CN115708798A - One-step hydrolysis preparation method of sheep placenta extract - Google Patents
One-step hydrolysis preparation method of sheep placenta extract Download PDFInfo
- Publication number
- CN115708798A CN115708798A CN202310032511.0A CN202310032511A CN115708798A CN 115708798 A CN115708798 A CN 115708798A CN 202310032511 A CN202310032511 A CN 202310032511A CN 115708798 A CN115708798 A CN 115708798A
- Authority
- CN
- China
- Prior art keywords
- homogenate
- sheep placenta
- placenta extract
- thawing
- step hydrolysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002826 placenta Anatomy 0.000 title claims abstract description 56
- 241001494479 Pecora Species 0.000 title claims abstract description 51
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 30
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- 238000005119 centrifugation Methods 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 20
- 238000010257 thawing Methods 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 238000005360 mashing Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 10
- 238000007710 freezing Methods 0.000 claims description 10
- 230000008014 freezing Effects 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 7
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000000413 hydrolysate Substances 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 2
- 238000005352 clarification Methods 0.000 claims description 2
- 230000003301 hydrolyzing effect Effects 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 6
- 239000000047 product Substances 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 description 17
- 230000001815 facial effect Effects 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 239000008055 phosphate buffer solution Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 4
- 239000006071 cream Substances 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 210000005059 placental tissue Anatomy 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000003020 moisturizing effect Effects 0.000 description 3
- 230000037303 wrinkles Effects 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 229920002385 Sodium hyaluronate Polymers 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000037394 skin elasticity Effects 0.000 description 2
- 229940010747 sodium hyaluronate Drugs 0.000 description 2
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(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- Chemical compound [Na+].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 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 2
- 230000002087 whitening effect Effects 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002233 benzalkonium bromide Drugs 0.000 description 1
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- ZCTXEAQXZGPWFG-UHFFFAOYSA-N imidurea Chemical compound O=C1NC(=O)N(CO)C1NC(=O)NCNC(=O)NC1C(=O)NC(=O)N1CO ZCTXEAQXZGPWFG-UHFFFAOYSA-N 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- -1 polyoxyethylene lauryl ether Polymers 0.000 description 1
- 229920000259 polyoxyethylene lauryl ether Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000013190 sterility testing Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
Images
Landscapes
- Cosmetics (AREA)
Abstract
The invention relates to the technical field of biological products, in particular to a one-step hydrolysis preparation method of a sheep placenta extract, which takes sheep placenta as a raw material, prepares homogenate, repeatedly freezes and freezes the homogenate until the cell breakage rate reaches 95%, then obtains supernatant through centrifugation, hydrolyzes the supernatant in one step, centrifuges the supernatant after the hydrolysis, and sterilizes the supernatant, thereby obtaining the sheep placenta extract with stable quality and better activity.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a one-step hydrolysis preparation method of a sheep placenta extracting solution.
Background
The placenta is the coating tissue stripped from the fetus after the production of the mammal, is rich in blood vessels and various stromal cells, has the functions of protecting and supporting the development of the embryo of the mammal, and the placenta extract is the product of the placenta tissue after hydrolysis and acidolysis, contains a plurality of amino acids and immunoregulation active substances, and can be used as the stock solution or additive of cosmetics.
At present, the preparation of the placenta extracting solution generally adopts a multi-time hydrolysis process, and the method easily causes the active ingredients in the placenta to be damaged, so that the prepared placenta extracting solution has poor activity. For example, chinese patent No. CN 101721458B discloses a method for preparing human placenta tissue fluid, which comprises obtaining a crude placenta tissue fluid by ultrasonic extraction, then sequentially performing acid hydrolysis and alkali hydrolysis, and performing fine filtration after hydrolysis to obtain a fine-filtered placenta tissue fluid. Thus, further improvements are needed.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a one-step hydrolysis preparation method of a sheep placenta extracting solution, the one-step hydrolysis preparation method of the sheep placenta extracting solution takes sheep placenta as a raw material, the preparation method is simple, and the prepared sheep placenta extracting solution is safe and effective and the quality is easy to control.
