invention discloses a method for preserving freeze-dried powder
capsules of phages. The method includes the following steps: S, performing
primary treatment on phages, selecting host bacteria with phages, inoculating the
phages into a fluid medium, performing shake culture for about 10 hours, and
collecting a phage-containing supernatant; S2, preparing a phage protectant, mixing
the phages and the protectant together, and fully mixng the phages and the protectant
together with stirring by a stirring rod; S3, preparing freeze-dried powder, placing a
mixed solution prepared in the S2 in a freezing chamber for freezing, performing
primary drying under a vacuum condition, then adding 10-20 parts of glycerin into
phage fluid subjected to primary drying, heating, and performing secondary drying
so as to obtain phage freeze-dried powder; and S4, preparing capsules, filling the
freeze-dried powder in the capsules in a sterile environment by using an
encapsulating machine, sealing the capsules, and preserving the sealed capsules in
the shade. A preservation effect of the present invention is excellent.
1
Description
Austracy
The present invention discloses a method for preserving freeze-dried powder capsules of phages. The method includes the following steps: S, performing primary treatment on phages, selecting host bacteria with phages, inoculating the phages into a fluid medium, performing shake culture for about 10 hours, and collecting a phage-containing supernatant; S2, preparing a phage protectant, mixing the phages and the protectant together, and fully mixng the phages and the protectant together with stirring by a stirring rod; S3, preparing freeze-dried powder, placing a mixed solution prepared in the S2 in a freezing chamber for freezing, performing primary drying under a vacuum condition, then adding 10-20 parts of glycerin into phage fluid subjected to primary drying, heating, and performing secondary drying so as to obtain phage freeze-dried powder; and S4, preparing capsules, filling the freeze-dried powder in the capsules in a sterile environment by using an encapsulating machine, sealing the capsules, and preserving the sealed capsules in the shade. A preservation effect of the present invention is excellent.
Description
Technical Field The present invention relates to the technical field of capsule preservation, and particularly relates to a method for preserving freeze-dried powder capsules of phages. Background Phages are viruses invading bacteria, and are also genetic materials endowing host bacteria with biological characters. The phages must be parasitic in viable bacteria, have strict host specificity, and depend on molecular structures and complementarity on phage adsorption organs and recipient bacterium surface receptors. The phages are populations that are the most common and most widespread in the viruses. A traditional preservation method is as follows: the phages are prepared into freeze-dried powder, and the freeze-dried powder is preserved in a freezing chamber at 4°C or preserved in liquid nitrogen. However, a preservation effect of such a method is poor. For example, a reference document with an application publication number of CN201610415919.6 discloses a method for preserving freeze-dried powder capsules of phages as follows: a protectant and phage fluid are mixed together and preserved at a temperature of 4°C. The preservation measure is still not perfect enough, and the preservation effect is poor.
Summary A technical problem to be solved in the present invention is to provide a method for preserving freeze-dried powder capsules of phages with an excellent preservation effect.
