CN105950567A - Preservation method for freeze-dried powder capsules of bacteriophage - Google Patents
Preservation method for freeze-dried powder capsules of bacteriophage Download PDFInfo
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- CN105950567A CN105950567A CN201610415919.6A CN201610415919A CN105950567A CN 105950567 A CN105950567 A CN 105950567A CN 201610415919 A CN201610415919 A CN 201610415919A CN 105950567 A CN105950567 A CN 105950567A
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Abstract
The invention provides a preservation method for freeze-dried powder capsules of bacteriophage. The method comprises the steps that bacteriophage liquid and a bacteriophage preservation protecting agent are mixed to be uniform to be prepared into freeze-dried bacteriophage powder, the freeze-dried powder is screened and then subpackaged into capsule shells to be sealed, and the bacteriophage capsules are obtained and preserved. The preservation method for the freeze-dried powder capsules of the bacteriophage is reasonable and convenient to apply, and the bacteriophage can be preserved at room temperature or refrigerated, is low in requirement for the preservation environment and good in adaptability and can be preserved for a long term.
Description
Technical field
The invention belongs to biotechnology and microbial product field, in particular it relates to a kind of phagocytosis
The freeze-dried powder capsule store method of body.
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, the preservation of prior art
The preservation effect of scheme pnagus medius is poor, and 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 the lyophilizing of a kind of phage
Powder capsule store method.
Technical scheme: the invention provides the freeze-dried powder capsule store method of a kind of phage, bag
Include following steps:
1) phage solution stands 4-8h in 2-7 DEG C, flocculent deposit occurs, passes through
8000-12000r/min is centrifuged 20-40min；
2) in above-mentioned precipitation, add phage buffer, by resolution of precipitate, obtain phagocytosis body fluid；
3) phage of phagocytosis body fluid volume 1-2.5 times preserves protective agent mix homogeneously, described
Phage preserves aminoacid described in protective agent by weight part, including following components: defatted milk
Powder 5-12 part, Polyethylene Glycol 2-8 part, algae egg albumen powder 0.2-0.6 part, sodium glutamate 0.3-0.8
Part, carboxymethyl cellulose 0.3-0.5 part, bentonite 0.08-0.15, aluminium hydroxide gel
0.03-0.18 part, Raffinose 0.3-1.2 part, magnesium sulfate 0.1-0.6 part, calcium chloride 0.6-1.2
Part, sodium sulfate 0.3-0.8 part, mannitol 0.5-1.2 part, dipotassium hydrogen phosphate 0.03-0.2 part,
Sodium dihydrogen phosphate 0.05-0.3 part, glycerol 0.02-0.08 part, dimethyl sulfoxide 0.01-0.04
Part and 100 parts of water；
4) cooling in advance: by above-mentioned mixed solution frozen cooling to 5 DEG C, and keep 30min；
Cool to-5 DEG C with the speed of 1 DEG C/min further, and keep 30min；Further with 1 DEG C
The speed of/min cools to-30 DEG C, and keeps 1h；
One-level is dried: by above-mentioned solution in-30 DEG C, the vacuum condition of 80-150mTorr pressure
Under be dried 12-18h；
Secondary drying: with the speed of 0.1 DEG C/min, heating said solution to 25 DEG C, then at upper
State and obtain phage lyophilized powder after keeping 4-8h under vacuum condition；
5) above-mentioned phage lyophilized powder is aseptically sub-packed in after 200-500 mesh sieve
After capsule shells seals, obtain phage capsule and preserve.
