CN102908365A - Preparation method of eggembryosin - Google Patents
Preparation method of eggembryosin Download PDFInfo
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- CN102908365A CN102908365A CN2011102193772A CN201110219377A CN102908365A CN 102908365 A CN102908365 A CN 102908365A CN 2011102193772 A CN2011102193772 A CN 2011102193772A CN 201110219377 A CN201110219377 A CN 201110219377A CN 102908365 A CN102908365 A CN 102908365A
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Abstract
The invention relates to a preparation method of eggembryosin. According to the invention, chicken fertilized eggs are selected and are subjected to surface sterilization; eggshells, yolks and embryoid bodies are removed; rest substances which are chick embryo components are frozen and crushed; pre-cooled normal saline is added for pulping; the obtained material is filtered by using raw-silk cloth with a specification of 20 meshes and a filter with a specification of 3mum; and an obtained filtrate is an eggembryosin solution. The eggembryosin comprises various biologically active substances such as amino acids, small-molecular peptides, growth regulatory factors, nucleic acid derivatives, and the like. The eggembryosin has relatively high nutritional value, beautification value and medicinal value. With the method, high-bioactivity eggembryosin large-scale extraction can be realized easily and fast.
Description
One. technical field:
The present invention relates to a kind of preparation method of embryo's element, especially relate to a kind of preparation method that has than the chick embryo element of high bioactivity.
Two. background technology:
Chick embryo element is one group of growth-stimulating factor with high bioactivity, nerve growth factor, active polypeptide, active polysaccharide, the small-molecule mixture of several amino acids and other nutrient substance that enriches, they have the elite composition that promotes growth and activation physiological function, energy human activin histiocyte, activation tissue organ function, the various immunity of balance the body, improve the ability of human body resist the disease, and delaying sanility, wherein contained growth-stimulating factor can increase moisture content of skin, promote epithelial metabolism, application through certain hour, can delay the skin corium aging, atrophy, make dermal layer of the skin full, and can increase water content, make epidermis smooth moist.In the short process of chicken oosperm hatching, huge variation has occured, in this change procedure, supervene a large amount of growth-stimulating factors, nerve growth factor, active polysaccharide, active polypeptide, several amino acids and other abundant nutrient substance, have high nutritive value and beauty treatment medical value.
In the prior art, the material that people extract embryo's element is Embryo Gallus domesticus, though can extract the chick embryo element with greater activity, process is complicated, length consuming time, instrument and equipment requires also higher.Select the chicken oosperm of hatching 7～11 days among the present invention, remove eggshell, Embryo Gallus domesticus, egg yolk, its residue is comprised that the Embryo Gallus domesticus composition of allantoic fluid, amniotic fluid, allantoic fluid allantocherion vasoganglion is as the material extraction chick embryo element, preparation process is simple and easy, and is consuming time short, equally also extracted the chick embryo element with greater activity.
Three. summary of the invention:
The objective of the invention is: improve to some extent on existing theory and technology basis, draw materials with in the past different, to hatch 7～11 days chicken oosperm as material, eggshell, egg yolk, Embryo Gallus domesticus are removed, with residue comprise allantoic fluid, amniotic fluid, allantoic fluid allantocherion vasoganglion etc. material extracted the chick embryo element that contains efficient bio-active through simple processing, provide a kind of simple and rapid scheme for producing embryo's element.
Technical scheme of the present invention is:
A kind of preparation method of embryo's element comprises selected germ cell and hatching, comprises following concrete steps:
A: selected chicken oosperm, surperficial conventional decontamination, conventional incubation 7～11 days.
B: the Embryo Gallus domesticus composition after will pulverizing is pulled an oar, and adds the physiological saline solution that 4～6 times of Embryo Gallus domesticus become partial volume pre-cooling to 0～4 ℃ during making beating in Embryo Gallus domesticus, stirs under 50～80 rev/mins mixing speed 6～10 minutes.
C: the Embryo Gallus domesticus composition of pulping is carried out multigelation 4～6 times, and freezing temperature is-20～-30 ℃, and each freezing time is 1～4 hour, melts under the room temperature condition.
D: the Embryo Gallus domesticus composition of multigelation is filtered through 20 order silks.The normal saline that adds again 4～6 times of volume pre-coolings after the filtration filters again through 3 μ filters, and the filtrate of acquisition is chick embryo element solution.
E: the loose lumps material that obtains after the filtrate lyophilization is described chick embryo element.
F: relative humidity less than 30% environment in, place hermetic container to keep in Dark Place resulting chick embryo element, with for subsequent use.