Description
To solve the above technical problem, the present invention provides the following technical solutions: the method for preserving freeze-dried powder capsules of phages includes the following steps: S1, performing primary treatment on phages, selecting host bacteria with phages, inoculating the phages into a fluid medium, performing shake culture for about 10 hours, and collecting a phage-containing supernatant; S2, preparing a phage protectant, selecting 15-20 parts of skim milk powder, 1-2 parts of glycerin, 1-5 parts of glucose, 0.6-1.2 parts of calcium chloride, 0.3-0.8 part of sodium sulfate, 0.5-1.2 parts of mannitol, 0.03-0.2 part of dipotassium phosphate, 0.05-0.3 part of sodium dihydrogen phosphate and 0.03-0.18 part of aluminum hydroxide glue, mixing the phages and the above mixed solution together, and enabling the phages and the solution to be fully mixed together with stirring of a stirring rod; S3, preparing freeze-dried powder, placing a mixed solution prepared in the S2 in a freezing chamber for freezing, performing primary drying under a vacuum condition, then adding 10-20 parts of glycerin into phage fluid subjected to primary drying, heating, and performing secondary drying so as to obtain phage freeze-dried powder; and S4, preparing capsules, filling the freeze-dried powder in the capsules in a sterile environment by using an encapsulating machine, sealing the capsules, and preserving the sealed capsules in the shade. Preferably, in the S2, the following components are selected: 15 parts of skim milk powder, 1.3 parts of glycerin, 4 parts of glucose, 0.6 part of calcium chloride, 0.36 part of sodium sulfate, 1 part of mannitol, 0.1 part of dipotassium phosphate, 0.18 part of sodium dihydrogen phosphate and 0.18 part of aluminum hydroxide glue. A ratio of the components of the protectant is changed, and a protection effect of the protectant is improved. Preferably, in the S2, the following components are selected: 17 parts of skim milk powder, 1.6 parts of glycerin, 4 parts of glucose, 1 part of calcium chloride,
Description
0.5 part of sodium sulfate, 1 part of mannitol, 0.03 part of dipotassium phosphate, 0.25 part of sodium dihydrogen phosphate and 0.06 part of aluminum hydroxide glue. Preferably, in the S2, the mixed solution is placed at a room temperature of °C and stirred at a rate of 2r/second by a stirrer, and stirring lasts for 15 minutes. Mixing uniformity is improved, and preservation life is prolonged. Preferably, in the S3, 10 parts of glycerin are added. Preferably, in the S3, 20 parts of glycerin are added. In the S3, when the 20 parts of glycerin are added, preservation life is obviously prolonged compared with preservation life when 10 parts of glycerin are added. Preferably, in the S3, 18 parts of glycerin are added. In the S3, when the 18 parts of glycerin are added, preservation life is slightly prolonged compared with preservation life when 10 parts of glycerin are added, but is shorter than preservation life when 20 parts of glycerin are added. Preferably, in the S3, a preliminary freezing temperature is maintained at 4°C; within 20-30 minutes after preservation, the temperature is continuously decreased and decreased to minus 30°C, and temperature drop lasts for 40 minutes. The temperature is prevented from directly decreasing from the room temperature to minus 30°C so as not to destruct host bacteria. Preferably, in the S3, the primary drying is performed in a vacuum environment at minus 30°C and under a pressure of 10-15 Pa. A drying effect is improved. Preferably, in the S4, before the capsules are filled, a 300-mesh screen is used for screening the freeze-dried powder, thereby ensuring fineness of the freeze-dried powder. Preferably, in the S4, the sealed capsules are preserved in an environment of 4°C. When the capsules are preserved at a low temperature, preservation life of the phage freeze-dried powder is prolonged.
Description
Compared with the prior art, the present invention has beneficial effects as follows: After the primary drying, lots of glycerin is added into the mixed solution, and the prepared capsules are placed in the environment of 4°C, so that the preservation life of the phage freeze-dried powder is prolonged, and the preservation effect is excellent.
