CN114983947A - Freeze-dried powder containing human collagen and preparation method thereof - Google Patents
Freeze-dried powder containing human collagen and preparation method thereof Download PDFInfo
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Abstract
The invention relates to freeze-dried powder containing human collagen and a preparation method thereof. The preparation method of the freeze-dried powder containing the human collagen comprises the steps of crosslinking pullulan polysaccharide and collagen with different molecular weights by sodium periodate, and controlling the heating speed, the temperature, the vacuum degree and the holding time of pre-freezing, primary sublimation and analysis drying stages in the freeze-drying process, wherein the moisture content of a final product is controlled to be 3-4%, and the freeze-dried powder comprises the following components: human collagen, pullulan, sodium periodate, mannitol and nicotinamide. The obtained lyophilized powder has long shelf life, and the activity of collagen can be maintained at high level.
Description
Technical Field
The invention relates to freeze-dried powder containing human collagen and a preparation process thereof.
Background
Human collagen: the human collagen produced by the gene recombination technology replaces the natural collagen. The recombinant human collagen is obtained by designing and artificially synthesizing a section of human collagen gene monomer based on tripeptide repeat sequence characteristics of human III type collagen alpha 1 chain collagen Gly-X-Y (glycine-X-Y), constructing an expression vector containing homodromous six-tandem human collagen gene monomer through a series of in vitro enzyme digestion connection, transferring Pichia pastoris, and performing high-density fermentation, separation and purification.
And (3) the pullulan: pullulan is a non-reducing polymeric substance. It has the properties of non-toxicity, safety, solubility, stability, lubricity, adhesiveness, coagulability, film coating property, decomposability, physical property improvement and the like, and is widely applied to the fields of food, medicine, light industry, chemical industry, petroleum and the like. The pullulan has good non-toxic effect, water solubility, dispersibility and hygroscopicity, so that the pullulan can be used as a viscous additive for cosmetics; is far lower in price than hyaluronic acid, but the effect is almost the same as that of hyaluronic acid. The amphiphilic graft or block polymer can form nanoparticles or nanobeams by self-assembly in aqueous solution, so that the pullulan polysaccharide which is good in biocompatibility, degradable and low in toxicity and the collagen are grafted, blended and subjected to crosslinking reaction.
The freeze-drying technology comprises the following steps: the vacuum freeze drying technology is to sublimate the water from ice directly at relatively low temperature to dry the sample without surface tension and deformation, and to dewater the material. Because the vacuum freeze-drying technology is carried out in a low-temperature and low-pressure environment, most biological reactions are stagnated, liquid water does not exist in the treatment process, and water is directly sublimated in a solid state, so that the original structure and shape of the material are protected to the greatest extent, and finally, a high-quality dried product with both appearance and internal quality is obtained.
Disclosure of Invention
The invention aims to provide freeze-dried powder containing human collagen and a preparation process thereof. The main component of the freeze-dried powder is a cross-linked product of human collagen and pullulan. The cross-linked product is pre-frozen, sublimed once and dried by analysis through low-temperature freeze-drying, the obtained freeze-dried powder can keep the activity and stability of the human collagen, and the product has the advantages of attractive appearance, no pungent smell, long storage life and excellent effect of treating skin wrinkles.
The technical purpose of the invention is realized by the following technical scheme:
preferably, the composition of the freeze-dried powder is as follows: 1g of freeze-dried powder comprises the following components in parts by weight: 10-12 parts of human collagen, 71-75 parts of pullulan polysaccharide, 1-2 parts of sodium periodate, 3-4 parts of mannitol and 2-3 parts of nicotinamide.
Preferably, the amount of the human collagen is 0.25g/100ml of the protein mixed solution.
Preferably, the pullulan polysaccharide is: the pullulan polysaccharide mixture with different molecular weights comprises the following components in percentage by mass: 100,000 g/mol: 170,000 g/mol: 530,000g/mol = 2: 2: 1.
preferably, the concentration of the aqueous sodium periodate solution used is 0.5 mol/L.
Preferably, the amount of mannitol used is 1.1g per 100ml of the protein mixed solution.
Preferably, the amount of nicotinamide used is 0.5g per 100ml of protein mixture solution.
