CN106619717A - Production method of deproteinized calf blood extractive intermediates - Google Patents
Production method of deproteinized calf blood extractive intermediates Download PDFInfo
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- CN106619717A CN106619717A CN201611176125.5A CN201611176125A CN106619717A CN 106619717 A CN106619717 A CN 106619717A CN 201611176125 A CN201611176125 A CN 201611176125A CN 106619717 A CN106619717 A CN 106619717A
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
The invention provides a production method of deproteinized calf blood extractive intermediates. The production method comprises the steps of isolating, grinding, denaturing, ultrafiltration, aseptic filling and the like. The raw blood collected herein is calf blood collected with 14 hours after birth of a calf, and has higher content of active ingredients than calf blood of the ordinary part; the production method has few steps and short production process, the purity of deproteinized calf blood extractive intermediates is fully guaranteed, and utilization value of calf blood is increased.
Description
Technical field：
The present invention relates to belong to deprotein extract production field, a kind of production of calf blood protein-removed extraction intermediate
Method.
Background technology：
At present existing calf blood protein-removed extraction intermediate mostly gathers the calf from after being born within 6 months, real
Border situation is that the ox age section ox source is less, thereby increases and it is possible to participates in the big ox blood in part, therefore affects the activity of the product.
And the production method of existing calf blood protein-removed extraction intermediate is all chemical leaching test, such as China substantially
Patent of invention:A kind of deproteinized calf blood extractive injection and its preparation side disclosed in CN200910246717.3
Method, its method adds sodium citrate for little ox blood, and the stirring that adds water heats up, and is cooled to -10～0 DEG C under stirring rapidly, is centrifuged, supernatant
PH value is adjusted, pepsin enzymolysis is added, is filtered, adjust pH value, add subtilopeptidase A enzymolysis, filtered；Filtrate adds
Ethanol water is stirred, and centrifugation, supernatant crosses activated-charcoal column, merges eluent and percolation liquid, is concentrated into without ethanol flavor, concentrate
Xylitol and epsilon-polylysine are added, is diluted with water, ultrafiltration；Ultrafiltrate dispenses filling rear ultraviolet sterilization, and packaging is obtained final product.It is such
Method is not only complicated, and needs to add substantial amounts of chemical substance, so as to destroy the pure property of calf blood protein-removed extraction.
The content of the invention：
For the above-mentioned deficiency of prior art, the technical problem to be solved is to provide a kind of deproteinized calf blood
The production method of extract intermediate, solves prior art complexity, and needs to add substantial amounts of chemical substance, so as to destroy
The problem of the pure property of calf blood protein-removed extraction.
The technology solution of the present invention is as follows：
A kind of production method of calf blood protein-removed extraction intermediate, including：
A, take blood：Whole bovine blood is gathered under cleaning ambient；
B, separation：Whole bovine blood constant temperature is placed after serum and clot natural separation into whole blood, centrifugation removes serum
After obtain clot, and clot cryogenic freezing is preserved；
C, mill：The clot of freezing is thawed to normal temperature and adds purified water, mixed liquor is obtained after being well mixed；
D, denaturation：Mixed liquor is added into the denaturation that heats up in retort, less than 50 DEG C are cooled to after denaturation carries out being centrifuged again
To centrifugate, by centrifugate freezen protective；
E, ultrafiltration：Ultrafiltration is carried out after centrifugate is thawed, collection obtains ultrafiltrate, and ultrafiltrate cryogenic freezing is preserved；
F, sterile filling：It is filling after aseptic filtration after ultrafiltrate is thawed, in obtaining calf blood protein-removed extraction
Between product, and by product in the middle of calf blood protein-removed extraction to 2～8 DEG C of preservations.