Detailed Description Terms "first" and "second" are used for the purposes of illustration only, but cannot be understood to indicate or imply relative importance or implicitly indicate the quantity of indicated technical feature. Thus, features defined with "first" and "second" may include one or more features clearly or implicitly. In illustration of the present application, a term "multiple" refers to two or more than two, unless otherwise specified. Embodiment 1 The present embodiment discloses a method for preserving freeze-dried powder capsules of phages, including the following steps: S1, primary treatment was performed on phages, host bacteria with phages were selected, the phages were inoculated into a fluid medium, shake culture was performed for about 10 hours, and a phage-containing supernatant was collected; S2, a phage protectant was prepared, 15 parts of skim milk powder, 1.3 parts of glycerin, 4 parts of glucose, 0.6 part of calcium chloride, 0.36 part of sodium sulfate, 1 part of mannitol, 0.1 part of dipotassium phosphate, 0.18 part of sodium dihydrogen phosphate and 0.18 part of aluminum hydroxide glue were selected, the phages and the above mixed solution were mixed together, and the phages and the solution were fully mixed together with stirring by a stirring rod; S3, freeze-dried powder was prepared, a mixed solution prepared in the S2 was placed in a freezing chamber for freezing, primary drying was performed under a vacuum condition, then 10 parts of glycerin were added into phage fluid
Description
subjected to primary drying, heating was performed, and secondary drying was performed so as to obtain phage freeze-dried powder; and S4, capsules were prepared, the freeze-dried powder was filled in the capsules in a sterile environment by using an encapsulating machine, the capsules were sealed, and the sealed capsules were preserved in an environment of 4°C. In the S2, the mixed solution is placed at a room temperature of 20°C and stirred at a rate of 2r/second by a stirrer, and stirring lasts for 15 minutes. Mixing uniformity is improved, and preservation life is prolonged. In the S3, a preliminary freezing temperature is maintained at 4°C; within -30 minutes after preservation, the temperature is continuously decreased and decreased to minus 30°C, and temperature drop lasts for 40 minutes. The temperature is prevented from directly decreasing from the room temperature to minus 30°C so as not to destruct host bacteria. In the S3, the primary drying is performed in a vacuum environment at minus °C and under a pressure of 10-15 Pa. A drying effect is improved. In the S4, before the capsules are filled, a 300-mesh screen is used for screening the freeze-dried powder, thereby ensuring fineness of the freeze-dried powder. After the primary drying, lots of glycerin is added to prolong the preservation life of the phage freeze-dried powder, and the preservation effect is excellent. Embodiment 2 S1, primary treatment was performed on phages, host bacteria with phages were selected, the phages were inoculated into a fluid medium, shake culture was performed for about 10 hours, and a phage-containing supernatant was collected; S2, a phage protectant was prepared, 17 parts of skim milk powder, 1.6 parts of glycerin, 4 parts of glucose, 1 part of calcium chloride, 0.5 part of sodium sulfate, 1 part of mannitol, 0.03 part of dipotassium phosphate, 0.25 part of sodium dihydrogen phosphate and 0.06 part of aluminum hydroxide glue were selected,
Description
the phages and the above mixed solution were mixed together, and the phages and the solution were fully mixed together with stirring by a stirring rod; S3, freeze-dried powder was prepared, a mixed solution prepared in the S2 was placed in a freezing chamber for freezing, primary drying was performed under a vacuum condition, then 10-20 parts of glycerin were added into phage fluid subjected to primary drying, heating was performed, and secondary drying was performed so as to obtain phage freeze-dried powder; and S4, capsules were prepared, the freeze-dried powder was filled in the capsules in a sterile environment by using an encapsulating machine, the capsules were sealed, and the sealed capsules were preserved in an environment of 4°C. A ratio of the components of the protectant is changed, and a protection effect of the protectant is improved. Embodiment 3 S, primary treatment was performed on phages, host bacteria with phages were selected, the phages were inoculated into a fluid medium, shake culture was performed for about 10 hours, and a phage-containing supernatant was collected; S2, a phage protectant was prepared, 15 parts of skim milk powder, 1.3 parts of glycerin, 4 parts of glucose, 0.6 part of calcium chloride, 0.36 part of sodium sulfate, 1 part of mannitol, 0.1 part of dipotassium phosphate, 0.18 part of sodium dihydrogen phosphate and 0.18 part of aluminum hydroxide glue were selected, the phages and the above mixed solution were mixed together, and the phages and the solution were fully mixed together with stirring by a stirring rod; S3, freeze-dried powder was prepared, a mixed solution prepared in the S2 was placed in a freezing chamber for freezing, primary drying was performed under a vacuum condition, then 20 parts of glycerin were added into phage fluid subjected to primary drying, heating was performed, and secondary drying was performed so as to obtain phage freeze-dried powder; and