Preferably, the pre-freezing time, temperature and pressure are that the temperature of the heat-conducting oil in the first stage reaches-15 ℃ in 5 minutes and is kept for 2 hours; the temperature of the second stage heat-conducting oil reaches-50 ℃ in 30 minutes, the temperature is kept for 4 hours, and the normal pressure is kept.
Preferably, the sublimation time, temperature and pressure used are: the temperature of the heat-conducting oil reaches-40 ℃ in 0.5 hour in the first stage, the vacuum degree is limited, and the heat-conducting oil is kept for 10 hours; the temperature of the second stage heat-conducting oil reaches-20 ℃ in 0.5 hour, the vacuum degree is 30 Pa, and the second stage heat-conducting oil is kept for 10 hours; the temperature of the heat-conducting oil in the third stage reaches-5 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the heat-conducting oil is kept for 10 hours; the temperature of the heat conducting oil in the fourth stage reaches 3 ℃ in 0.5 hour, the vacuum degree is 50 Pa, and the heat conducting oil is kept for 3 hours.
Preferably, the drying time, temperature and pressure used are: the temperature of the heat conducting oil reaches 15 ℃ in 0.5 hour at the first stage, the vacuum degree is 50 Pa, and the heat conducting oil is kept for 4 hours; the temperature of the second stage heat-conducting oil reaches 20 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the second stage heat-conducting oil is kept for 3 hours.
In conclusion, the invention has the following beneficial effects:
1. the activity and stability of the human collagen can be maintained through the crosslinking effect of the human collagen and the pluronic polysaccharide, and the appearance of the product is improved through additives (mannitol and nicotinamide). The reaction principle is that after cross-linking polymerization of human collagen and pullulan, sodium periodate is added for further cross-linking reaction, firstly, the amino group of the human collagen and the oxidized carbonyl group are subjected to Schiff base reaction to generate a new amido bond; secondly, after the sodium periodate is crosslinked, when the hydroxyl on the pullulan is not oxidized in time, the hydroxyl is connected with the carboxyl terminal of the human collagen to form an ester bond; the reaction process comprises 2 steps; after the human collagen is crosslinked by the sodium periodate, two pieces of human collagen are added on the molecular chain of the pullulan through graft blending, so that the molecular chain of a crosslinking molecular system is increased, the space chain structure after crosslinking is changed, and the degradation resistance of the human collagen crosslinking system is improved, thereby influencing the physical and chemical properties and the biological compatibility of the human collagen crosslinking system. If the sodium periodate and the pullulan are mixed firstly, two adjacent hydroxyl groups of the pullulan are oxidized to generate aldehyde groups in the sodium periodate oxidation process and cannot generate a crosslinking reaction with the human collagen, so that the crosslinking effect of the human collagen is obviously reduced, and although the high-molecular-weight pullulan can better perform the crosslinking reaction with the human collagen, the high-molecular-weight pullulan is beneficial to improving the binding rate of the human collagen, the formed compactness of the pullulan makes the water binding force in a crosslinking substance stronger, so that the water is more difficult to lose in the subsequent freeze-drying reaction, an irregular blocky structure is formed, and the pullulan is difficult to form powder. In order to meet the combination rate of the pullulan polysaccharide and the human collagen at the same time, and ensure that a cross-linked product of the pullulan polysaccharide and the human collagen is easier to form a powdery structure and more uniform in crystallization, a pullulan polysaccharide mixture with different molecular weights is adopted;
2. vacuum drying is generally divided into sublimation and drying, wherein sublimation drying mainly aims at free water in materials; the desorption drying is mainly to remove the adsorbed water strongly bonded to the solid. The vacuum degree and temperature during the drying process directly influence the drying process and the final moisture of the product, and the moisture in turn influences the biological activity of the collagen. According to the invention, reasonable pre-freezing and sublimation temperatures and vacuum degrees are selected, a freeze-drying curve is optimized, the product stability is enhanced while the product properties are improved, and the biological activity of collagen is kept at a higher level.
Drawings
FIG. 1 is a flow chart of a freeze-dried powder preparation process containing human collagen.
Detailed Description
Example 1
The embodiment provides freeze-dried powder containing human collagen and a preparation process thereof, and the formula comprises the following components:
1g pullulan (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 2: 2: 1), 0.25g human collagen solution, 0.1g sodium periodate, 1.1g mannitol, 0.5g niacinamide.