In order to achieve the technical effect, the invention adopts the following technical scheme:
a one-step hydrolysis preparation method of sheep placenta extract comprises the following steps:
s1: sheep placenta was pre-treated to isolate cotyledons:
s2: preparing the cotyledons into homogenate;
s3: repeatedly freezing and thawing the homogenate until the cell breakage rate reaches 95 percent;
s4: centrifuging the homogenate obtained after repeated freeze thawing, and collecting a supernatant after the centrifugation is finished;
s5: filtering the supernatant obtained in the step S4 to obtain clear filtrate, and hydrolyzing the clear filtrate at the temperature of 60-80 ℃ to obtain hydrolysate;
s6: centrifuging the hydrolysate, and sterilizing the supernatant to obtain sheep placenta extractive solution.
Further, the preprocessing of S1 includes: taking fresh sheep placenta or unfreezing the frozen sheep placenta, separating and rinsing cotyledons, and shearing the rinsed cotyledons into massive tissues with the diameter of 2-3 mm.
Further, the rinsing conditions are: rinsing for 2-4 times, and rinsing by using 100-2000 ml of PBS solution precooled at 4 ℃ in volume for each rinsing.
Further, the S2 specifically includes:
s21: pre-washing the homogenizer, wherein the pre-washing comprises washing by adopting PBS (phosphate buffer solution) and then washing by using normal saline, and specifically comprises the following steps: firstly, 150-200ml of PBS precooled at 4 ℃ is added into a mashing cup of a homogenizer, the homogenizer is operated for 2-3 min, then the PBS is poured out, and the mashing cup is repeatedly washed for 2-3 times by adopting the PBS; then 100-200 ml of pre-cooled normal saline at 4 ℃ is added, the homogenizer is operated for 2-3 min, and the normal saline is poured out.
S22: putting the rinsed cotyledon into a mashing cup of a homogenizer, adding 4 ℃ precooled normal saline according to 2-3 ml/g, rotating a speed regulating knob, slowly accelerating until the speed is 2/3 of the highest speed, mashing for 2 minutes, stopping the machine, pouring the homogenate into a container, adding 4 ℃ precooled normal saline according to 2-3 ml/g, washing the mashing cup, and pouring the washing liquid into a beaker.
Further, the S3 specifically is: and (3) subpackaging the homogenate prepared in the step (2), freezing and storing the homogenate at the temperature of-30 to-20 ℃ for 24-26 h, taking out the homogenate to be thawed, repeatedly freezing and thawing for 4-5 times, and storing the homogenate in a refrigerator at the temperature of 4 ℃ for later use. Preferably, the freezing temperature is-30 ℃.
Preferably, the thawing process specifically comprises: taking out the frozen homogenate, thawing the homogenate by 1/5 to 1/3 of the volume at room temperature below 37 ℃, then placing the homogenate in a water bath below 37 ℃ until the volume of the homogenate is more than 95 percent of the volume of the thaw or the thaw is completed, and shaking the homogenate every 5 to 10min in the thawing process, wherein the shaking can be manual shaking or mechanical shaking.
Further, the clarification filtration is carried out by adopting a filter membrane, the aperture of the selected filter membrane is 0.8 μm, and the filter membrane is soaked in sterilized water for 20min and then is soaked in normal saline for 10min before use.
Further, the hydrolysis in S5 is specifically: preheating the water bath kettle to 60-80 ℃, preferably 80 ℃, putting the filtrate into a container, transferring the container into the water bath kettle for water bath to carry out hydrolysis, wherein the treatment time is 20-40 min, preferably 30min.
Further, the sterilization in S6 adopts a high-pressure steam sterilization method, specifically: the sterilization temperature is 121 ℃, and the sterilization is carried out for 2 to 3 times, 20 to 30min each time.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a one-step hydrolysis preparation method of sheep placenta extract, which takes sheep placenta as a raw material, prepares homogenate, repeatedly freezes and freezes the homogenate until the cell breakage rate reaches 95%, then obtains supernatant through centrifugation and carries out one-step hydrolysis on the supernatant, then centrifugally separates the supernatant after hydrolysis, and sterilizes the supernatant, thus obtaining the sheep placenta extract with stable quality and better activity.