Description
S4, capsules were prepared, the freeze-dried powder was filled in the capsules in a sterile environment by using an encapsulating machine, the capsules were sealed, and the sealed capsules were preserved in an environment of 4°C. In the S3, 20 parts of glycerin are added; and when the 20 parts of glycerin are added, preservation life is obviously prolonged compared with preservation life when 10 parts of glycerin are added. Embodiment 4 S1, primary treatment was performed on phages, host bacteria with phages were selected, the phages were inoculated into a fluid medium, shake culture was performed for about 10 hours, and a phage-containing supernatant was collected; S2, a phage protectant was prepared, 15 parts of skim milk powder, 1.3 parts of glycerin, 4 parts of glucose, 0.6 part of calcium chloride, 0.36 part of sodium sulfate, 1 part of mannitol, 0.1 part of dipotassium phosphate, 0.18 part of sodium dihydrogen phosphate and 0.18 part of aluminum hydroxide glue were selected, the phages and the above mixed solution were mixed together, and the phages and the solution were fully mixed together with stirring by a stirring rod; S3, freeze-dried powder was prepared, a mixed solution prepared in the S2 was placed in a freezing chamber for freezing, primary drying was performed under a vacuum condition, then 18 parts of glycerin were added into phage fluid subjected to primary drying, heating was performed, and secondary drying was performed so as to obtain phage freeze-dried powder; and S4, capsules were prepared, the freeze-dried powder was filled in the capsules in a sterile environment by using an encapsulating machine, the capsules were sealed, and the sealed capsules were preserved in an environment of 4°C. In the S3, 18 parts of glycerin are added; and when the 18 parts of glycerin are added, preservation life is obviously prolonged compared with preservation life when 10 parts of glycerin are added, but is shorter than preservation life when 20 parts of glycerin are added. Embodiment 5
'7
Description
S1, primary treatment was performed on phages, host bacteria with phages were selected, the phages were inoculated into a fluid medium, shake culture was performed for about 10 hours, and a phage-containing supernatant was collected; S2, a phage protectant was prepared, 15 parts of skim milk powder, 1.3 parts of glycerin, 4 parts of glucose, 0.6 part of calcium chloride, 0.36 part of sodium sulfate, 1 part of mannitol, 0.1 part of dipotassium phosphate, 0.18 part of sodium dihydrogen phosphate and 0.18 part of aluminum hydroxide glue were selected, the phages and the above mixed solution were mixed together, and the phages and the solution were fully mixed together with stirring by a stirring rod; S3, freeze-dried powder was prepared, a mixed solution prepared in the S2 was placed in a freezing chamber for freezing, primary drying was performed under a vacuum condition, then 20 parts of glycerin were added into phage fluid subjected to primary drying, heating was performed, and secondary drying was performed so as to obtain phage freeze-dried powder; and S4, capsules were prepared, the freeze-dried powder was filled in the capsules in a sterile environment by using an encapsulating machine, the capsules were sealed, and the sealed capsules were preserved at normal temperature. After 20 parts of glycerin are added, preservation life at room temperature environment is equal to the preservation life in embodiment 3. For those skilled in the art, apparently, the present invention is not limited to details of the above exemplary embodiment, and may be realized in other specific forms without deviating from the spirit or essential features of the present invention. Therefore, for every point, the embodiments shall be regarded to be exemplary and non-restrictive. The scope of the present invention is limited by appended claims, rather than the above description. Therefore, all modifications in the meaning and scope of equivalent elements of the claims are included in the present invention. The above embodiments only represent the implementation modes of the present invention. The protection scope of the present invention is not limited to
Description
the above embodiments. Several variations and improvements may be made by those skilled in the art without deviating from the concept of the present invention. These variations and improvements belong to the protection scope of the present invention.
