CN117821362A - Embryo bovine serum freeze-dried powder and preparation method thereof - Google Patents
Embryo bovine serum freeze-dried powder and preparation method thereof Download PDFInfo
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- 239000000843 powder Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 239000012888 bovine serum Substances 0.000 title description 2
- 210000001161 mammalian embryo Anatomy 0.000 title description 2
- 239000000284 extract Substances 0.000 claims abstract description 86
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 84
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 82
- 230000009182 swimming Effects 0.000 claims abstract description 79
- 238000007710 freezing Methods 0.000 claims abstract description 66
- 230000008014 freezing Effects 0.000 claims abstract description 58
- 239000012091 fetal bovine serum Substances 0.000 claims abstract description 57
- 239000008176 lyophilized powder Substances 0.000 claims abstract description 35
- 241000736131 Sphingomonas Species 0.000 claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 30
- 230000004151 fermentation Effects 0.000 claims abstract description 30
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 28
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 28
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 28
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000012894 fetal calf serum Substances 0.000 claims abstract description 25
- 239000011259 mixed solution Substances 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 18
- 238000004108 freeze drying Methods 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 239000000706 filtrate Substances 0.000 claims description 31
- 239000000047 product Substances 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 238000001035 drying Methods 0.000 claims description 23
- 239000012528 membrane Substances 0.000 claims description 22
- 210000004712 air sac Anatomy 0.000 claims description 21
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 20
- 101710130006 Beta-glucanase Proteins 0.000 claims description 20
- 108010059892 Cellulase Proteins 0.000 claims description 20
- 241000252230 Ctenopharyngodon idella Species 0.000 claims description 20
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 20
- 229940106157 cellulase Drugs 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 17
- 238000004140 cleaning Methods 0.000 claims description 11
- 239000013078 crystal Substances 0.000 abstract description 6
- 238000001556 precipitation Methods 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 6
- 206010018910 Haemolysis Diseases 0.000 abstract description 5
- 230000002776 aggregation Effects 0.000 abstract description 5
- 238000004220 aggregation Methods 0.000 abstract description 5
- 230000008588 hemolysis Effects 0.000 abstract description 5
- 230000003647 oxidation Effects 0.000 abstract description 5
- 238000007254 oxidation reaction Methods 0.000 abstract description 5
- 239000004480 active ingredient Substances 0.000 abstract description 4
- 238000006116 polymerization reaction Methods 0.000 abstract description 4
- 239000007983 Tris buffer Substances 0.000 abstract description 2
- 239000002274 desiccant Substances 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 16
- 239000000243 solution Substances 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 108010007119 flavourzyme Proteins 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
Abstract
The invention belongs to the technical field of biology, and particularly discloses a lyophilized powder of fetal bovine serum and a preparation method thereof, wherein the preparation method of the lyophilized powder of fetal bovine serum comprises the following steps: (1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution; (2) And (3) placing the mixed solution in a freeze dryer, and freeze-drying to obtain the fetal bovine serum freeze-dried powder. According to the invention, the swimming bladder extract, the sphingomonas fermentation product extract, the glycerol, the tris and the hyaluronic acid are creatively added in the freeze-drying process of the fetal calf serum, so that the fetal calf serum can be effectively protected, the generation of ice crystals is avoided, the occurrence of hemolysis is avoided, meanwhile, the aggregation or precipitation in the freezing process is avoided, the oxidation and the polymerization of active ingredients are prevented, the stability of the fetal calf serum is further effectively improved, and the fetal calf serum freeze-drying agent has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a fetal bovine serum freeze-dried powder and a preparation method thereof.
Background
Fetal bovine serum is a serum isolated from fetal bovine blood and is commonly used in cell culture experiments. The appearance of the liquid is light yellow clear, non-hemolysis and non-foreign matter slightly viscous liquid. The nutrients, hormones and growth factors in the fetal bovine serum are critical for the growth and proliferation of cells. During cell culture, the fetal bovine serum can provide nutrients required by cells and support the growth and proliferation of cells.