Preferably, taking in blood step, what blood collection was not taken food within being born 14 hours newly calves, and gathers environment
Should be in superclean bench.The serum separated of calf blood in birth 14 hours be cell culture medium main component it
One, with higher economic worth.And whole bovine blood separates the clot obtained after serum still containing higher active component, this
Invention obtains calf blood protein-removed extraction intermediate from separation and Extraction in the original clot for abandoning, and on the one hand takes full advantage of little
The all components of ox blood, improve its utilization rate；On the other hand also to a certain degree improve in the middle of calf blood protein-removed extraction
The active component of body.
Preferably, in separating step, constant temperature places temperature control at 25～35 DEG C, and standing time is 30 minutes, centrifugation
Rotating speed is 2000～4000 turns, centrifugation time 10～20 minutes, and Cord blood temperature is not less than -5 DEG C.
Preferably, milling in step, mixed using grinding mill during mixing, grinding mill rotating speed is 2000～3000
Rev/min, 50L mixed liquors of often milling need to mill 10 minutes, and the volume of the purified water of addition is 3 with the volume ratio of clot：1.
Preferably, in denaturing step, warming temperature is 80 ± 2 DEG C, holding time heating-up time is 30 minutes, and bar is centrifuged
Part is 40 ± 2 DEG C, and centrifugal rotational speed is 3000～5000 revs/min, and centrifugation time is 10～20 minutes, and Cord blood temperature is not low
In -5 DEG C.
Preferably, in ultrafiltration step, the membrane retention molecular weight that ultrafiltration is used is 10KD.
Preferably, in sterile filling step, pore size filter is 0.22 μm.
The beneficial effects of the present invention is：
Compared with prior art, the present invention has the advantages that：1st, the raw material blood that the present invention is gathered comes from just
The bovine blood gathered within 14 hours after the birth of birth, than the active component content in the bovine blood of common process more
It is high；2nd, the present invention little ox blood is progressively processed using pure physical means, so as to obtain calf blood protein-removed extraction in
Mesosome, not only step is few, and the technological process of production is short, more due to using pure physical means, deproteinized calf blood being completely secured and having carried
The pure property of thing intermediate is taken, the value of little ox blood is improve.
Specific embodiment：
Embodiment 1：A kind of production method of calf blood protein-removed extraction intermediate, including：
A, take blood：Whole bovine blood is gathered under cleaning ambient；
B, separation：Whole bovine blood constant temperature is placed after serum and clot natural separation into whole blood, centrifugation removes serum
After obtain clot, and clot cryogenic freezing is preserved；
C, mill：The clot of freezing is thawed to normal temperature and adds purified water, mixed liquor is obtained after being well mixed；
D, denaturation：Mixed liquor is added into the denaturation that heats up in retort, less than 50 DEG C are cooled to after denaturation carries out being centrifuged again
To centrifugate, by centrifugate freezen protective；
E, ultrafiltration：Ultrafiltration is carried out after centrifugate is thawed, collection obtains ultrafiltrate, and ultrafiltrate cryogenic freezing is preserved；
F, sterile filling：It is filling after aseptic filtration after ultrafiltrate is thawed, in obtaining calf blood protein-removed extraction
Between product, and by product in the middle of calf blood protein-removed extraction to 2～8 DEG C of preservations.
In taking blood step, what blood collection was not taken food within being born 1 hour newly calves, and collection environment should be in ultra-clean work
Make in platform.
In separating step, constant temperature places temperature control at 25 DEG C, and standing time is 30 minutes, and centrifugal rotational speed is 2000 turns,
Centrifugation time 10 minutes, Cord blood temperature is not less than -5 DEG C.
Mill in step, mixed using grinding mill during mixing, grinding mill rotating speed is 2000 revs/min, and often mill 50L
Mixed liquor needs to mill 10 minutes, and the volume of the purified water of addition is 3 with the volume ratio of clot：1.
In denaturing step, warming temperature is 80 ± 2 DEG C, and holding time heating-up time is 30 minutes, and centrifugal condition is 40 ± 2
DEG C, centrifugal rotational speed is 3000 revs/min, and centrifugation time is 10～20 minutes, and Cord blood temperature is not less than -5 DEG C.
In ultrafiltration step, the membrane retention molecular weight that ultrafiltration is used is 10KD.
