CN105950569A - High-quality phage preservation protective agent and preparation method and application thereof - Google Patents
High-quality phage preservation protective agent and preparation method and application thereof Download PDFInfo
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- CN105950569A CN105950569A CN201610416073.8A CN201610416073A CN105950569A CN 105950569 A CN105950569 A CN 105950569A CN 201610416073 A CN201610416073 A CN 201610416073A CN 105950569 A CN105950569 A CN 105950569A
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Abstract
The invention provides a high-quality phage preservation protective agent and preparation and application methods thereof. According to the high-quality phage preservation protective agent, the components are reasonable, skimmed milk powder, albumin powder, yeast powder, neutral clay and the like form a framework of the whole preservation protective agent, the preservation protective agent contains various saccharides and salts, the components are reasonable, the raw materials are wide in source, easy to obtain and low in cost, the preservation protective agent can be used for preservation of various phages, and the adaptability is good. According to the preparation method of high-quality phage preservation protective agent, the method is reasonable, the process is simple and easy to operate, the efficiency is high, the application cost is low, and operation is simple and easy to achieve. The high-quality phage preservation protective agent has an extremely high application prospect, use is convenient, and application is convenient and rapid.
Description
Technical field
The invention belongs to biotechnology and microbial product field, in particular it relates to a kind of Gao Pin
Matter phage preserve protective agent, and this high-quality phage preserve protectant preparation method and
Application process.
Background technology
Phage is the total of the virus of the microorganisms such as a class energy bacterial infection, actinomycetes, spirillum
Claim, some antibacterials are had the specificity of height, when these antibacterials of Phage Infection, can be
Antibacterial breeds and kills antibacterial, and it does not has toxicity to animals and plants.Along with molecular biology skill
The development of art, phage is widely used for multiple fields such as medical diagnosis on disease, water process.Due to
Gene can be inserted the characteristic in host DNA by phage so that it also becomes important
Molecule and genetics research instrument.Utilize phage, a lot of exquisite experiment can be designed.
But in above process, be required for phage is preserved, in the preservation of phage
During, generally require addition preservation protective agent thus ensure the activity of phage.Prior art
In protective agent usually preservation effect poor, the holding time of phage is short, and survival rate is low, phagocytosis
Body is easily contaminated, and must preserve under cryogenic, seriously constrains phagocytosis
The development of body application.
Summary of the invention
Goal of the invention: in order to overcome above deficiency, the invention provides a kind of high-quality phage
Preserve protective agent and this high-quality phage preserves protectant methods for making and using same.
Technical scheme: the invention provides a kind of high-quality phage and preserve protective agent, with weight
Component meter, including following components:
Defatted milk powder 5-12 part
Albumin powder 3-5 part
Yeast powder 0.5-1.2 part
Neutral clay 0.02-0.1 part
Charcoal powder 0.01-0.03 part
Propolis 0.02-0.05 part
Lactose 1-1.8 part
Maltose 0.8-2 part
Sodium sulfate 0.2-0.3 part
Calcium chloride 0.5-1.5 part
Magnesium chloride 0.3-0.8 part
Dipotassium hydrogen phosphate 0.02-0.3 part
Sodium dihydrogen phosphate 0.04-0.15 part
Glycerol 1-3 part
Arlacel-80 0.01-0.08 part
100 parts of water.