Drawings
Fig. 1 is a comparative graph of skin spots before and after the test of volunteer No. 1 (experimental group one) provided in example 5 of the present invention (left is before the test, right is after the test);
fig. 2 is a comparison graph of skin wrinkle detection before and after the test of volunteer No. 2 (experimental group two) provided in example 5 of the present invention (left is before the test, right is after the test);
fig. 3 is a comparative chart of the skin purple test of volunteers No. 3 (experimental group three) before and after the test (left is before the test and right is after the test) provided in example 5 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents or apparatus used are those which are not indicated by the manufacturer, are commercially available reagents and materials unless otherwise specified, and are not indicated by specific conditions in the examples, and are carried out according to conventional conditions or conditions recommended by the manufacturer.
Example 1
This example is a first preparation example of the present invention, and provides a method for preparing a sheep placenta extract, where the method includes:
s1: sheep placenta was pre-treated to isolate cotyledons:
taking out the frozen sheep placenta from-20 ℃, putting the sheep placenta into a refrigerator at 4 ℃ or thawing the sheep placenta at room temperature, separating cotyledons, rinsing the cotyledons by PBS prepared in advance and pre-cooled at 4 ℃, and rinsing the cotyledons for 3 to 4 times each time in 150 to 250 ml;
the placental cotyledons were then trimmed to a size of 2-3 mm in diameter using surgical scissors, weighed and recorded as weight M1, and then stored at 4 ℃ until homogenized.
S2: cotyledons were prepared as a homogenate comprising:
s21: pre-washing the homogenizer: adding 150-200ml of PBS precooled at 4 ℃ into a mashing cup of a homogenizer, starting the homogenizer to run for 1 minute, stopping the homogenizer for 2 minutes, pouring out the PBS, and repeating the process for 2 times.
Then 100ml of pre-cooled normal saline at 4 ℃ is added, the machine is started to run for 1 minute, then the machine is stopped for 2 minutes, and the normal saline is poured out.
S22: and (3) putting the rinsed cotyledon into a mashing cup of a homogenizer to prepare homogenate, wherein the specific operation is as follows:
placing the block tissues in a mashing cup, adding 4 ℃ pre-cooled physiological saline according to 2ml/g, rotating a speed regulating knob, slowly accelerating until the speed is 2/3 of the highest speed, mashing for 2 minutes, stopping the machine, pouring the homogenate into a beaker, adding 4 ℃ pre-cooled physiological saline according to 2ml/g, washing the mashing cup, and pouring the washing liquid into the beaker.
S3: repeatedly freezing and thawing the homogenate until the cell breakage rate reaches 95 percent, specifically comprising the following steps: and subpackaging the homogenate into 15ml centrifuge tubes, wherein 10ml of homogenate is placed in each tube, and the tubes are frozen and stored in a refrigerator below-30 to-20 ℃ for 24 hours and then thawed. The homogenate is thawed at room temperature below 37 deg.C, and after 1/4 part of homogenate is thawed, it can be quickly thawed in water bath at temperature below 37 deg.C to promote cell rupture, and the homogenate should not contact the bottle mouth, and should not be too large, and should not be shaken every 5 min during thawing.
After homogenate is completely or 90% is thawed, the homogenate is immediately sent to a refrigerator with the temperature of-30 ℃ for freezing storage and is placed in order. The process is freeze thawing once, and the freeze thawing is carried out for 4 times for standby and is to be centrifuged.
S4: centrifuging the homogenate after repeated freeze-thawing, and collecting the supernatant after the centrifugation is finished, comprising:
before centrifugation, a rotary head of the centrifuge is wiped by gauze dipped with benzalkonium bromide or alcohol, the centrifuge is precooled at 4 ℃, then the thawed homogenate is taken out, the centrifuge is shut down after centrifugation operation is carried out for 20 minutes at 4000-8000 rpm, and supernatant obtained by centrifugation is collected.
S5: and (3) filtering the supernatant obtained in the step (S4) to obtain clear filtrate, wherein during filtering, the supernatant is clarified and filtered by using a 500ml culture medium filter (connected with a vacuum pump) which is prepared in advance, the aperture of a filter membrane is 0.8 mu m, collecting the filtrate, freezing and storing 100ul of small samples for detection, and soaking the filter membrane in sterile water for 20min and normal saline for 10min before use.