Claims (5)
1. A method for preserving freeze-dried powder capsules of phages, comprising the following steps: S, performing primary treatment on phages, selecting host bacteria with phages, inoculating the phages into a fluid medium, performing shake culture for about 10 hours, and collecting a phage-containing supernatant; S2, preparing a phage protectant, selecting 15-20 parts of skim milk powder, 1-2 parts of glycerin, 1-5 parts of glucose, 0.6-1.2 parts of calcium chloride, 0.3-0.8 part of sodium sulfate, 0.5-1.2 parts of mannitol, 0.03-0.2 part of dipotassium phosphate, 0.05-0.3 part of sodium dihydrogen phosphate and 0.03-0.18 part of aluminum hydroxide glue, mixing the phages and the above mixed solution together, and fully mixing the phages and the solution together with stirring by a stirring rod; S3, preparing freeze-dried powder, placing a mixed solution prepared in the S2 in a freezing chamber for freezing, performing primary drying under a vacuum condition, then adding 10-20 parts of glycerin into phage fluid subjected to primary drying, heating, and performing secondary drying so as to obtain phage freeze-dried powder; and S4, preparing capsules, filling the freeze-dried powder in the capsules in a sterile environment by using an encapsulating machine, sealing the capsules, and preserving the sealed capsules in an environment of 4°C.
2. The method for preserving freeze-dried powder capsules of phages according to claim 1, wherein in the S2, the mixed solution is placed at a room temperature of 20°C and stirred at a rate of 2r/second by a stirrer, and stirring lasts for 15 minutes.
3. The method for preserving freeze-dried powder capsules of phages according to claim 1, wherein in the S3, a preliminary freezing temperature is maintained at 4°C; within 20-30 minutes after preservation, the temperature is continuously decreased and decreased to minus 30°C, and temperature drop lasts for 40 minutes.
Claims
4. The method for preserving freeze-dried powder capsules of phages according to claim 1, wherein in the S3, the primary drying is performed in a vacuum environment at minus 30°C and under a pressure of 10-15 Pa.
5. The method for preserving freeze-dried powder capsules of phages according to claim 1, wherein in the S4, before the capsules are filled, a 300-mesh screen is used for screening the freeze-dried powder.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010001657.5A CN110982792A (en) | 2020-01-02 | 2020-01-02 | Method for storing freeze-dried powder capsules of bacteriophage |
| CN202010001657.5 | 2020-01-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2020100856A4 true AU2020100856A4 (en) | 2020-07-02 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2020100856A Active AU2020100856A4 (en) | 2020-01-02 | 2020-05-27 | Method for preserving freeze-dried powder capsules of phages |
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| Country | Link |
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| CN (1) | CN110982792A (en) |
| AU (1) | AU2020100856A4 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115252899A (en) * | 2022-09-13 | 2022-11-01 | 济南之羽医疗科技有限公司 | Freeze-dried powder containing recombinant human collagen and preparation method thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528460B (en) * | 2021-07-14 | 2024-06-21 | 江苏省农业科学院 | Phage composite powder and preparation method and application thereof |
| KR20230148045A (en) * | 2022-04-15 | 2023-10-24 | 씨제이제일제당 (주) | Novel excipient to maintain the long-term storage stability of bacteriophages |
-
2020
- 2020-01-02 CN CN202010001657.5A patent/CN110982792A/en not_active Withdrawn
- 2020-05-27 AU AU2020100856A patent/AU2020100856A4/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115252899A (en) * | 2022-09-13 | 2022-11-01 | 济南之羽医疗科技有限公司 | Freeze-dried powder containing recombinant human collagen and preparation method thereof |
| CN115252899B (en) * | 2022-09-13 | 2024-03-26 | 济南之羽医疗科技有限公司 | Freeze-dried powder containing recombinant human collagen and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN110982792A (en) | 2020-04-10 |
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