High-quality phage of the present invention preserves protective agent, and component is reasonable, wherein defatted milk
Powder, albumin powder, yeast powder, neutral clay etc. form the protectant skeleton of whole preservation, anti-
Stopping carbonization and the oxidation of lower-molecular substance, protection active substance not temperature influence, makes guarantor simultaneously
Protect agent form porous, loose sponge and there is certain water content, in frozen process
The original framework of middle holding, thus increase dissolubility.Additionally, various saccharides and salt make phage
Keep existing state and moisture is risen mitigation, and keeping electrolyte concentration.Phage
Preserve protective agent can make under phage cryopreservation from things such as solution damage and ice crystal damages
The damage of reason factor, and maintain the high survival rate of phage and activity, make the work of phage
Move and be in half static or resting state.Additionally, protective agent is with the water molecules in solution, send out
Raw hydration, the crystallization process of reduction water makes the viscosity of solution increase thus reduces the shape of ice crystal
Becoming, cryoprotective agent can pass through to maintain inside and outside phage certain molar concentration, fall simultaneously
Do not freeze inside and outside low the concentration of electrolyte in solution, makes cell from the damage of solute.Additionally,
Protective agent can also be used for the preservation of multiple phage at ambient temperature, and adaptability is good.
Further, above-mentioned high-quality phage preserves protective agent, by weight part, also
Including the vitamin of 0.1-0.5 part, described vitamin by weight part, including following components:
Vitamin C 1-3 part
Vitamin E 3-8 part
Vitamin A 0.5-0.8 part
Vitamin B2 0.2-0.5 part.
By increasing vitamin, increase the protectant nutritional labeling of whole preservation and antioxygen is turned into
With, make phage keep existing state and moisture is risen mitigation.
Further, above-mentioned high-quality phage preserves protective agent, and described glycerol is
The glycerol of 20%-40%.The glycerol good fluidity of 20%-40%, it is easy to application.
Further, above-mentioned high-quality phage preserves protective agent, and described water is deionization
Water or purified water.Deionized water or purified water can ensure that high-quality phage preserves protective agent matter
Measure stable, controlled.
The present invention also provides for a kind of high-quality phage and preserves protectant preparation method, including with
Lower step:
1) by defatted milk powder, albumin powder, lactose, maltose, sodium sulfate, calcium chloride and
Magnesium chloride joins in the water of 1/2 weight, is warmed up to 20-30 DEG C, magnetic agitation mixing standby
With；
2) by yeast powder, neutral clay, charcoal powder, propolis, dipotassium hydrogen phosphate and di(2-ethylhexyl)phosphate
Hydrogen sodium joins in the water of 1/2 weight, standby after magnetic agitation mixing；
3) by step 1) mixed liquor join step 2) solution that obtains mixes, magnetic
Power stirs；
4) to blend step 3) solution in drip glycerol and Arlacel-80, magnetic agitation again
20-30min；
5) regulating step 4) add in 115-121 DEG C after pH to the 5.5-7.5 of solution that obtains
Heat sterilization 20-30min, obtains described high-quality phage and preserves protective agent.
High-quality phage of the present invention preserves protectant preparation method, and technique is the easiest
OK, efficiency is high, and application cost is low, simple to operate, be easily achieved, and application is convenient.
Further, above-mentioned high-quality phage preserves protectant preparation method, heat sterilization
After liquid is cooled to room temperature, aseptically add the mixing of vitamin magnetic agitation.Rear interpolation is tieed up
Raw element, it is ensured that the composition of vitamin is not destroyed.
Further, above-mentioned high-quality phage preserves protectant preparation method, described in stir
Mixing speed is 50-200r/min.Mild condition, it is easy to accomplish.
Further, above-mentioned high-quality phage preserves protectant preparation method, uses dilute
Hydrochloric acid and/or sodium hydrate regulator solution pH.Raw material sources are wide, regulate very convenient, and
Application cost is low.
Further, above-mentioned high-quality phage preserves protectant preparation method, described room
Temperature is 15-25 DEG C.Mild condition, it is easy to accomplish.
The present invention also provides for a kind of high-quality phage and preserves protectant application process, described height
When quality phage preserves protective agent application, phagocytosis body fluid preserves protective agent with high-quality phage
According to the volume ratio mix homogeneously of 1:1-2.5, after room temperature preservation or vacuum lyophilization
2-7 DEG C carries out frozen.It is easy to use that high-quality phage preserves protective agent, may be used for multiple
The use that different phagies is frozen, adaptability is good, can be right in room temperature or stored refrigerated
Ambient temperature requires low, does not requires that and must could preserve by low temperature, even if the room of 15-25 DEG C
The preservation of phage can also be carried out under the conditions of temperature, may be used for multiple in the case of preservation, tool
There is high application prospect.