After filtration, the water bath kettle is preheated to 80 ℃ in advance, and the filtrate is transferred into the water bath kettle for hydrolysis, wherein the treatment time is 30min.
S6: centrifuging the hydrolysate under the following conditions: 4000rpm for 15 minutes; separating the supernatant at the temperature of 8 ℃, and performing high-pressure steam sterilization on the supernatant at the sterilization temperature of 121 ℃ for 20-30 min each time for 2-3 times to obtain the sheep placenta extract.
S7: diluting the obtained sheep placenta extractive solution, filtering, calculating the dilution volume according to M1, and diluting to obtain sheep placenta extractive solution with concentration of 0.2g/ml (mass fraction), which can be directly used for preparing skin care product.
S8: subpackaging and detecting, namely sampling the sheep placenta extract extracted from each batch, detecting the protein content, osmotic pressure, aseptic detection and endotoxin, and then subpackaging the sheep placenta extract qualified by detection by adopting a brown penicillin bottle.
The detection result shows that the protein content range is 0.1 plus or minus 0.03 mg/ml; the osmotic pressure is 250 +/-30 Osml/kg; sterility testing should be negative; and if the endotoxin detection is lower than 0.5EU, the prepared sheep placenta extract is qualified and can be further subpackaged and used.
Example 2
The present embodiment is a second preparation embodiment of the present invention, and provides a skin introduction repairing liquid and a preparation method thereof, where the skin introduction repairing liquid includes a sheep placenta extracting solution prepared by the method of the present invention and sodium hyaluronate, and specifically:
the skin introduction repair solution comprises the sheep placenta extracting solution with the concentration of 0.2g/ml (mass fraction) prepared in the embodiment 1, the sheep placenta extracting solution with the concentration of 0.2g/ml (mass fraction) is added into a sodium hyaluronate solution according to the mass ratio of 1.
Example 3
The present embodiment provides a sheep placenta extract facial mask and a preparation method thereof, in which the sheep placenta extract facial mask includes a facial mask matrix and a facial mask solution, the facial mask matrix may be prepared by using cosmetic cotton or silk, the facial mask solution includes a first component and a second component, and the first component and the second component respectively include the following components in parts by mass:
a first component: 12 parts of polyvinyl alcohol, 10 parts of glycerol, 1 part of imidazolidinyl urea, 5 parts of ethanol, 7 parts of polyoxyethylene lauryl ether, a proper amount of essence and a preservative, and purified water is used for complementing the system to 100 parts.
A second component: the sheep placenta extract was prepared in example 1 at a concentration of 0.2g/ml (mass fraction), and was added in an amount of 10% by volume of the first component.
The preparation method specifically comprises the following steps: the facial mask is prepared by preparing a facial mask matrix from cosmetic cotton or silk, uniformly mixing a first component and a second component to prepare a facial mask liquid, and adsorbing the facial mask liquid on the facial mask matrix at 4 ℃, so that the facial mask can be directly applied in use, and the effects of skin whitening, repairing and anti-aging can be achieved by long-term facial application.
Example 4
This embodiment is a fourth preparation example of the present invention, and provides a sheep placenta extract moisturizing cream and a preparation method thereof, in which the sheep placenta extract moisturizing cream prepared in the above example 1 and having a concentration of 0.2g/ml (mass fraction) is used as a raw material, and is prepared into a cream product according to a mass ratio of 1.
Example 5
This example is an application example of the present invention in which skin care products were prepared as in examples 2-4, respectively, and volunteers were organized for use testing.
Among 30 testers, the testers were divided into 3 groups of 10 people each, the first group sought to whiten skin and lighten spots, the second group sought to refine pores and reduce wrinkles, and the third group sought to tighten skin and increase skin elasticity.
In the test process, the skin repair liquid prepared in example 2 was introduced by using an introduction instrument 1 time per week for 3 consecutive weeks as a test period, and the mask pack of example 3 and the moisturizing cream of example 4 were used in combination after introduction. The skin of each subject was examined using a VISIA medical skin detector and recorded prior to introduction.