The freeze-dried powder capsule store method of phage of the present invention, method is reasonable, application side
Just, phage stock solution adds and preserves protective agent, preserve with lyophilized powder form, owing to freezing is done
Dry carrying out at low temperatures, therefore phage will not occur degeneration or lose biologos, in material
The loss of some volatile ingredients the least, be dried and carry out under vacuo, oxygen is few, therefore one
The most oxidizable material is protected, and phagocytosis physical ability keeps original character, after drying
Lyophilized powder loose porous, in spongy, after adding solvent, phage can be dissolved rapid and complete,
Recover original character the most immediately.Lyophilized powder is stored in instant packed in capsule and stores fortune
Defeated, and can be stored at room temperature or cold preservation, Conservation environment is required low, adaptability is good, can
Preserve with long-term.Preserve defatted milk powder, Polyethylene Glycol, algae egg albumen powder, carboxymethyl in protective agent
Cellulose, bentonite form the protectant skeleton of whole preservation, prevent the carbonization of lower-molecular substance
And oxidation, protection active substance not temperature influence simultaneously, make protective agent form porous, dredge
Pine sponge and there is certain water content, during frozen, keep original framework,
Thus increase dissolubility.Additionally, various saccharides and salt make phage keep existing state and right
Moisture plays mitigation, and keeps electrolyte concentration.Phage preserves protective agent and can make to bite
From the damage of the physical factors such as solution damage and ice crystal damage under thalline cryopreservation, and tie up
Hold the high survival rate of phage and activity, make the activity of phage be in half static or static shape
State.Additionally, protective agent is with the water molecules in solution, there is hydration, weaken water
Crystallization process makes the viscosity of solution increase thus reduces the formation of ice crystal, and cryoprotective agent can simultaneously
With by maintaining certain molar concentration inside and outside phage, electricity in the solution that do not freezes inside and outside reduction
Solve the concentration of matter, make cell from the damage of solute.Additionally, protective agent can also be at room temperature bar
For the preservation of multiple phage under part, adaptability is good.
Further, the freeze-dried powder capsule store method of above-mentioned phage, described phage is protected
Deposit protective agent by weight part, also include the antioxidant of 0.2-0.6 part, described antioxidation
Agent by weight part, including following components: vitamin C 2-8 part, vitamin E 3-10
Part, thiourea 3-7 part, potassium iodide 2-5 part, sodium thiosulfate 0.3-1 part and ammonium molybdate 0.5-2
Part.By increasing antioxidant, make phage keep the existing state of temperature for a long time, preserve effect
The best.
Further, the freeze-dried powder capsule store method of above-mentioned phage, described phage glue
Capsule is in dry environments in room temperature or 2-7 DEG C of preservation.Preservation effect is more preferably under the dry condition,
Holding time is more long.May be used for the use that multiple different phage is frozen, adaptability is good,
Can require low to ambient temperature in room temperature or stored refrigerated, not requiring that must low temperature
Can preserve, even if the preservation of phage can also be carried out under the room temperature condition of 15-25 DEG C, can
For the preservation in the case of multiple, there is high application prospect.
Further, the freeze-dried powder capsule store method of above-mentioned phage, described step exists
Complete under the damp condition of 4-6%.Under the damp condition of 4-6%, Bacteriolytic activity is optimal, preserves effect
The most best.
Further, the freeze-dried powder capsule store method of above-mentioned phage, described phage is delayed
Rush liquid by weight part, including following components: Tri(Hydroxymethyl) Amino Methane Hydrochloride 8-15
Part, sodium chloride 0.05-0.2 part, potassium chloride 1-5 part, magnesium sulfate 5-20 part, phosphoric acid hydrogen two
Potassium 1-3 part, sodium dihydrogen phosphate 8-12 part, gelatin 0.05-0.1 part, 100 parts of water, and
And regulation pH to 7-7.5.Phage buffer composition is reasonable, easy to use, may be used for
The dissolving of phage and resuspended, and do not affect the activity of phage.
Further, the freeze-dried powder capsule store method of above-mentioned phage, described glycerol is
The glycerol of 20%-40%.The glycerol good fluidity of 20%-40%, it is easy to application.
Further, the freeze-dried powder capsule store method of above-mentioned phage, described water is for going
Ionized water or purified water.Deionized water or purified water can ensure that novel phage preserves protective agent
With phage buffer steady quality, controlled.
Further, the preparation method of the freeze-dried powder capsule store method of above-mentioned phage, make
With dilute hydrochloric acid and/or sodium hydrate regulator solution pH.Raw material sources are wide, regulate very convenient,
And application cost is low.