The freeze-dried powder is an aseptic powder injection prepared by freezing the liquid medicine into a solid state under an aseptic environment, vacuumizing and sublimating and drying the water. The preparation method of the freeze-dried powder is a freeze-drying technology, which is also called a vacuum freeze-drying technology, and the preparation of the freeze-dried powder of the fetal bovine serum is not yet available at present, and in the freeze-drying process of the fetal bovine serum, water in the serum can form ice crystals. These ice crystals may puncture erythrocytes in serum, leading to hemolysis and thus to serious influence on the quality of serum, leading to destruction of components in serum, and the frozen and dried fetal bovine serum may show turbidity or precipitation, possibly due to aggregation or precipitation of certain components during freezing, and oxidation and polymerization of the fetal bovine serum during freeze drying, leading to color change.
In view of this, the present application is presented.
Disclosure of Invention
The invention provides the lyophilized powder of the fetal bovine serum and the preparation method thereof, which can effectively prevent the formation of ice crystals, avoid the generation of hemolysis, simultaneously avoid aggregation or precipitation in the freezing process, and prevent the oxidation and polymerization of active ingredients, thereby effectively improving the stability of the lyophilized powder of the fetal bovine serum and having wide application prospect.
The invention solves the technical problems by adopting the following technical scheme:
a preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
(2) And (3) placing the mixed solution in a freeze dryer, and freeze-drying to obtain the fetal bovine serum freeze-dried powder.
As a preferred embodiment of the present invention, the mass ratio of the fetal bovine serum, the swimming bladder extract, the sphingomonas fermentation product extract, the glycerol, the tris (hydroxymethyl) aminomethane and the hyaluronic acid is 100: (0.5-3): (0.5-2): (0.2-1): (0.1 to 0.6): (0.1 to 0.5).
As a preferred embodiment of the present invention, the mass ratio of the fetal bovine serum, the swimming bladder extract, the sphingomonas fermentation product extract, the glycerol, the tris (hydroxymethyl) aminomethane and the hyaluronic acid is 100: (0.8-2): (0.8 to 1.5): (0.3 to 0.8): (0.2 to 0.5): (0.1 to 0.3).
As a preferred embodiment of the present invention, the mass ratio of the fetal bovine serum, the swimming bladder extract, the sphingomonas fermentation product extract, the glycerol, the tris (hydroxymethyl) aminomethane and the hyaluronic acid is 100:2:1:0.6:0.4:0.2.
as a preferred embodiment of the invention, the preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning, drying and crushing the swimming bladder of the grass carp to 200-500 meshes to obtain swimming bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water for enzymolysis to obtain an enzymolysis liquid;
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting the pH to 5.4-5.8, and drying to obtain the swimming bladder extract.
As a preferred embodiment of the invention, the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1: (0.01-0.05): (0.01-0.05): (4-10).
As a preferred embodiment of the present invention, the mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide, the N-hydroxysuccinimide, the filtrate is 10: (0.5-1): (0.5-1).
As a preferred embodiment of the present invention, the freeze-drying is specifically:
vacuum freezing for 2-5 h at the vacuum degree of 0.5-0.55 mbar and the temperature of-60-70 ℃; vacuum freezing at the temperature of-45-55 ℃ for 10-15 hours under the vacuum degree of 0.5-0.55 mbar; vacuum freezing at the temperature of 18-22 ℃ for 10-15 h under the vacuum degree of 0.25-0.3 mbar; vacuum degree is 0.25-0.3 mbar, and vacuum freezing is carried out for 10-15 h at 0 ℃; vacuum degree is 0.25-0.3 mbar, and vacuum freezing is carried out for 4-10 hours at 8-12 ℃.
As a preferred embodiment of the present invention, the freeze-drying is specifically:
vacuum freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum degree 0.26mbar, vacuum freezing at 10deg.C for 6h.
The invention also provides the lyophilized powder of the fetal bovine serum, which is prepared by adopting the preparation method.