After 2 test periods (6 weeks) were completed, the volunteers of each experimental group were examined using a VISIA skin examination apparatus and the corresponding data was analyzed and statistically examined. The test results are: the effective rate of the effect of whitening and fading the spots of the experiment group I reaches 40 percent, the effective rate of the experiment group II is 30 percent for improving wrinkles and pores, and the effective rate of the experiment group III is 20 percent for tightening and increasing the skin elasticity.
And a second test result: referring to fig. 1 to 3, this embodiment further provides a VISIA detection contrast chart of some volunteers before and after the test.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.
Claims (10)
1. A one-step hydrolysis preparation method of a sheep placenta extract is characterized by comprising the following steps:
s1: sheep placenta was pretreated to isolate cotyledons:
s2: preparing the cotyledons into homogenate;
s3: repeatedly freezing and thawing the homogenate until the cell breakage rate reaches 95 percent;
s4: centrifuging the homogenate after repeated freeze thawing, and collecting the supernatant after the centrifugation is finished;
s5: filtering the supernatant obtained in the step S4 to obtain clear filtrate, and hydrolyzing the clear filtrate at the temperature of 60-80 ℃ to obtain hydrolysate;
s6: centrifuging the hydrolysate, and sterilizing the supernatant to obtain sheep placenta extractive solution.
2. The one-step hydrolysis preparation method of sheep placenta extract as claimed in claim 1, wherein: the pretreatment of S1 comprises the following steps: taking fresh sheep placenta or unfreezing the frozen sheep placenta, separating and rinsing cotyledons, and shearing the rinsed cotyledons into massive tissues with the diameter of 2-3 mm.
3. The method for preparing sheep placenta extract by one-step hydrolysis as claimed in claim 2, wherein the rinsing conditions are as follows: rinsing for 2-4 times, and rinsing by using 100-2000 ml of PBS solution precooled at 4 ℃ in volume for each rinsing.
4. The one-step hydrolysis preparation method of the sheep placenta extract according to claim 1, wherein the step S2 specifically comprises:
s21: pre-washing the homogenizer;
s22: putting the rinsed cotyledon into a mashing cup of a homogenizer, adding 4 ℃ precooled physiological saline according to 2-3 ml/g, rotating a speed regulating knob, slowly accelerating until the highest speed is 2/3, mashing for 2 minutes, stopping the machine, pouring the homogenate into a container, adding 4 ℃ precooled physiological saline according to 2-3 ml/g, washing the mashing cup, and pouring the washing liquid into a beaker.
5. The one-step hydrolysis preparation method of the sheep placenta extract according to claim 1, wherein the step S21 is specifically: firstly, 150-200ml of PBS precooled at 4 ℃ is added into a mashing cup of a homogenizer, the homogenizer is operated for 2-3 min, then the PBS is poured out, and the mashing cup is repeatedly washed for 2-3 times by adopting the PBS; then 100-200 ml of pre-cooled physiological saline at 4 ℃ is added, the homogenizer is operated for 2-3 min, and the physiological saline is poured out.
6. The one-step hydrolysis preparation method of the sheep placenta extract according to claim 1, wherein the S3 is specifically: and (3) subpackaging the homogenate prepared in the step (2), freezing and storing the homogenate for 24-26 h at the temperature of-30 to-20 ℃, taking out the homogenate for thawing, repeatedly freezing and thawing for 4-5 times, and storing the homogenate in a refrigerator at the temperature of 4 ℃.
7. The one-step hydrolysis preparation method of sheep placenta extract as claimed in claim 6, wherein: the defrosting process specifically comprises the following steps: taking out the frozen homogenate, thawing the homogenate by 1/5 to 1/3 of the volume at room temperature below 37 ℃, then placing the homogenate in a water bath below 37 ℃ until the thawing is more than 95 percent or completely thawed, and shaking the homogenate every 5 to 10min in the thawing process.
8. The one-step hydrolysis preparation method of the sheep placenta extract as claimed in claim 1, wherein the method comprises the steps of: the clarification filtration adopts a filter membrane for filtration, and the aperture of the filter membrane is 0.8 mu m.
9. The method for preparing the sheep placenta extract by one-step hydrolysis as claimed in claim 1, wherein the hydrolysis in S5 is specifically: preheating the water bath to 60-80 deg.c, loading the filtrate into container and transferring to water bath for hydrolysis for 20-40 min.