Hatching parameter in the described conventional incubation method is: 1. incubation temperature: 36～38 ℃; 2. hatch humidity: relative humidity is about 40～60%: 3. ventilation: keep the airborne oxygen content of hatching box more than 21%; 4. egg-turning: egg-turning was 1 time in per 1～2 hour; 5. hatch to 7～11 days, discard eggshell, Embryo Gallus domesticus, egg yolk behind the taking-up incubated egg.
The method that described Embryo Gallus domesticus composition is pulverized is: will remove the Embryo Gallus domesticus composition behind eggshell, Embryo Gallus domesticus, the egg yolk, main component comprises the material of allantoic fluid, amniotic fluid, allantoic fluid allantocherion vasoganglion, place low temperature refrigerator-20～-30 ℃ freezing, with size reduction machinery it is crushed to 50～90 orders after freezing 1～10 hour.
Described multigelation is that the Embryo Gallus domesticus after the making beating is become to be placed in the low temperature refrigerator, after sample being refrigerated to-20～-30 ℃, freezing 1～4 hour, then place when naturally being melted up to the frozen water coexistence under the room temperature condition, place again low temperature refrigerator, sample is refrigerated to-20～-30 ℃, freezing 1～4 hour, so multigelation was 3～5 times.
Described filter method is that the Embryo Gallus domesticus composition behind the multigelation is filtered through 20 order silks, and then filters through 3 μ filters.
Described freeze-drying method is: resulting ultrafiltrate is placed aseptic rustless steel pallet, charging thickness＜1.5 centimetre, put in advance sterilization, in the freeze dryer lyophilizing cabin after the pre-cooling, regulate the lyophilizing parameter, above-mentioned ultrafiltrate evenly is refrigerated to below-40 ℃, then start successively vacuum pump and lobe pump in the freeze dryer vacuum system, regulate vacuum below 6.5～13Pa, kept 2～3 hours, then start the heating system in the freeze dryer, attention and maintenance vacuum are beyond 6.5～13Pa in the heating process, and the material maximum temperature must not surpass 35 ℃ during heating, be stabilized in 35 ℃ when being heated to temperature of charge, vacustat is when 8Pa is following, kept 1～3 hour, i.e. shutdown, discharging, resulting loose lumps thing is chick embryo element.
Positive beneficial effect of the present invention is:
1. the present invention utilizes simple slurrying, multiple times of filtration technology to prepare chick embryo element, and is simple to operate, saves cost.
2. the preparation method of chick embryo element of the present invention has kept the various bioactive substances in the Embryo Gallus domesticus.The chick embryo element of producing is the nutrition body not only, can also promote the metabolism of body tissue, and regulating action is played in the immunity of body.
3. the chick embryo element that obtains of the preparation method of chick embryo element of the present invention is one group of growth-stimulating factor with high bioactivity, nerve growth factor, active polysaccharide, active polypeptide, the small-molecule mixture of several amino acids and other nutrient substance that enrich, they have the elite composition of growth promotion and activation physiological function, energy human activin histiocyte, activation tissue organ function, the various immunity of balance the body, improve the ability of human body resist the disease, and delaying sanility, wherein contained " allantoin " and growth-stimulating factor can increase moisture content of skin, promote epithelial metabolism, application through certain hour, delaying sanility, atrophy, make dermal layer of the skin full, and can increase the epidermis water content, make epidermis smooth moist.The high bioactivity chick embryo element that obtains can be widely used in preparing the industries such as high-activity biological medicine, functional food and cosmetics.
4. the chick embryo element that obtains of the preparation method of chick embryo element of the present invention has preferably oxidation resistance, can improve significantly the activity of SOD in the D-galactose subacute aging model mouse blood, reduces MDA concentration in the serum.
5. the chick embryo element that obtains of the preparation method of chick embryo element of the present invention can significantly improve or adjust the metabolism of senile cell, improves the vitality of cell, delays the process of body and skin aging.