Beneficial effect: compared with prior art, the invention have the advantages that of the present invention
The freeze-dried powder capsule store method of phage, method is reasonable, and application is convenient, and phage is permissible
Being stored at room temperature or cold preservation, require low to Conservation environment, adaptability is good, and can be long-term
Preserve.
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 freeze-dried powder capsule store method of mycobacterium phage, step 4% humidity
Under the conditions of complete, comprise the following steps:
1) mycobacterium phage solution stands 4h in 2 DEG C, flocculent deposit occurs, passes through
8000r/min is centrifuged 40min；
2) in above-mentioned precipitation, add phage buffer, by resolution of precipitate, obtain phagocytosis body fluid；
Described phage buffer by weight part, including following components: trishydroxymethylaminomethane
Hydrochlorate 8 parts, 0.05 part of sodium chloride, 1 part of potassium chloride, 5 parts of magnesium sulfate, phosphoric acid hydrogen
Dipotassium 1 part, sodium dihydrogen phosphate 8 parts, 0.05 part of gelatin and deionized water 100 parts, and
And use dilute hydrochloric acid and/or sodium hydroxide regulation pH to 7；
3) the isopyknic phage of phagocytosis body fluid preserves protective agent mix homogeneously, described phage
Preserve aminoacid described in protective agent by weight part, including following components: defatted milk powder 5
Part, Polyethylene Glycol 2 parts, algae egg albumen powder 0.2 part, sodium glutamate 0.3 part, carboxymethyl fibre
Dimension element 0.3 part, bentonite 0.08 part, aluminium hydroxide gel 0.03 part, Raffinose 0.3 part,
0.1 part of magnesium sulfate, 0.6 part of calcium chloride, 0.3 part of sodium sulfate, 0.5 part of mannitol, phosphorus
Acid hydrogen dipotassium 0.03 part, sodium dihydrogen phosphate 0.05 part, 20% glycerol 0.02 part, dimethyl
Sulfoxide 0.01 part and deionized water 100 parts；
Additionally, also include the antioxidant of 0.2 part, described antioxidant by weight part,
Including following components: vitamin C 2 parts, vitamin E 3 parts, thiourea 3 parts, potassium iodide 2
Part, 0.3 part of sodium thiosulfate and ammonium molybdate 0.5 part；
4) cooling in advance: by above-mentioned mixed solution frozen cooling to 5 DEG C, and keep 30min；
Cool to-5 DEG C with the speed of 1 DEG C/min further, and keep 30min；Further with 1 DEG C
The speed of/min cools to-30 DEG C, and keeps 1h；
One-level is dried: by above-mentioned solution in-30 DEG C, under the vacuum condition of 80mTorr pressure dry
Dry 12h；
Secondary drying: with the speed of 0.1 DEG C/min, heating said solution to 25 DEG C, then at upper
State and obtain phage lyophilized powder after keeping 4h under vacuum condition；
5) above-mentioned phage lyophilized powder is aseptically sub-packed in capsule after 200 mesh sieves
After sealing in shell, obtaining phage capsule, described phage capsule is in dry environments in 2 DEG C of guarantors
Deposit.