In sterile filling step, pore size filter is 0.22 μm.
The correlated traits detection of table 1, the calf blood protein-removed extraction of the production of embodiment 1
Embodiment 2：A kind of production method of calf blood protein-removed extraction intermediate, including：
A, take blood：Whole bovine blood is gathered under cleaning ambient；
B, separation：Whole bovine blood constant temperature is placed after serum and clot natural separation into whole blood, centrifugation removes serum
After obtain clot, and clot cryogenic freezing is preserved；
C, mill：The clot of freezing is thawed to normal temperature and adds purified water, mixed liquor is obtained after being well mixed；
D, denaturation：Mixed liquor is added into the denaturation that heats up in retort, less than 50 DEG C are cooled to after denaturation carries out being centrifuged again
To centrifugate, by centrifugate freezen protective；
E, ultrafiltration：Ultrafiltration is carried out after centrifugate is thawed, collection obtains ultrafiltrate, and ultrafiltrate cryogenic freezing is preserved；
F, sterile filling：It is filling after aseptic filtration after ultrafiltrate is thawed, in obtaining calf blood protein-removed extraction
Between product, and by product in the middle of calf blood protein-removed extraction to 2～8 DEG C of preservations.
In taking blood step, what blood collection was not taken food for 2 hours from being born newly calves, and collection environment should be in superclean bench
It is interior.
In separating step, constant temperature places temperature control at 35 DEG C, and standing time is 30 minutes, and centrifugal rotational speed is 4000 turns,
Centrifugation time 20 minutes, Cord blood temperature is not less than -5 DEG C.
Mill in step, mixed using grinding mill during mixing, grinding mill rotating speed is 3000 revs/min, and often mill 50L
Mixed liquor needs to mill 10 minutes, and the volume of the purified water of addition is 3 with the volume ratio of clot：1.
In denaturing step, warming temperature is 80 ± 2 DEG C, and holding time heating-up time is 30 minutes, and centrifugal condition is 40 ± 2
DEG C, centrifugal rotational speed is 5000 revs/min, and centrifugation time is 10～20 minutes, and Cord blood temperature is not less than -5 DEG C.
In ultrafiltration step, the membrane retention molecular weight that ultrafiltration is used is 10KD.
In sterile filling step, pore size filter is 0.22 μm.
The correlated traits detection of table 2, the calf blood protein-removed extraction of the production of embodiment 2
Embodiment 3：A kind of production method of calf blood protein-removed extraction intermediate, including：
A, take blood：Whole bovine blood is gathered under cleaning ambient；
B, separation：Whole bovine blood constant temperature is placed after serum and clot natural separation into whole blood, centrifugation removes serum
After obtain clot, and clot cryogenic freezing is preserved；
C, mill：The clot of freezing is thawed to normal temperature and adds purified water, mixed liquor is obtained after being well mixed；
D, denaturation：Mixed liquor is added into the denaturation that heats up in retort, less than 50 DEG C are cooled to after denaturation carries out being centrifuged again
To centrifugate, by centrifugate freezen protective；
E, ultrafiltration：Ultrafiltration is carried out after centrifugate is thawed, collection obtains ultrafiltrate, and ultrafiltrate cryogenic freezing is preserved；
F, sterile filling：It is filling after aseptic filtration after ultrafiltrate is thawed, in obtaining calf blood protein-removed extraction
Between product, and by product in the middle of calf blood protein-removed extraction to 2～8 DEG C of preservations.
In taking blood step, what blood collection was not taken food for 14 hours from being born newly calves, and collection environment should be in ultra-clean work
In platform.
In separating step, constant temperature places temperature control at 30 DEG C, and standing time is 30 minutes, and centrifugal rotational speed is 3000 turns,
Centrifugation time 15 minutes, Cord blood temperature is not less than -5 DEG C.
Mill in step, mixed using grinding mill during mixing, grinding mill rotating speed is 2500 revs/min, and often mill 50L
Mixed liquor needs to mill 10 minutes, and the volume of the purified water of addition is 3 with the volume ratio of clot：1.