Four. the specific embodiment:
Embodiment one: selected chicken oosperm, and surperficial conventional decontamination, conventional incubation 9 days, selected chicken oosperm are in 3～7 days puerperal, profile rule, not damaged.Hatching parameter in the conventional incubation method is: 1. incubation temperature: 37 ℃, hatching humidity about 60%; 3. ventilation: keep the airborne oxygen content of hatching box more than 21%; 4. egg-turning: egg-turning was 1 time in per 2 hours; 5. hatch to 9 days, discard eggshell, Embryo Gallus domesticus, egg yolk behind the taking-up incubated egg.Embryo Gallus domesticus composition after pulverizing is pulled an oar, in Embryo Gallus domesticus, add the physiological saline solution that 4 times of Embryo Gallus domesticus become partial volume pre-cooling to 4 ℃ during making beating, under 80 rev/mins mixing speed, stirred 10 minutes, make slurry.The Embryo Gallus domesticus composition of pulping is carried out multigelation 6 times, and freezing temperature is-20～-30 ℃, and each freezing time is 4 hours, melts under the room temperature condition.20 order silks filter behind the multigelation.The normal saline that adds 6 times of volume pre-coolings after filtering filters again through 3 μ filters, and the filtrate of acquisition is chick embryo element solution.Resulting ultrafiltrate is placed aseptic rustless steel pallet, 1.3 centimetres of charging thickness, put in advance sterilization, in the freeze dryer lyophilizing cabin after the pre-cooling, regulate the lyophilizing parameter, above-mentioned ultrafiltrate evenly is refrigerated to below-40 ℃, then start successively vacuum pump and lobe pump in the freeze dryer vacuum system, regulate vacuum below 13Pa, kept 3 hours, then start the heating system in the freeze dryer, attention and maintenance vacuum are beyond 13Pa in the heating process, and the material maximum temperature must not surpass 35 ℃ during heating, be stabilized in 35 ℃ when being heated to temperature of charge, vacustat is when 8Pa is following, kept 3 hours, i.e. shutdown, discharging, resulting loose lumps thing is chick embryo element.Relative humidity less than 30% environment in, place hermetic container to keep in Dark Place resulting chick embryo element, with for subsequent use.
Embodiment two: selected chicken oosperm, and surperficial conventional decontamination, conventional incubation 8 days, selected chicken oosperm are in 3～7 days puerperal, profile rule, not damaged.Hatching parameter in the conventional incubation method is: 1. incubation temperature: 38 ℃; Hatching relative humidity: about 50%; 3. ventilation: keep the airborne oxygen content of hatching box more than 21%; 4. egg-turning: egg-turning was 1 time in per 1 hour; 5. hatch to 8 days, discard eggshell, Embryo Gallus domesticus, egg yolk behind the taking-up incubated egg.Embryo Gallus domesticus composition after pulverizing is pulled an oar, in Embryo Gallus domesticus, add the physiological saline solution that 4 times of Embryo Gallus domesticus become partial volume pre-cooling to 0 ℃ during making beating, under 50 rev/mins mixing speed, stir and made slurry in 6 minutes.The Embryo Gallus domesticus composition of pulping is carried out 4 times getting multigelation, and freezing temperature is-20～-30 ℃, and each freezing time is 3 hours, melts under the room temperature condition.The Embryo Gallus domesticus composition of multigelation is filtered through 20 order silks.The normal saline that adds 5 times of volume pre-coolings after filtering filters again through 3 μ filters, and the filtrate of acquisition is chick embryo element solution.Resulting ultrafiltrate is placed aseptic rustless steel pallet, 1.1 centimetres of charging thickness, put in advance sterilization, in the freeze dryer lyophilizing cabin after the pre-cooling, regulate the lyophilizing parameter, above-mentioned ultrafiltrate evenly is refrigerated to below 40 ℃, then start successively vacuum pump and lobe pump in the freeze dryer vacuum system, regulate vacuum below 12Pa, kept 2 hours, then start the heating system in the freeze dryer, attention and maintenance vacuum are below 12Pa in the heating process, and the material maximum temperature must not surpass 35 ℃ during heating, be stabilized in 35 ℃ when being heated to temperature of charge, vacustat is when 8Pa is following, kept 2 hours, i.e. shutdown, discharging, resulting loose lumps thing is chick embryo element.Relative humidity less than 30% environment in, place hermetic container to keep in Dark Place resulting chick embryo element, with for subsequent use.
Chick embryo element determination of activity experiment is as follows:
1. experiment material
1.1. medicine and reagent: chick embryo element meets the quality standard that researcher proposes:
D-galactose (D-gal) and all the other reagent are commercially available domestic biochemical reagents or analytical pure chemical reagent; SOD, MDA measures test kit, and Nanjing is built up bio-engineering research and is provided; Tetraethoxypropane (TAP), Switzerland FluKa product.
1.2. animal: healthy Kunming mouse, body weight 20 ± 2g, male and female half and half are provided by Medical University Of Tianjin zoopery center.