The sample is prepared according to the formula, and the method specifically comprises the following steps:
(1) 1g of pullulan powder (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 2: 2: 1) is dissolved in 100ml of deionized water at the temperature of 2 ℃ and is uniformly stirred, and the solution is placed at the temperature of 4 ℃ for 24 hours to fully dissolve the pullulan solution to prepare solution a;
(2) dissolving 0.25g of human collagen solution in 100ml of deionized water at 10 ℃ to prepare human collagen solution b;
(3) adding 1.1g of mannitol and 0.5g of nicotinamide into the solution b described in (2), and stirring for dissolving to obtain a solution c;
(4) dissolving 0.1g of sodium periodate in 1 ml of deionized water to prepare a solution d;
(5) and mixing the solution a and the solution c, stirring for 20 min, adding the solution c, continuing stirring for 20 min, and placing the dissolved solution in a hot water bath at 37 ℃ for reaction for 12 h in a dark place. Then putting the reaction product into a dialysis bag (MD 50 (8000)) for dialysis for 12 h to remove residual sodium periodate in the reaction;
(6) placing the cross-linked product dialyzed in the step (5) in a freeze-drying device, and performing pre-freezing, sublimation, resolution and drying, wherein the specific steps are as follows;
pre-freezing, namely keeping the temperature of the heat-conducting oil at the first stage at-15 ℃ for 2 hours within 5 minutes;
the temperature of the heat conducting oil in the second stage reaches-30 ℃ in 30 minutes, and is kept for 4 hours;
sublimation:
the temperature of the heat-conducting oil reaches-40 ℃ in 0.5 hour in the first stage, the vacuum degree is limited, and the heat-conducting oil is kept for 10 hours; the temperature of the second stage heat-conducting oil reaches-20 ℃ in 0.5 hour, the vacuum degree is 30 Pa, and the second stage heat-conducting oil is kept for 10 hours; the temperature of the heat-conducting oil in the third stage reaches-5 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the heat-conducting oil is kept for 10 hours; the temperature of the heat-conducting oil in the fourth stage reaches 3 ℃ in 0.5 hour, the vacuum degree is 50 Pa, and the heat-conducting oil is kept for 3 hours;
and (3) resolving and drying:
the temperature of the heat conducting oil reaches 15 ℃ in 0.5 hour at the first stage, the vacuum degree is 50 Pa, and the heat conducting oil is kept for 4 hours; the temperature of the second stage heat-conducting oil reaches 20 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the second stage heat-conducting oil is kept for 3 hours;
the freeze-dried solid was lightly pressed to a uniform powder.
Example 2
The embodiment provides freeze-dried powder containing human collagen and a preparation process thereof, and the formula comprises the following components:
1g pullulan (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 2: 1: 1), 0.25g human collagen solution, 0.1g sodium periodate, 1.1g mannitol, 0.5g niacinamide.
The production process of the freeze-dried powder in the embodiment is the same as that of the embodiment 1.
Example 3
The embodiment provides freeze-dried powder containing human collagen and a preparation process thereof, and the formula comprises the following components:
1g pullulan (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 1: 2: 1), 0.25g human collagen solution, 0.1g sodium periodate, 1.1g mannitol, 0.5g niacinamide.
The production process of the freeze-dried powder in the embodiment is the same as that of the embodiment 1.
Example 4
The embodiment provides freeze-dried powder containing human collagen and a preparation process thereof, and the formula comprises the following components:
1g of pullulan (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 1: 1: 1), 0.25g of human collagen solution, 0.1g of sodium periodate, 1.1g of mannitol, 0.5g of nicotinamide.
The production process of the freeze-dried powder in the embodiment is the same as that of the embodiment 1.
Example 5
The embodiment provides freeze-dried powder containing human collagen and a preparation process thereof, and the formula comprises the following components:
1g of pullulan (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 1: 1: 2), 0.25g of human collagen solution, 0.1g of sodium periodate, 1.1g of mannitol, 0.5g of nicotinamide.
The production process of the freeze-dried powder in the embodiment is the same as that of the embodiment 1.