The invention has the beneficial effects that: (1) According to the invention, the swimming bladder extract, the sphingomonas fermentation product extract, the glycerol, the tris and the hyaluronic acid are creatively added in the freeze-drying process of the fetal calf serum, so that the fetal calf serum can be effectively protected, the generation of ice crystals is avoided, the occurrence of hemolysis is avoided, meanwhile, the aggregation or precipitation in the freezing process is avoided, the oxidation and the polymerization of active ingredients are prevented, the stability of the fetal calf serum is further effectively improved, and the fetal calf serum freeze-drying agent has a wide application prospect; (2) The swimming bladder extract prepared by the invention can effectively avoid oxidation, aggregation or precipitation of the fetal bovine serum, protect active ingredients of the fetal bovine serum, prevent the influence of ice crystals generated during freezing on the fetal bovine serum, and effectively improve the stability of the lyophilized powder of the fetal bovine serum.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
In the present invention, the specific dispersing and stirring treatment method is not particularly limited.
The reagents or instruments used in the invention are not pointed out by manufacturers, are all conventional products which can be obtained commercially, and the raw materials used in each comparative example and the raw materials used in the experiment parallel to each example are all the same commercial products except for specific descriptions.
Example 1
A preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
the mass ratio of the fetal calf serum to the swimming bladder extract to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100:2:1:0.6:0.4:0.2.
(2) Placing the mixed solution in a freeze dryer, and vacuum-freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 10deg.C for 6 hr to obtain lyophilized powder of fetal bovine serum.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis solution; the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1:0.03:0.02:5.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting pH to 5.6, and drying to obtain swimming bladder extract. The mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide to the filtrate is 10:0.8:0.8.
example 2
A preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
the mass ratio of the fetal calf serum to the swimming bladder extract to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100:0.5:2:0.2:0.6:0.1.
(2) Placing the mixed solution in a freeze dryer, and vacuum-freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 10deg.C for 6 hr to obtain lyophilized powder of fetal bovine serum.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis solution; the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1:0.03:0.02:5.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting pH to 5.6, and drying to obtain swimming bladder extract. The mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide to the filtrate is 10:0.8:0.8.
example 3
A preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
the mass ratio of the fetal calf serum to the swimming bladder extract to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100:3:0.5:1:0.1:0.5.
(2) Placing the mixed solution in a freeze dryer, and vacuum-freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 10deg.C for 6 hr to obtain lyophilized powder of fetal bovine serum.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis solution; the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1:0.03:0.02:5.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting pH to 5.6, and drying to obtain swimming bladder extract. The mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide to the filtrate is 10:0.8:0.8.
example 4
A preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
the mass ratio of the fetal calf serum to the swimming bladder extract to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100:0.8:1.5:0.3:0.5:0.1.
(2) Placing the mixed solution in a freeze dryer, and vacuum-freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 10deg.C for 6 hr to obtain lyophilized powder of fetal bovine serum.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis solution; the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1:0.03:0.02:5.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting pH to 5.6, and drying to obtain swimming bladder extract. The mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide to the filtrate is 10:0.8:0.8.
example 5
A preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
the mass ratio of the fetal calf serum to the swimming bladder extract to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100:2:0.8:0.8:0.2:0.3.
(2) Placing the mixed solution in a freeze dryer, and vacuum-freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 10deg.C for 6 hr to obtain lyophilized powder of fetal bovine serum.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis solution; the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1:0.03:0.02:5.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting pH to 5.6, and drying to obtain swimming bladder extract. The mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide to the filtrate is 10:0.8:0.8.
example 6
A preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
the mass ratio of the fetal calf serum to the swimming bladder extract to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100:2:1:0.6:0.4:0.2.
(2) Placing the mixed solution in a freeze dryer, and vacuum-freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 10deg.C for 6 hr to obtain lyophilized powder of fetal bovine serum.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis solution; the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1:0.05:0.01:4.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting pH to 5.6, and drying to obtain swimming bladder extract. The mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide to the filtrate is 10:0.5:0.5.
example 7
A preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
the mass ratio of the fetal calf serum to the swimming bladder extract to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100:2:1:0.6:0.4:0.2.
(2) Placing the mixed solution in a freeze dryer, and vacuum-freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 10deg.C for 6 hr to obtain lyophilized powder of fetal bovine serum.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis solution; the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1:0.01:0.05:10.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting pH to 5.6, and drying to obtain swimming bladder extract. The mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide to the filtrate is 10:1:1.
comparative example 1
Comparative example 1 differs from example 1 in that comparative example 1 was not added with swim bladder extract, all other things being equal.