Beneficial effect: compared with prior art, the invention have the advantages that of the present invention
High-quality phage preserve protective agent, its component is reasonable, and raw material sources are wide, it is easy to obtain,
Low cost, can maintain the high survival rate of phage and activity, and adaptability is good.This
Bright described high-quality phage preserves protectant preparation method, and method is reasonable, and technique is simple
Easy, efficiency is high, and application cost is low, simple to operate, be easily achieved.There is high application
Prospect, to storage temperature require low and easy to use, may be used for multiple in the case of guarantor
Depositing, it is convenient to apply.
Accompanying drawing explanation
Fig. 1 is that use embodiment 1-4 high-quality phage of the present invention preserves protectant biting
Thalline survival rate figure.
Detailed description of the invention
To be further elucidated with the present invention by several specific embodiments below, these embodiments are only
Illustrate that problem, be not a kind of restriction.
Embodiment 1
A kind of high-quality phage preserves protective agent, by weight part, including following components:
Defatted milk powder 5 parts
3 parts of albumin powder
Yeast powder 0.5 part
Neutral clay 0.02 part
Charcoal powder 0.01 part
Propolis 0.02 part
Lactose 1 part
Maltose 0.8 part
0.2 part of sodium sulfate
0.5 part of calcium chloride
0.3 part of magnesium chloride
Dipotassium hydrogen phosphate 0.02 part
Sodium dihydrogen phosphate 0.04 part
20% glycerol 1 part
Arlacel-80 0.01 part
Deionized water 100 parts；
Additionally, above-mentioned high-quality phage preserves protective agent, by weight part, also include
The vitamin of 0.1 part, described vitamin by weight part, including following components:
Vitamin C 1 part
Vitamin E 3 parts
Vitamin A 0.5 part
Vitamin B2 0.2 part.
Above-mentioned high-quality phage preserves protectant preparation method, comprises the following steps:
1) by defatted milk powder, albumin powder, lactose, maltose, sodium sulfate, calcium chloride and
Magnesium chloride joins in the water of 1/2 weight, is warmed up to 20 DEG C, under the mixing speed of 50r/min
Magnetic agitation is standby after mixing；
2) by yeast powder, neutral clay, charcoal powder, propolis, dipotassium hydrogen phosphate and di(2-ethylhexyl)phosphate
Hydrogen sodium joins in the water of 1/2 weight, under the mixing speed of 50r/min after magnetic agitation mixing
Standby；
3) by step 1) mixed liquor join step 2) solution that obtains mixes, 50r/min
Mixing speed under magnetic agitation uniform；
4) to blend step 3) solution in drip glycerol and Arlacel-80 again, 50r/min's
Magnetic agitation 20min under mixing speed；
5) dilute hydrochloric acid and/or sodium hydroxide regulating step 4 are used) pH of solution that obtains arrives
In 115 DEG C of heat sterilization 20min after 5.5, after heat sterilization liquid is cooled to 15 DEG C, at aseptic bar
Adding vitamin under part, under the mixing speed of 50r/min, magnetic agitation mixing, obtains described Gao Pin
Matter phage preserves protective agent.
Above-mentioned phage preserves the protective agent preservation for vibrio parahaemolyticus phage, during application,
Phagocytosis body fluid preserves protective agent with high-quality phage and mixs homogeneously according to the volume ratio of 1:1,
After vacuum lyophilization, 2 DEG C carry out frozen.