10. The one-step hydrolysis preparation method of the sheep placenta extract as claimed in claim 1, wherein the method comprises the steps of: and the sterilization in the S6 adopts a high-pressure steam sterilization method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310032511.0A CN115708798A (en) | 2023-01-10 | 2023-01-10 | One-step hydrolysis preparation method of sheep placenta extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310032511.0A CN115708798A (en) | 2023-01-10 | 2023-01-10 | One-step hydrolysis preparation method of sheep placenta extract |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115708798A true CN115708798A (en) | 2023-02-24 |
Family
ID=85236275
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310032511.0A Pending CN115708798A (en) | 2023-01-10 | 2023-01-10 | One-step hydrolysis preparation method of sheep placenta extract |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115708798A (en) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298709A (en) * | 2000-11-03 | 2001-06-13 | 内蒙古草原兴发股份有限公司 | Process for preparing polypeptide-nucleic acid nutritive liquid of sheep placenta |
| JP2006232693A (en) * | 2005-02-23 | 2006-09-07 | Nitta Gelatin Inc | Animal-derived extract composition, method for producing the same, and cosmetic composition |
| CN1843377A (en) * | 2006-02-13 | 2006-10-11 | 北京凯诗伦生物技术有限公司 | Sheep placenta essence and its preparing process |
| CN1944459A (en) * | 2006-10-25 | 2007-04-11 | 中国辐射防护研究院 | Preparation method of placenta polypeptide for injection |
| KR20080096219A (en) * | 2007-04-27 | 2008-10-30 | 동덕제약주식회사 | Method of preparing composition for cosmetics and medicine from porcine placenta |
| CN101721458A (en) * | 2009-10-30 | 2010-06-09 | 葛龙海 | Method for preparing human placenta tissue fluid |
| CN105456294A (en) * | 2014-09-05 | 2016-04-06 | 中国辐射防护研究院 | Preparing method of placenta peptide liquor for livestock and placenta peptide liquor for livestock |
| JP2017100979A (en) * | 2015-11-30 | 2017-06-08 | 株式会社協和 | Method for producing placenta extract |
| CN107213106A (en) * | 2017-06-29 | 2017-09-29 | 佛山市南海区普罗圣塔生物科技有限公司 | Essence, facial mask liquid and preparation method thereof |
| CN109568254A (en) * | 2018-12-18 | 2019-04-05 | 江西润泽药业有限公司 | A kind of Plancenta Histolysate's injection and preparation method thereof |
| CN110721146A (en) * | 2019-08-01 | 2020-01-24 | 浙江中林泰网络科技有限公司 | Hydrolyzed embryo protein wrinkle-removing liquid and preparation method thereof |
| CN111067839A (en) * | 2020-01-16 | 2020-04-28 | 广东丽姿生物科技有限公司 | Conditioning repairing agent and preparation method thereof |
| CN111467292A (en) * | 2020-04-03 | 2020-07-31 | 焕生汇生物基因技术(北京)有限公司 | Preparation method of human placenta extract supernatant |
| US20220287952A1 (en) * | 2021-03-15 | 2022-09-15 | Guangzhou Saliai Biological Genetic Engineering Co., Ltd. | Compositions containing exosomes from animal placenta, methods for producing the same and uses thereof |
-
2023
- 2023-01-10 CN CN202310032511.0A patent/CN115708798A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298709A (en) * | 2000-11-03 | 2001-06-13 | 内蒙古草原兴发股份有限公司 | Process for preparing polypeptide-nucleic acid nutritive liquid of sheep placenta |
| JP2006232693A (en) * | 2005-02-23 | 2006-09-07 | Nitta Gelatin Inc | Animal-derived extract composition, method for producing the same, and cosmetic composition |
| CN1843377A (en) * | 2006-02-13 | 2006-10-11 | 北京凯诗伦生物技术有限公司 | Sheep placenta essence and its preparing process |
| CN1944459A (en) * | 2006-10-25 | 2007-04-11 | 中国辐射防护研究院 | Preparation method of placenta polypeptide for injection |
| KR20080096219A (en) * | 2007-04-27 | 2008-10-30 | 동덕제약주식회사 | Method of preparing composition for cosmetics and medicine from porcine placenta |
| CN101721458A (en) * | 2009-10-30 | 2010-06-09 | 葛龙海 | Method for preparing human placenta tissue fluid |