Example 6
The appearance and the water content of the freeze-dried powder produced in the embodiments 1 to 5 are inspected, and the influence of pullulan with different molecular weights and proportions on the freeze-dried powder is inspected. The water content testing method comprises the following steps: heating and drying the freeze-dried powder for 24h at the temperature of 40 ℃, and weighing the powder mass before and after drying: calculating the formula: (mass before drying-mass after drying)/mass before drying.
The collagen detection method is a Woessner method:
although pullulan with high molecular weight can better perform cross-linking reaction with human collagen, the compactness of pullulan not only reduces the water content in the cross-linked product, but also makes water more difficult to lose in the subsequent freeze-drying reaction and difficult to form powder.
Example 7
The embodiment provides freeze-dried powder containing human collagen and a preparation process thereof, and the formula comprises the following components:
1g of pullulan (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 2: 2: 1), 0.2g of human collagen solution, 0.1g of sodium periodate, 1.1g of mannitol, 0.5g of nicotinamide.
The production process of the lyophilized powder in this embodiment is the same as that in embodiment 1.
Example 8
The embodiment provides a freeze-dried powder containing human collagen and a preparation process thereof, and the formula comprises the following components:
1g pullulan (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 2: 2: 1), 0.3g human collagen solution, 0.1g sodium periodate, 1.1g mannitol, 0.5g niacinamide.
The production process of the freeze-dried powder in the embodiment is the same as that of the embodiment 1.
Example 9
The embodiment provides freeze-dried powder containing human collagen and a preparation process thereof, and the formula comprises the following components:
1g of pullulan (100,000 g/mol: 170,000 g/mol: 530,000g/mol = 2: 2: 1), 0.35g of human collagen solution, 0.1g of sodium periodate, 1.1g of mannitol, 0.5g of nicotinamide.
The production process of the lyophilized powder in this embodiment is the same as that in embodiment 1.
Example 10
The appearance of the freeze-dried powder produced in examples 1 and 7, 8 and 9, and the collagen content were examined.
By changing the addition amount of the collagen, the content of the collagen is detected by the Woessner method, and although the collagen content in the freeze-dried powder is increased by adding excessive collagen, the increasing trend is not obvious, and the excessive collagen is lost during dialysis.
Example 11
In this example, the formulation and production process of the lyophilized powder were the same as in example 1.
Taking samples at different freeze drying stages to test the water content, investigating the volatilization condition of water in the vacuum freeze drying process,
the above experimental data show that: the water is mainly lost in the first stage of sublimation.
Comparative example 1
In this comparative example, the formulation and production process of the lyophilized powder were the same as in example 1.
Pre-freezing is not performed during the freeze-drying process.
Comparative example 2
In this comparative example, the formulation and production process of the lyophilized powder were the same as in example 1.
The sublimation is as follows: the temperature of the heat-conducting oil reaches-35 ℃ in 0.5 hour in the first stage, the vacuum degree is limited, and the heat-conducting oil is kept for 10 hours; the temperature of the second stage heat-conducting oil reaches-20 ℃ in 0.5 hour, the vacuum degree is 30 Pa, and the second stage heat-conducting oil is kept for 10 hours; the temperature of the heat-conducting oil in the third stage reaches-5 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the heat-conducting oil is kept for 10 hours; the temperature of the heat-conducting oil in the fourth stage reaches 3 ℃ in 0.5 hour, the vacuum degree is 50 Pa, and the heat-conducting oil is kept for 3 hours;
comparative example 3
In this comparative example, the formulation and production process of the lyophilized powder were the same as in example 1.
The sublimation is as follows: the temperature of the heat conducting oil reaches-45 ℃ in 0.5 hour at the first stage, the vacuum degree is limited to be vacuum, and the heat conducting oil is kept for 10 hours; the temperature of the heat-conducting oil in the second stage reaches-20 ℃ in 0.5 hour, the vacuum degree is 30 Pa, and the temperature is kept for 10 hours; the temperature of the heat-conducting oil in the third stage reaches-5 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the heat-conducting oil is kept for 10 hours; the temperature of the heat conducting oil in the fourth stage reaches 3 ℃ in 0.5 hour, the vacuum degree is 50 Pa, and the heat conducting oil is kept for 3 hours;
comparative example 4
In this comparative example, the formulation and production process of the lyophilized powder were the same as in example 1. Adopting direct freeze drying sublimation: the temperature of the heat conducting oil reaches-40 ℃ in 1 hour, the vacuum degree is limited to be vacuum, and the heat conducting oil is kept for 40 hours. After which it reaches 25 ℃ in 1 hour.