A preparation method of lyophilized powder of fetal bovine serum comprises the following steps:
(1) Uniformly mixing fetal bovine serum, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
the mass ratio of the fetal bovine serum to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100:1:0.6:0.4:0.2.
(2) Placing the mixed solution in a freeze dryer, and vacuum-freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 10deg.C for 6 hr to obtain lyophilized powder of fetal bovine serum.
Comparative example 2
Comparative example 2 is different from example 1 in that the preparation method of the swimming bladder extract is different and the other are the same.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis solution; the mass ratio of the swimming bladder powder to the cellulase to the beta-glucanase to the water is 1:0.03:0.02:5.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate, and drying to obtain swimming bladder extract.
Comparative example 3
Comparative example 3 is different from example 1 in that the preparation method of the swimming bladder extract is different and the other are the same.
The preparation method of the swimming bladder extract comprises the following steps:
(1) Cleaning and drying the swim bladder of the grass carp, and crushing the grass carp to 400 meshes to obtain swim bladder powder;
(2) Adding swimming bladder powder, pectase and flavourzyme into water, and performing enzymolysis for 4 hours at 50 ℃ to obtain an enzymolysis liquid; the mass ratio of the swimming bladder powder to the pectase to the flavor protease to the water is 1:0.03:0.02:5.
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting pH to 5.6, and drying to obtain swimming bladder extract. The mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide to the filtrate is 10:0.8:0.8.
comparative example 4
Comparative example 4 differs from example 1 in that comparative example 4 was not added with Sphingomonas fermentation product extract, all other things being equal.
Test case
The lyophilized powders of examples and comparative examples were reconstituted with 9 times of water by mass, the viscosity was measured, and the viscosity was measured by standing at 30℃for 2 months.
TABLE 1
As can be seen from table 1, the lyophilized powder of fetal bovine serum according to the present invention has excellent stability.
As can be seen from comparative examples 1 to 7, example 1 is the best mode for carrying out the present invention, and the stability is the best; the mass ratio of the fetal bovine serum to the swimming bladder extract to the sphingomonas fermentation product extract to the glycerol to the tris (hydroxymethyl) aminomethane to the hyaluronic acid is 100: (0.5-3): (0.5-2): (0.2-1): (0.1 to 0.6): (0.1 to 0.5), the stability is further improved.
As can be seen from comparative examples 1 and 1-3, the swimming bladder extract prepared by the method provided by the invention has significantly improved stability, and the swimming bladder extract prepared by different preparation methods of the swimming bladder extract has different stability improvements, and compared with other methods, the swimming bladder extract prepared by the preparation method provided by the invention has significantly improved stability.
Comparative example 1 and comparative example 4 it can be seen that the present invention further improves stability by adding Sphingomonas fermentation product extract.
Finally, it should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, and that those skilled in the art will understand that changes can be made to the technical solutions of the invention or equivalents thereof without departing from the spirit and scope of the technical solutions of the invention.
Claims (10)
1. The preparation method of the lyophilized powder of the fetal bovine serum is characterized by comprising the following steps:
(1) Uniformly mixing fetal calf serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid to obtain a mixed solution;
(2) And (3) placing the mixed solution in a freeze dryer, and freeze-drying to obtain the fetal bovine serum freeze-dried powder.
2. The method for preparing the lyophilized powder of fetal bovine serum according to claim 1, wherein the mass ratio of fetal bovine serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid is 100: (0.5-3): (0.5-2): (0.2-1): (0.1 to 0.6): (0.1 to 0.5).
3. The method for preparing the lyophilized powder of fetal bovine serum according to claim 1, wherein the mass ratio of fetal bovine serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid is 100: (0.8-2): (0.8 to 1.5): (0.3 to 0.8): (0.2 to 0.5): (0.1 to 0.3).
4. The method for preparing the lyophilized powder of fetal bovine serum according to claim 1, wherein the mass ratio of fetal bovine serum, swimming bladder extract, sphingomonas fermentation product extract, glycerol, tris (hydroxymethyl) aminomethane and hyaluronic acid is 100:2:1:0.6:0.4:0.2.