| CN105456294A (en) * | 2014-09-05 | 2016-04-06 | 中国辐射防护研究院 | Preparing method of placenta peptide liquor for livestock and placenta peptide liquor for livestock |
| JP2017100979A (en) * | 2015-11-30 | 2017-06-08 | 株式会社協和 | Method for producing placenta extract |
| CN107213106A (en) * | 2017-06-29 | 2017-09-29 | 佛山市南海区普罗圣塔生物科技有限公司 | Essence, facial mask liquid and preparation method thereof |
| CN109568254A (en) * | 2018-12-18 | 2019-04-05 | 江西润泽药业有限公司 | A kind of Plancenta Histolysate's injection and preparation method thereof |
| CN110721146A (en) * | 2019-08-01 | 2020-01-24 | 浙江中林泰网络科技有限公司 | Hydrolyzed embryo protein wrinkle-removing liquid and preparation method thereof |
| CN111067839A (en) * | 2020-01-16 | 2020-04-28 | 广东丽姿生物科技有限公司 | Conditioning repairing agent and preparation method thereof |
| CN111467292A (en) * | 2020-04-03 | 2020-07-31 | 焕生汇生物基因技术(北京)有限公司 | Preparation method of human placenta extract supernatant |
| US20220287952A1 (en) * | 2021-03-15 | 2022-09-15 | Guangzhou Saliai Biological Genetic Engineering Co., Ltd. | Compositions containing exosomes from animal placenta, methods for producing the same and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| 姜惠敏: "羊胎盘抗氧化肽的制备及其在抗衰老化妆品中的应用" * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106967674B (en) | Separation culture method of sheep rumen epithelial cells | |
| CN109207545B (en) | Collagen extraction method | |
| JPH01111008A (en) | Production of collagen tissue structure and said structure | |
| CN113583109B (en) | Jellyfish active protein and preparation method and application thereof | |
| CN104382825A (en) | Extraction method of micromolecular sheep placenta extract and application of micromolecular sheep placenta extract in field of skin-care products | |
| CN113041206A (en) | Essence containing mesenchymal stem cell factor and preparation method thereof | |
| CN112516058A (en) | Amino acid-loaded modified amniotic membrane mask and preparation method thereof | |
| CN111117954A (en) | Separation culture method of umbilical cord-derived primary mesenchymal stem cells | |
| CN111467292A (en) | Preparation method of human placenta extract supernatant | |
| CN105963787A (en) | Preparation method of new biological patches | |
| CN114832015A (en) | Facial repair injection based on PRP and stem cell exosome and preparation method thereof | |
| CN115708798A (en) | One-step hydrolysis preparation method of sheep placenta extract | |
| CN112426402A (en) | Application based on fibroblast exosomes and preparation method | |
| CN115581754B (en) | Application of ginger extracellular vesicles in preparation of medicine for promoting proliferation of hair follicle stem cells | |
| US4687518A (en) | Method for manufacturing pyrogen-free collagen gels useful as contact lenses | |
| CN111518743A (en) | Method for constructing human dermal fibroblast in-vitro thermal injury model and application thereof | |
| CN113069407A (en) | Preparation method of stem cell filtrate and stem cell factor mixed solution | |
| CN113621555A (en) | Preparation method of skin fibroblast | |
| CN110946879B (en) | Acellular biological amniotic membrane containing loose layer and preparation method thereof | |
| CN116772523B (en) | Freeze-drying method of collagen sponge | |
| CN114457009A (en) | Preparation method and application of stem cell extract | |
| CN114561355B (en) | Acute and rapid separation method for spinal cord scar tissue cells | |
| CN109837262A (en) | A kind of people's umbilical cord Wharton jelly tissue digestion enzyme and preparation method thereof | |
| CN114540296A (en) | Preparation method of composite exosome and application of composite exosome in directionally enhancing angiogenesis capacity | |
| CN118787664A (en) | Umbilical cord mesenchymal stem cell exosome, and preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20230224 |