Example 12
Inspecting the appearance and the water content of the freeze-dried powder produced in the example 1 and the comparative examples 1, 2 and 3;
the above experimental data show that: not only the freeze-dried powder which is directly sublimated after pre-freezing is in an irregular block shape, but also the water content is 15%; if the drying is carried out by direct temperature reduction, the moisture is lost too fast due to the rapid change of the temperature difference, so that the caking is caused, and the powdery structure cannot be formed.
(1) Test for investigating toxicity of freeze-dried powder to human body
MTT (trade name thiazole blue) colorimetric method is adopted, and is a method for detecting relative survival rate of cells. The selected instrument is an enzyme linked immunosorbent assay (ELISA) detector, the measured wavelength is selected at 490 ran, and the light absorption value can indirectly reflect the number of living cells. Since exogenous MTT can be reduced to formazan by succinate dehydrogenase in mitochondria of living cells, the toxicity of material leaching solutions can be judged by the light absorption value reflected by formazan. The larger the amount of MTT crystal formation, the larger the number of surviving mouse cells within a certain range of cell number. Firstly preparing a leaching liquor and a cell suspension, inoculating a cell culture solution into a 96-well plate, putting the 96-well plate into an incubator at 37 ℃ for culturing for 1 day, removing a culture medium, adding a material leaching liquor and a reference sample, respectively, removing and adding the liquid after 1, 3 and 5 days, adding PBS (phosphate buffer solution) for washing for 1 time, adding 10uL of MTT (methyl thiazolyl tetrazolium) into the mixed solution, waiting for 2 hours until purple crystals appear at the bottom of the well plate, adding 100uL of dimethyl sulfoxide to dissolve the purple crystals, and measuring a light absorption value. The blank OD was adjusted to 0. The relative survival rate of the cells in the experimental group is obtained by comparing the value of the OD1 in the experimental group with that of the OD2 in the control group.
Survival rate = (OD 1-OD 2)/OD 1
TABLE 1 testing of the toxicity of lyophilized powders to humans
The collagen and pullulan used have no harm to mouse cells.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (9)
1. A freeze-dried powder containing human collagen is characterized in that: 1g of freeze-dried powder comprises the following components in parts by weight: 10-12 parts of human collagen, 71-75 parts of pullulan, 1-2 parts of sodium periodate, 3-4 parts of mannitol and 2-3 parts of nicotinamide; the preparation method comprises the steps of dissolving mannitol and nicotinamide, dissolving human collagen solution in water to prepare human collagen mixed solution, adding pullulan solution and the human collagen mixed solution for cross-linking and mixing, adding sodium periodate solution for further cross-linking reaction, and molding the obtained cross-linked product by regulating and controlling the heating speed, temperature, vacuum degree and holding time of pre-freezing, primary sublimation and desorption drying stages in the freeze drying process to prepare the freeze-dried powder containing the human collagen.
2. Lyophilized powder containing human collagen according to claim 1, characterized in that: the pullulan used is one or more of 100,000g/mol, 170,000g/mol and 530,000g/mol in molecular weight.
3. Lyophilized powder containing human collagen according to claim 1, characterized in that: the sodium periodate concentration used was 0.5 mol/L.
4. Lyophilized powder containing human collagen according to claim 1, characterized in that: the human collagen mixed solution is 100ml of water solution containing 0.2-0.35 g of human collagen.
5. Lyophilized powder containing human collagen according to claim 1, characterized in that: the human collagen mixed solution is 100ml of water solution containing additives: 1g to 1.2g of mannitol and 0.5g to 0.6g of nicotinamide.
6. Lyophilized powder containing human collagen according to claim 1, characterized in that: the pre-freezing is divided into two sections: the temperature of the first stage reaches-15 ℃ in 5 minutes and is kept for 2 hours; the second stage temperature reached-30 ℃ over 30 minutes and was held for 4 hours.