Embodiment 2
A kind of freeze-dried powder capsule store method of coliphage, step 6% humidity
Under the conditions of complete, comprise the following steps:
1) coliphage solution stands 8h in 7 DEG C, flocculent deposit occurs, passes through
12000r/min is centrifuged 20min；
2) in above-mentioned precipitation, add phage buffer, by resolution of precipitate, obtain phagocytosis body fluid；
Described phage buffer by weight part, including following components: trishydroxymethylaminomethane
Hydrochlorate 15 parts, 0.2 part of sodium chloride, 5 parts of potassium chloride, 20 parts of magnesium sulfate, phosphoric acid hydrogen
Dipotassium 3 parts, sodium dihydrogen phosphate 12 parts, 0.1 part of gelatin and purified water 100 parts, and
Use dilute hydrochloric acid and/or sodium hydroxide regulation pH to 7.5；
3) phagocytosis body fluid volume 2.5 times phage preserve protective agent mix homogeneously, described in bite
Thalline preserves aminoacid described in protective agent by weight part, including following components: defatted milk powder
12 parts, Polyethylene Glycol 8 parts, algae egg albumen powder 0.6 part, sodium glutamate 0.8 part, carboxymethyl
Cellulose 0.5 part, bentonite 0.15 part, aluminium hydroxide gel 0.18 part, Raffinose 1.2
Part, 0.6 part of magnesium sulfate, 1.2 parts of calcium chloride, 0.8 part of sodium sulfate, 1.2 parts of mannitol,
Dipotassium hydrogen phosphate 0.2 part, sodium dihydrogen phosphate 0.3 part, 40% glycerol 0.08 part, dimethyl
Sulfoxide 0.04 part and purified water 100 parts；
Additionally, also include the antioxidant of 0.6 part, described antioxidant by weight part,
Including following components: vitamin C 8 parts, vitamin E 10 parts, thiourea 7 parts, potassium iodide
5 parts, 1 part of sodium thiosulfate and ammonium molybdate 2 parts；
4) cooling in advance: by above-mentioned mixed solution frozen cooling to 5 DEG C, and keep 30min；
Cool to-5 DEG C with the speed of 1 DEG C/min further, and keep 30min；Further with 1 DEG C
The speed of/min cools to-30 DEG C, and keeps 1h；
One-level is dried: by above-mentioned solution in-30 DEG C, under the vacuum condition of 150mTorr pressure dry
Dry 18h；
Secondary drying: with the speed of 0.1 DEG C/min, heating said solution to 25 DEG C, then at upper
State and obtain phage lyophilized powder after keeping 8h under vacuum condition；
5) above-mentioned phage lyophilized powder is aseptically sub-packed in capsule after 500 mesh sieves
After sealing in shell, obtaining phage capsule, described phage capsule is in dry environments in 7 DEG C of guarantors
Deposit.
Embodiment 3
A kind of freeze-dried powder capsule store method of lactobacillus phage, step 5% humidity bar
Complete under part, comprise the following steps:
1) lactobacillus phage solution stands 5h in 4 DEG C, flocculent deposit occurs, passes through
10000r/min is centrifuged 30min；
2) in above-mentioned precipitation, add phage buffer, by resolution of precipitate, obtain phagocytosis body fluid；
Described phage buffer by weight part, including following components: trishydroxymethylaminomethane
Hydrochlorate 12 parts, 0.09 part of sodium chloride, 3 parts of potassium chloride, 12 parts of magnesium sulfate, phosphoric acid
Hydrogen dipotassium 2 parts, sodium dihydrogen phosphate 10 parts, 0.08 part of gelatin and deionized water 100 parts,
And use dilute hydrochloric acid and/or sodium hydroxide regulation pH to 7.2；
3) phage of phagocytosis body fluid volume 2 times preserves protective agent mix homogeneously, described phagocytosis
Body preserves aminoacid described in protective agent by weight part, including following components: defatted milk powder 8
Part, Polyethylene Glycol 6 parts, algae egg albumen powder 0.4 part, sodium glutamate 0.6 part, carboxymethyl fibre
Dimension element 0.4 part, bentonite 0.1 part, aluminium hydroxide gel 0.12 part, Raffinose 0.8 part,
0.4 part of magnesium sulfate, 0.8 part of calcium chloride, 0.6 part of sodium sulfate, 0.9 part of mannitol, phosphorus
Acid hydrogen dipotassium 0.1 part, sodium dihydrogen phosphate 0.2 part, 30% glycerol 0.05 part, dimethyl are sub-
Sulfone 0.02 part and deionized water 100 parts；
Additionally, also include the antioxidant of 0.4 part, described antioxidant by weight part,
Including following components: vitamin C 5 parts, vitamin E 5 parts, thiourea 5 parts, potassium iodide 3
Part, 0.8 part of sodium thiosulfate and ammonium molybdate 1 part；
4) cooling in advance: by above-mentioned mixed solution frozen cooling to 5 DEG C, and keep 30min；
Cool to-5 DEG C with the speed of 1 DEG C/min further, and keep 30min；Further with 1 DEG C
The speed of/min cools to-30 DEG C, and keeps 1h；
One-level is dried: by above-mentioned solution in-30 DEG C, under the vacuum condition of 100mTorr pressure dry
Dry 16h；
Secondary drying: with the speed of 0.1 DEG C/min, heating said solution to 25 DEG C, then at upper
State and obtain phage lyophilized powder after keeping 6h under vacuum condition；
5) above-mentioned phage lyophilized powder is aseptically sub-packed in capsule after 300 mesh sieves
After sealing in shell, obtaining phage capsule, described phage capsule is in dry environments in 5 DEG C of guarantors
Deposit.