Embodiment 2
A kind of high-quality phage preserves protective agent, by weight part, including following components:
Defatted milk powder 12 parts
5 parts of albumin powder
Yeast powder 1.2 parts
Neutral clay 0.1 part
Charcoal powder 0.03 part
Propolis 0.05 part
Lactose 1.8 parts
Maltose 2 parts
0.3 part of sodium sulfate
1.5 parts of calcium chloride
0.8 part of magnesium chloride
Dipotassium hydrogen phosphate 0.3 part
Sodium dihydrogen phosphate 0.15 part
40% glycerol 3 parts
Arlacel-80 0.08 part
Purified water 100 parts；
Additionally, above-mentioned high-quality phage preserves protective agent, by weight part, also include
The vitamin of 0.5 part, described vitamin by weight part, including following components:
Vitamin C 3 parts
Vitamin E 8 parts
Vitamin A 0.8 part
Vitamin B2 0.5 part.
Above-mentioned high-quality phage preserves protectant preparation method, comprises the following steps:
1) by defatted milk powder, albumin powder, lactose, maltose, sodium sulfate, calcium chloride and
Magnesium chloride joins in the water of 1/2 weight, is warmed up to 30 DEG C, the mixing speed of 200r/min
Lower magnetic agitation is standby after mixing；
2) by yeast powder, neutral clay, charcoal powder, propolis, dipotassium hydrogen phosphate and di(2-ethylhexyl)phosphate
Hydrogen sodium joins in the water of 1/2 weight, under the mixing speed of 200r/min after magnetic agitation mixing
Standby；
3) by step 1) mixed liquor join step 2) solution that obtains mixes,
Under the mixing speed of 200r/min, magnetic agitation is uniform；
4) to blend step 3) solution in drip glycerol and Arlacel-80 again, 200r/min's
Magnetic agitation 30min under mixing speed；
5) dilute hydrochloric acid and/or sodium hydroxide regulating step 4 are used) pH of solution that obtains arrives
In 121 DEG C of heat sterilization 30min after 7.5, after heat sterilization liquid is cooled to 25 DEG C, at aseptic bar
Adding vitamin under part, under the mixing speed of 200r/min, magnetic agitation mixing, obtains described Gao Pin
Matter phage preserves protective agent.
Above-mentioned phage preserves the protective agent preservation for coliphage, during application, bites
Thalline liquid preserves protective agent with high-quality phage and mixs homogeneously according to the volume ratio of 1:2.5,
After vacuum lyophilization, 7 DEG C carry out frozen.
Embodiment 3
A kind of high-quality phage preserves protective agent, by weight part, including following components:
Defatted milk powder 8 parts
4 parts of albumin powder
Yeast powder 1 part
Neutral clay 0.06 part
Charcoal powder 0.02 part
Propolis 0.03 part
Lactose 1.5 parts
Maltose 1 part
0.25 part of sodium sulfate
0.9 part of calcium chloride
0.6 part of magnesium chloride
Dipotassium hydrogen phosphate 0.2 part
Sodium dihydrogen phosphate 0.1 part
30% glycerol 2 parts
Arlacel-80 0.016 part
Purified water 100 parts；
Additionally, above-mentioned high-quality phage preserves protective agent, by weight part, also include
The vitamin of 0.3 part, described vitamin by weight part, including following components:
Vitamin C 2 parts
Vitamin E 6 parts
Vitamin A 0.7 part
Vitamin B2 0.3 part.
Above-mentioned high-quality phage preserves protectant preparation method, comprises the following steps:
1) by defatted milk powder, albumin powder, lactose, maltose, sodium sulfate, calcium chloride and
Magnesium chloride joins in the water of 1/2 weight, is warmed up to 25 DEG C, the mixing speed of 100r/min
Lower magnetic agitation is standby after mixing；
2) by yeast powder, neutral clay, charcoal powder, propolis, dipotassium hydrogen phosphate and di(2-ethylhexyl)phosphate
Hydrogen sodium joins in the water of 1/2 weight, under the mixing speed of 100r/min after magnetic agitation mixing
Standby；
3) by step 1) mixed liquor join step 2) solution that obtains mixes,
Under the mixing speed of 100r/min, magnetic agitation is uniform；
4) to blend step 3) solution in drip glycerol and Arlacel-80 again, 100r/min's
Magnetic agitation 25min under mixing speed；
5) dilute hydrochloric acid and/or sodium hydroxide regulating step 4 are used) pH of solution that obtains arrives
In 118 DEG C of heat sterilization 25min after 7, after heat sterilization liquid is cooled to 20 DEG C, at aseptic condition
Lower addition vitamin, under the mixing speed of 100r/min, magnetic agitation mixing, obtains described high-quality
Phage preserves protective agent.