5. the method for preparing the lyophilized powder of fetal bovine serum according to claim 1, wherein the method for preparing the swim bladder extract comprises the following steps:
(1) Cleaning, drying and crushing the swimming bladder of the grass carp to 200-500 meshes to obtain swimming bladder powder;
(2) Adding swimming bladder powder, cellulase and beta-glucanase into water for enzymolysis to obtain an enzymolysis liquid;
(3) Ultrafiltering the enzymolysis liquid with ultrafilter membrane with molecular weight cut-off of 1000Da, and ultrafiltering with ultrafilter membrane with molecular weight cut-off of 5000Da to obtain filtrate;
(4) Adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the filtrate, adjusting the pH to 5.4-5.8, and drying to obtain the swimming bladder extract.
6. The method for preparing the lyophilized powder of fetal bovine serum according to claim 5, wherein the mass ratio of the swim bladder powder to the cellulase to the beta-glucanase to the water is 1: (0.01-0.05): (0.01-0.05): (4-10).
7. The method for preparing the lyophilized powder of fetal bovine serum according to claim 5, wherein the mass ratio of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and filtrate is 10: (0.5-1): (0.5-1).
8. The method for preparing the lyophilized powder of fetal bovine serum according to claim 1, wherein the lyophilization specifically comprises:
vacuum freezing for 2-5 h at the vacuum degree of 0.5-0.55 mbar and the temperature of-60-70 ℃; vacuum freezing at the temperature of-45-55 ℃ for 10-15 hours under the vacuum degree of 0.5-0.55 mbar; vacuum freezing at the temperature of 18-22 ℃ for 10-15 h under the vacuum degree of 0.25-0.3 mbar; vacuum degree is 0.25-0.3 mbar, and vacuum freezing is carried out for 10-15 h at 0 ℃; vacuum degree is 0.25-0.3 mbar, and vacuum freezing is carried out for 4-10 hours at 8-12 ℃.
9. The method for preparing the lyophilized powder of fetal bovine serum according to claim 1, wherein the lyophilization specifically comprises:
vacuum freezing at-65deg.C for 4 hr at vacuum degree of 0.54 mbar; vacuum freezing at-50deg.C for 12 hr at vacuum degree of 0.52 mbar; vacuum freezing at-20deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum freezing at 0deg.C for 12 hr at vacuum degree of 0.26 mbar; vacuum degree 0.26mbar, vacuum freezing at 10deg.C for 6h.
10. The lyophilized powder of fetal bovine serum is characterized by being prepared by the preparation method of any one of claims 1-9.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116801861A (en) * | 2020-12-18 | 2023-09-22 | 欧莱雅 | Extracts of Sphingomonas bacteria |
| KR102619727B1 (en) * | 2023-06-02 | 2024-01-02 | 큐티스바이오 주식회사 | Liposome composition comprising Sphingomonas olei culture extract |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116801861A (en) * | 2020-12-18 | 2023-09-22 | 欧莱雅 | Extracts of Sphingomonas bacteria |
| KR102619727B1 (en) * | 2023-06-02 | 2024-01-02 | 큐티스바이오 주식회사 | Liposome composition comprising Sphingomonas olei culture extract |
Non-Patent Citations (4)
| Title |
|---|
| S. GERVASON 等: "Attenuation of negative effects of senescence in human skin using an extract from Sphingomonas hydrophobicum: development of new skin care solution", 《INTERNATIONAL JOURNAL OF COSMETIC SCIENCE》, vol. 41, 17 June 2019 (2019-06-17), pages 391 - 397, XP071470269, DOI: 10.1111/ics.12534 * |
| 屈义 等: "鳙鱼鱼鳔黏多糖的提取及其抗氧化性活性评", 《广东海洋大学学报》, vol. 38, no. 1, 28 February 2018 (2018-02-28), pages 47 - 53 * |
| 张颖 等: "鞘氨醇单胞菌的特性及应用研究进展", 《化学与生物工程》, vol. 38, no. 3, 31 December 2021 (2021-12-31), pages 6 - 131 * |
| 赵乃昕 等: "《医学细菌名称及分类鉴定》", 31 March 2013, 济南：山东大学出版社, pages: 383 - 386 * |
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