2. method and result
2.1. the impact on model of blood dificiency mouse red blood cell and hemoglobin: get 16 of Kunming mouses, body weight 17～20g, male and female half and half, be divided at random two groups, measure first each Mus administration proerythrocyte (RBC) and hemoglobin (Hb) value, then after the afterbody blood-letting is sent out method and is caused Lost blood anemia model (RBC, Hb all are starkly lower than normal value), difference gavage chick embryo element every day or normal saline, successive administration 15 days, last during to chick embryo element 2 after, RBC, Hb value are surveyed in afterbody blood sampling, and detailed results is referring to table 1:
Table 1 chick embryo element is on the impact of model of blood dificiency mouse red blood cell and hemoglobin (x ± s)
| Group | Number of animals (n) | Dosage (g/kg) | RBC increment (ten thousand/mm3) | Hb rise in value (g%) |
| The chick embryo element group | 8 | 10.0 | 356.41±36.68** | 4.05±0.62** |
| The normal saline group | 8 | 10.0 | 224.73±39.32 | 3.31±0.48 |
Compare with normal saline: * * P＜0.01
As seen from Table 1, take blood deficiency mice RBC and the Hb value of chick embryo element after 15 days and be significantly higher than to give birth to and bury saline group (P＜0.01), illustrate that this product has preferably blood tonification effect.
2.2.D-galactose subacute aging model facture: get 24 of mices, male and female half and half are divided into chick embryo element group, model group and normal group at random.Front two treated animal every Mus nape every day subcutaneous injection 5%D-galactose 0.4ml, the latter, injected 9 days as blank continuously with approach injection equal-volume normal saline.In the 10th day (continuation injection of d-galactose), chick embryo element group mice gavages chick embryo element by body weight 5g/kg, and model group and normal group all gavage the equal-volume normal saline, and successive administration and modeling are 30 days again.Last gavage and injection of d-galactose were weighed after 2 hours, and eye socket is got blood, were used for biochemical measurement; Get liver and weigh, make homogenate, for subsequent use; Peel off skin and tail tendon, for subsequent use.
2.3. superoxide dismutase in the blood (SOD) determination of activity: SOD measures and to carry out according to test kit description operation requirements and computational methods, detailed results is referring to table 2:
Table 2 chick embryo element is on the impact of SOD activity in the aging model mouse blood (x ± s)
| Group | Number of animals (n) | 5%D-gal (ml/ only) | Chick embryo element (g/kg) | Whole blood SOD (μ/ml) |
| The chick embryo element group | 8 | 0.4 | 4.5 | 387.44±49.78** |
| Model group | 8 | 0.4 | - | 298.3±47.02 |
| Normal group | 8 | - | - | 506.2±72.66 |
Compare with model group: * * P＜0.01
As shown in Table 3, can obviously the raise vigor of SOD in the mouse blood of chick embryo element is with the poor heteropole of model group significantly (P＜0.01).
2.4. Serum MDA (MDA) is measured: eye socket is got blood, and separation of serum carries out according to test kit description operation requirements and computational methods, detailed results is referring to table 3:
Table 3: chick embryo element is on the impact of MDA content in the aging model mice serum (x ± s)
| Group | Number of animals (n) | 5%D-gal (ml/ only) | Chick embryo element (g/kg) | MDA(nM/ml) |
| The chick embryo element group | 8 | 0.4 | 4.5 | 5.18±0.93** |
| Model group | 8 | 0.4 | - | 10.62±0.94 |
| Normal group | 8 | - | - | 3.15±0.51 |
Compare with model group: * * P＜0.01
The result shows, MDA content is significantly higher than the normal group level and knits (P＜0.01) in the model group mice serum, behind the oral administration chick embryo element 40 days, MDA content obviously descends (P＜0.01) in the serum, illustrates that chick embryo element is to the obvious inhibitory action of being formed with of MDA.
Originally experimental results show that chick embryo element has preferably oxidation resistance, can improve significantly the activity of SOD in D-galactose subacute aging model mice 22. blood, reduce MDA concentration in the serum, the results suggest chick embryo element can significantly improve or adjust the new border metabolism of senile cell, improve the vitality of cell, delay the process of body and skin aging.MDA is the ageing products of interior free yl reaction, and they increase with advancing age, and superoxide dismutase (SOD) then is the main enzyme that body is removed free radical, and it with age and activity decreased.
Above-mentioned detailed description of the preparation method of this kind cancer therapy drug being carried out with reference to embodiment; illustrative rather than determinate; can list several embodiment according to institute's limited range; therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (6)
1. the preparation method of embryo's element comprises selected germ cell and hatching, it is characterized in that, comprises following concrete steps:
A: selected chicken oosperm, surperficial conventional decontamination, conventional incubation 7～11 days, selected chicken oosperm are in 3～7 days puerperal, profile rule, not damaged.Routine disinfection is carried out on the germ cell surface that hatching is good, removes eggshell, idiosome and egg yolk, and surplus materials is put into refrigerator-freezer, under≤-20 ℃ temperature conditions, freezing 1～10 hour, then the Embryo Gallus domesticus composition is pulverized.