7. Lyophilized powder containing human collagen according to claim 1, characterized in that: the first sublimation is divided into four sections: the temperature of the first stage reaches-40 ℃ in 0.5 hour, the vacuum degree is limited to vacuum, and the vacuum is kept for 10 hours; the temperature of the second stage reaches-20 ℃ in 0.5 hour, the vacuum degree is 30 Pa, and the second stage is kept for 10 hours; the temperature of the third stage reaches-5 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the temperature is kept for 10 hours; the temperature of the fourth stage reaches 3 ℃ in 0.5 hour, the vacuum degree is 50 Pa, and the fourth stage is kept for 3 hours.
8. The lyophilized powder containing human collagen according to claim 1, wherein: the analysis and drying are divided into two sections: the temperature of the first stage reaches 15 ℃ in 0.5 hour, the vacuum degree is 50 Pa, and the first stage is kept for 4 hours; the temperature of the second stage is up to 20 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the second stage is kept for 3 hours.
9. A method for preparing lyophilized powder containing human collagen according to any one of claims 1 to 8, comprising the steps of:
(1) 1-1.2 g of pullulan powder (one or more of molecular weight of 100,000g/mol, 170,000g/mol and 530,000 g/mol) is dissolved in 100ml of deionized water at 2-3 ℃, uniformly stirred and placed at 5-6 ℃ for 24 hours to fully dissolve the pullulan solution to prepare a solution a;
(2) dissolving 0.2-0.35 g of human collagen solution in 100ml of deionized water at 10 ℃ to prepare human collagen solution b;
(3) adding 1-1.2 g of mannitol and 0.4-0.6 g of nicotinamide into the solution b described in the step (2), and stirring for dissolving to obtain a solution c;
(4) dissolving 0.1-0.11 g of sodium periodate in 1 ml of deionized water to prepare a solution d;
(5) mixing the solution a and the solution c, stirring for 20 min, adding the solution c, continuing stirring for 20 min, and placing the dissolved solution in a hot water bath at 37 ℃ for reaction for 12 h in a dark place; then putting the reaction product into a dialysis bag (MD 50 (8000)) for dialysis for 12 h to remove residual sodium periodate in the reaction;
(6) the cross-linked mixed product is put into a freeze dryer for second-stage pre-freezing,
the temperature of the first stage reaches-15 ℃ in 5 minutes and is kept for 2 hours; the temperature of the second stage reaches-30 ℃ within 30 minutes, and is kept for 4 hours;
(7) changing the temperature and pressure, and carrying out four-stage one-time sublimation:
the temperature of the first stage reaches minus 35 ℃ to minus 45 ℃ in 0.5 hour, the vacuum degree is limited to vacuum, and the vacuum is kept for 10 hours; the temperature of the second stage reaches-20 ℃ in 0.5 hour, the vacuum degree is 30 Pa, and the second stage is kept for 10 hours; the temperature of the third stage reaches-5 ℃ in 0.5 hour, the vacuum degree is 40 Pa, and the temperature is kept for 10 hours; the temperature of the fourth stage reaches 3 ℃ in 0.5 hour, the vacuum degree is 50 Pa, and the fourth stage is kept for 3 hours;
(8) changing the temperature and the pressure, and carrying out two-stage analysis and drying:
the temperature of the first stage reaches 15 ℃ in 0.5 hour, the vacuum degree is 50 Pa, and the first stage is kept for 4 hours; the temperature of the second stage reaches 20 ℃ within 0.5 hour, the vacuum degree is 40 Pa, and the second stage is kept for 3 hours; and slightly extruding the freeze-dried solid into uniform powder to obtain the freeze-dried powder product.
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| 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 |
| CN115844761A (en) * | 2022-12-26 | 2023-03-28 | 晟薇药业(上海)有限公司 | Freeze-dried mask with smoothie appearance experience and preparation method thereof |
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| 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 |
| CN115844761A (en) * | 2022-12-26 | 2023-03-28 | 晟薇药业(上海)有限公司 | Freeze-dried mask with smoothie appearance experience and preparation method thereof |
| CN115844761B (en) * | 2022-12-26 | 2024-03-22 | 晟薇药业(上海)有限公司 | Freeze-dried mask with smoothie appearance experience and preparation method thereof |
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