Embodiment 4
A kind of freeze-dried powder capsule store method of vibrio alginolyticus phage, step 6% humidity
Under the conditions of complete, comprise the following steps:
1) vibrio alginolyticus phage solution stands 4h in 2 DEG C, flocculent deposit occurs, passes through
9000r/min is centrifuged 40min；
2) in above-mentioned precipitation, add phage buffer, by resolution of precipitate, obtain phagocytosis body fluid；
Described phage buffer by weight part, including following components: trishydroxymethylaminomethane
Hydrochlorate 8 parts, 0.05 part of sodium chloride, 5 parts of potassium chloride, 20 parts of magnesium sulfate, phosphoric acid hydrogen
Dipotassium 2 parts, sodium dihydrogen phosphate 10 parts, 0.08 part of gelatin and purified water 100 parts, and
And use dilute hydrochloric acid and/or sodium hydroxide regulation pH to 7；
3) phagocytosis body fluid volume 1.5 times phage preserve protective agent mix homogeneously, described in bite
Thalline preserves aminoacid described in protective agent by weight part, including following components: defatted milk powder
5 parts, Polyethylene Glycol 2 parts, algae egg albumen powder 0.6 part, sodium glutamate 0.8 part, carboxymethyl
Cellulose 0.3 part, bentonite 0.08 part, aluminium hydroxide gel 0.03 part, Raffinose 1 part,
0.2 part of magnesium sulfate, 0.8 part of calcium chloride, 0.3 part of sodium sulfate, 0.5 part of mannitol, phosphorus
Acid hydrogen dipotassium 0.2 part, 0.3 part of 40% glycerol of sodium dihydrogen phosphate 0.08 part, dimethyl are sub-
Sulfone 0.02 part and purified water 100 parts；
Additionally, also include the antioxidant of 0.3 part, described antioxidant by weight part,
Including following components: vitamin C 2 parts, vitamin E 3 parts, thiourea 7 parts, potassium iodide 5
Part, 0.5 part of sodium thiosulfate and ammonium molybdate 2 parts；
4) cooling in advance: by above-mentioned mixed solution frozen cooling to 5 DEG C, and keep 30min；
Cool to-5 DEG C with the speed of 1 DEG C/min further, and keep 30min；Further with 1 DEG C
The speed of/min cools to-30 DEG C, and keeps 1h；
One-level is dried: by above-mentioned solution in-30 DEG C, under the vacuum condition of 100mTorr pressure dry
Dry 15h；
Secondary drying: with the speed of 0.1 DEG C/min, heating said solution to 25 DEG C, then at upper
State and obtain phage lyophilized powder after keeping 7h under vacuum condition；
5) above-mentioned phage lyophilized powder is aseptically sub-packed in capsule after 300 mesh sieves
After sealing in shell, obtaining phage capsule, described phage capsule is in dry environments in 20 DEG C
Preserve.
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 rate of more than 60% in 9 months.