In denaturing step, warming temperature is 80 ± 2 DEG C, and holding time heating-up time is 30 minutes, and centrifugal condition is 40 ± 2
DEG C, centrifugal rotational speed is 4000 revs/min, and centrifugation time is 15 minutes, and Cord blood temperature is not less than -5 DEG C.
In ultrafiltration step, the membrane retention molecular weight that ultrafiltration is used is 10KD.
In sterile filling step, pore size filter is 0.22 μm.
The correlated traits detection of table 3, the calf blood protein-removed extraction of the production of embodiment 3
Presently preferred embodiments of the present invention is the foregoing is only, those of ordinary skill in the art are not making creative work
Under the premise of the every other embodiment that obtained, should all belong to the scope of protection of the invention.
Claims (7)
1. a kind of production method of calf blood protein-removed extraction intermediate, it is characterised in that：Including：
A, take blood：Whole bovine blood is gathered under cleaning ambient；
B, separation：Whole bovine blood constant temperature is placed after serum and clot natural separation into whole blood, centrifugation removed and obtained after serum
To clot, and clot cryogenic freezing is preserved；
C, mill：The clot of freezing is thawed to normal temperature and adds purified water, mixed liquor is obtained after being well mixed；
D, denaturation：Mixed liquor is added and is heated up denaturation in retort, be cooled to after denaturation less than 50 DEG C carry out again centrifugation obtain from
Heart liquid, by centrifugate freezen protective；
E, ultrafiltration：Ultrafiltration is carried out after centrifugate is thawed, collection obtains ultrafiltrate, and ultrafiltrate cryogenic freezing is preserved；
F, sterile filling：It is filling after aseptic filtration after ultrafiltrate is thawed, obtain in the middle of calf blood protein-removed extraction
Product, and by product in the middle of calf blood protein-removed extraction to 2～8 DEG C of preservations.
2. as described in claim 1 a kind of production method of calf blood protein-removed extraction intermediate, it is characterised in that：Institute
State and take in blood step, what blood collection was not taken food within being born 14 hours newly calves, collection environment should be in superclean bench
It is interior.
3. as described in claim 1 a kind of production method of calf blood protein-removed extraction intermediate, it is characterised in that：Institute
In stating separating step, constant temperature places temperature control at 25～35 DEG C, and standing time is 30 minutes, and centrifugal rotational speed is 2000～4000
Turn, centrifugation time 10～20 minutes, Cord blood temperature is not less than -5 DEG C.
4. as described in claim 1 a kind of production method of calf blood protein-removed extraction intermediate, it is characterised in that：Institute
State and mill in step, mixed using grinding mill during mixing, grinding mill rotating speed is 2000～3000 revs/min, and often mill 50L
Mixed liquor needs to mill 10 minutes, and the volume of the purified water of addition is 3 with the volume ratio of clot：1.
5. as described in claim 2 a kind of production method of calf blood protein-removed extraction intermediate, it is characterised in that：Institute
In stating denaturing step, warming temperature is 80 ± 2 DEG C, and holding time heating-up time is 30 minutes, and centrifugal condition is 40 ± 2 DEG C, from
Heart rotating speed is 3000～5000 revs/min, and centrifugation time is 10～20 minutes, and Cord blood temperature is not less than -5 DEG C.
6. a kind of production method of calf blood protein-removed extraction intermediate as described in claim 1, is characterized in that：It is described
In ultrafiltration step, the membrane retention molecular weight that ultrafiltration is used is 10KD.
7. a kind of production method of calf blood protein-removed extraction intermediate as described in claim 1, is characterized in that：It is described
In sterile filling step, pore size filter is 0.22 μm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110812442A (en) * | 2019-12-06 | 2020-02-21 | 湖北威仕生物药业股份有限公司 | Preparation method and application of five-ginseng-astragalus oral solution |
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Application publication date: 20170510 |