Above-mentioned phage preserves the protective agent preservation for vibrio alginolyticus phage, during application, bites
Thalline liquid preserves protective agent with high-quality phage and mixs homogeneously according to the volume ratio of 1:2, very
After vacuum freecing-dry, 4 DEG C carry out frozen.
Embodiment 4
A kind of high-quality phage preserves protective agent, by weight part, including following components:
Defatted milk powder 5 parts
3 parts of albumin powder
Yeast powder 1.2 parts
Neutral clay 0.1 part
Charcoal powder 0.02 part
Propolis 0.03 part
Lactose 1.8 parts
Maltose 2 parts
0.2 part of sodium sulfate
0.5 part of calcium chloride
-0.8 part of magnesium chloride
Dipotassium hydrogen phosphate 0.1 part
Sodium dihydrogen phosphate 0.04 part
20% glycerol 3 parts
Arlacel-80 0.08 part
Deionized water 100 parts；
Additionally, above-mentioned high-quality phage preserves protective agent, by weight part, also include
The vitamin of 0.2 part, described vitamin by weight part, including following components:
Vitamin C 3 parts
Vitamin E 8 parts
Vitamin A 0.5 part
Vitamin B2 0.2 part.
Above-mentioned high-quality phage preserves protectant preparation method, comprises the following steps:
1) by defatted milk powder, albumin powder, lactose, maltose, sodium sulfate, calcium chloride and
Magnesium chloride joins in the water of 1/2 weight, is warmed up to 30 DEG C, the mixing speed of 120r/min
Lower magnetic agitation is standby after mixing；
2) by yeast powder, neutral clay, charcoal powder, propolis, dipotassium hydrogen phosphate and di(2-ethylhexyl)phosphate
Hydrogen sodium joins in the water of 1/2 weight, under the mixing speed of 120r/min after magnetic agitation mixing
Standby；
3) by step 1) mixed liquor join step 2) solution that obtains mixes,
Under the mixing speed of 120r/min, magnetic agitation is uniform；
4) to blend step 3) solution in drip glycerol and Arlacel-80 again, 120r/min's
Magnetic agitation 20min under mixing speed；
5) dilute hydrochloric acid and/or sodium hydroxide regulating step 4 are used) pH of solution that obtains arrives
In 121 DEG C of heat sterilization 20min after 6, after heat sterilization liquid is cooled to 25 DEG C, at aseptic condition
Lower addition vitamin, under the mixing speed of 120r/min, magnetic agitation mixing, obtains described high-quality
Phage preserves protective agent.
Above-mentioned phage preserves the protective agent preservation for mycobacterium phage, during application, bites
Thalline liquid preserves protective agent with high-quality phage and mixs homogeneously according to the volume ratio of 1:1.5,
In 20 DEG C of preservations.
After using the protectant preservation of phage 1,3,6,9 months of embodiment 1-4
Pollution of Phage situation and survival rate are investigated respectively, and as shown in table 1, phage is the most not
Pollute, and as it is shown in figure 1, phage kept the survival of more than 62.5% in 9 months
Rate.
Table 1 Pollution of Phage situation
The above is only several embodiments of invention, it is noted that for the art
Those of ordinary skill for, on the premise of without departing from inventive principle, it is also possible to make some
Improving, these improvement also should be regarded as protection scope of the present invention.