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 (8)
1. the freeze-dried powder capsule store method of a phage, it is characterised in that: comprise the following steps:
1) phage solution stands 4-8h in 2-7 DEG C, flocculent deposit occurs, is centrifuged 20-40min through 8000-12000r/min；
2) in above-mentioned precipitation, add phage buffer, by resolution of precipitate, obtain phagocytosis body fluid；
null3) phage of phagocytosis body fluid volume 1-2.5 times preserves protective agent mix homogeneously，Described phage preserves aminoacid described in protective agent by weight part，Including following components: defatted milk powder 5-12 part、Polyethylene Glycol 2-8 part、Algae egg albumen powder 0.2-0.6 part、Sodium glutamate 0.3-0.8 part、Carboxymethyl cellulose 0.3-0.5 part、Bentonite 0.08-0.15、Aluminium hydroxide gel 0.03-0.18 part、Raffinose 0.3-1.2 part、Magnesium sulfate 0.1-0.6 part、Calcium chloride 0.6-1.2 part、Sodium sulfate 0.3-0.8 part、Mannitol 0.5-1.2 part、Dipotassium hydrogen phosphate 0.03-0.2 part、Sodium dihydrogen phosphate 0.05-0.3 part、Glycerol 0.02-0.08 part、Dimethyl sulfoxide 0.01-0.04 part and 100 parts of water；
4) cooling in advance: by above-mentioned mixed solution frozen cooling to 5 DEG C, and keep 30min；Cool to-5 DEG C with the speed of 1 DEG C/min further, and keep 30min；Further cool to-30 DEG C with the speed of 1 DEG C/min, and keep 1h；
One-level is dried: by above-mentioned solution in-30 DEG C, be dried 12-18h under the vacuum condition of 80-150mTorr pressure；
Secondary drying: with the speed of 0.1 DEG C/min, heating said solution to 25 DEG C, obtain phage lyophilized powder after keeping 4-8h under above-mentioned vacuum condition；
5), after above-mentioned phage lyophilized powder is aseptically sub-packed in capsule shells sealing after 200-500 mesh sieve, obtains phage capsule and preserve.
The freeze-dried powder capsule store method of phage the most according to claim 1; it is characterized in that: described phage preserves protective agent by weight part; also include the antioxidant of 0.2-0.6 part; described antioxidant by weight part, including following components: vitamin C 2-8 part, vitamin E 3-10 part, thiourea
3-7 part, potassium iodide 2-5 part, sodium thiosulfate 0.3-1 part and ammonium molybdate 0.5-2 part.
The freeze-dried powder capsule store method of phage the most according to claim 1, it is characterised in that: described phage capsule is in dry environments in room temperature or 2-7 DEG C of preservation.
The freeze-dried powder capsule store method of phage the most according to claim 1, it is characterised in that: described step completes under the damp condition of 4-6%.
The freeze-dried powder capsule store method of phage the most according to claim 1, it is characterized in that: described phage buffer by weight part, including following components: Tri(Hydroxymethyl) Amino Methane Hydrochloride 8-15 part, sodium chloride 0.05-0.2 part, potassium chloride 1-5 part, magnesium sulfate 5-20 part, dipotassium hydrogen phosphate
1-3 part, sodium dihydrogen phosphate 8-12 part, gelatin 0.05-0.1 part, 100 parts of water, and regulate pH to 7-7.5.
The freeze-dried powder capsule store method of phage the most according to claim 1, it is characterised in that: described glycerol is the glycerol of 20%-40%.
The freeze-dried powder capsule store method of phage the most according to claim 1 or 5, it is characterised in that: described water is deionized water or purified water.
The preparation method of the freeze-dried powder capsule store method of phage the most according to claim 5, it is characterised in that: use dilute hydrochloric acid and/or sodium hydrate regulator solution pH.
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| CN114181913A (en) * | 2021-12-14 | 2022-03-15 | 厦门昶科生物工程有限公司 | Phage freeze-dried powder preparation and production method thereof |
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| CN110452887A (en) * | 2019-07-18 | 2019-11-15 | 广东海大集团股份有限公司畜牧水产研究中心 | A kind of bacteriophage protective agent and its application |
| CN114181913A (en) * | 2021-12-14 | 2022-03-15 | 厦门昶科生物工程有限公司 | Phage freeze-dried powder preparation and production method thereof |
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Application publication date: 20160921 |