Claims (10)
1. a high-quality phage preserves protective agent, it is characterised in that: by weight part, including following components:
Defatted milk powder 5-12 part
Albumin powder 3-5 part
Yeast powder 0.5-1.2 part
Neutral clay 0.02-0.1 part
Charcoal powder 0.01-0.03 part
Propolis 0.02-0.05 part
Lactose 1-1.8 part
Maltose 0.8-2 part
Sodium sulfate 0.2-0.3 part
Calcium chloride 0.5-1.5 part
Magnesium chloride 0.3-0.8 part
Dipotassium hydrogen phosphate 0.02-0.3 part
Sodium dihydrogen phosphate 0.04-0.15 part
Glycerol 1-3 part
Arlacel-80 0.01-0.08 part
100 parts of water.
High-quality phage the most according to claim 1 preserves protective agent, it is characterised in that: by weight part, also include the vitamin of 0.1-0.5 part,
Described vitamin by weight part, including following components:
Vitamin C 1-3 part
Vitamin E 3-8 part
Vitamin A 0.5-0.8 part
Vitamin B2 0.2-0.5 part.
High-quality phage the most according to claim 1 preserves protective agent, it is characterised in that: described glycerol is the glycerol of 20%-40%.
High-quality phage the most according to claim 1 preserves protective agent, it is characterised in that: described water is deionized water or purified water.
5. preserve protectant preparation method according to the high-quality phage described in any one of right 1-4, it is characterised in that: comprise the following steps:
1) defatted milk powder, albumin powder, lactose, maltose, sodium sulfate, calcium chloride and magnesium chloride are joined in the water of 1/2 weight, be warmed up to 20-30 DEG C, standby after magnetic agitation mixing；
2) yeast powder, neutral clay, charcoal powder, propolis, dipotassium hydrogen phosphate and sodium dihydrogen phosphate are joined in the water of 1/2 weight, standby after magnetic agitation mixing；
3) mixed liquor of step 1) is joined step 2) solution that obtains mixes, magnetic agitation is uniform；
4) to blend step 3) solution in drip glycerol and Arlacel-80, magnetic agitation 20-30min again；
5) regulating step 4) after pH to the 5.5-7.5 of solution that obtains in 115-121 DEG C of heat sterilization 20-30min, obtain described high-quality phage and preserve protective agent.
High-quality phage the most according to claim 5 preserves protectant preparation method, it is characterised in that: after heat sterilization liquid is cooled to room temperature, aseptically add the mixing of vitamin magnetic agitation.
7. preserve protectant preparation method according to the high-quality phage described in claim 5 or 6, it is characterised in that: described mixing speed is 50-200r/min.
High-quality phage the most according to claim 5 preserves protectant preparation method, it is characterised in that: use dilute hydrochloric acid and/or sodium hydrate regulator solution pH.
High-quality phage the most according to claim 6 preserves protectant preparation method, it is characterised in that: described room temperature is 15-25 DEG C.
The most according to claim 1 or 5, high-quality phage preserves protectant application process; it is characterized in that: when described high-quality phage preserves protective agent application; phagocytosis body fluid preserves protective agent with high-quality phage and mixs homogeneously according to the volume ratio of 1:1-2.5, and after room temperature preservation or vacuum lyophilization, 2-7 DEG C carries out frozen.
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| CN109837251A (en) * | 2017-11-27 | 2019-06-04 | 菲吉乐科(南京)生物科技有限公司 | A kind of bacteriophage protective agent and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109837251A (en) * | 2017-11-27 | 2019-06-04 | 菲吉乐科(南京)生物科技有限公司 | A kind of bacteriophage protective agent and its preparation method and application |
| CN109837251B (en) * | 2017-11-27 | 2022-01-28 | 菲吉乐科(南京)生物科技有限公司 | Phage protective agent and preparation method and application thereof |
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Application publication date: 20160921 |