B: the Embryo Gallus domesticus composition after will pulverizing is pulled an oar, and adds the physiological saline solution that 4～6 times of Embryo Gallus domesticus become partial volume pre-cooling to 0～4 ℃ during making beating in Embryo Gallus domesticus, stirs under 50～80 rev/mins mixing speed 6～10 minutes.
C: the Embryo Gallus domesticus composition of pulping is carried out multigelation 4～6 times, and freezing temperature is-20～-30 ℃, and each freezing time is 1～4 hour, melts under the room temperature condition.
D: the Embryo Gallus domesticus composition of multigelation is filtered through 20 order silks.
The normal saline that adds again 4～6 times of volume pre-coolings after the filtration filters again through 3 μ filters, and the filtrate of acquisition is chick embryo element solution.
E: the loose lumps material that obtains after the filtrate lyophilization is described chick embryo element.
F: relative humidity less than 30% environment in, place hermetic container to keep in Dark Place resulting chick embryo element, with for subsequent use.
2. according to claims 1 described chick embryo element preparation method, it is characterized in that: the hatching parameter in the described conventional incubation method is:
1. incubation temperature: 36～38 ℃; 2. hatch humidity: relative humidity is 40～60%; 3. ventilation: keep the airborne oxygen content of hatching box more than 21%; 4. egg-turning: egg-turning was 1 time in per 1～2 hour; 5. hatch to 7～11 days, discard eggshell, Embryo Gallus domesticus, egg yolk behind the taking-up incubated egg.
3. according to claims 1 described chick embryo element preparation method, it is characterized in that: the method that described Embryo Gallus domesticus composition is pulverized is with the Embryo Gallus domesticus composition behind removal eggshell, Embryo Gallus domesticus, the egg yolk, main component comprises the material of allantoic fluid, amniotic fluid, allantoic fluid allantocherion vasoganglion, place low temperature refrigerator-20～-30 ℃ freezing, with size reduction machinery it is crushed to 50～90 orders after freezing 1～10 hour.
4. according to claims 1 described chick embryo element preparation method, it is characterized in that: described multigelation is that the Embryo Gallus domesticus after the making beating is become to be placed in the low temperature refrigerator, after sample being refrigerated to-20～-30 ℃, freezing 1～4 hour, then place when naturally being melted up to the frozen water coexistence under the room temperature condition, place again low temperature refrigerator, sample is refrigerated to-20～-30 ℃, freezing 1～4 hour, so multigelation was 3～5 times.
5. according to claims 1 described chick embryo element preparation method, it is characterized in that: described filter method is that the Embryo Gallus domesticus composition behind the multigelation is filtered through 20 order silks, and then filters through 3 μ filters.
6. according to claims 1 described chick embryo element preparation method, it is characterized in that: described freeze-drying method is: resulting ultrafiltrate is placed aseptic rustless steel pallet, charging thickness＜1.5 centimetre, put in advance sterilization, in the freeze dryer lyophilizing cabin after the pre-cooling, regulate the lyophilizing parameter, above-mentioned ultrafiltrate evenly is refrigerated to below-40 ℃, then start successively vacuum pump and lobe pump in the freeze dryer vacuum system, regulate vacuum below 6.5～13Pa, kept 2～3 hours, then start the heating system in the freeze dryer, attention and maintenance vacuum are below 6.5～13Pa in the heating process, the material maximum temperature must not be above 35 ℃ during heating, be stabilized in 35 ℃ when being heated to temperature of charge, vacustat kept 1～3 hour when 8Pa is following, i.e. shutdown, discharging, resulting loose lumps thing is chick embryo element.
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| CN110812306A (en) * | 2018-08-10 | 2020-02-21 | 浙江楚沅生物科技有限公司 | Skin care and repair composition |
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| CN106562903A (en) * | 2016-08-31 | 2017-04-19 | 宁夏清华园知识产权研究与开发有限公司 | Beauty maintaining and young keeping product prepared by incubating eggs |
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| CN106562995A (en) * | 2016-10-26 | 2017-04-19 | 沈会强 | Preparation method for preparing swan embryos and application of swan embryos |
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| CN111955744A (en) * | 2020-07-20 | 2020-11-20 | 安徽农业大学 | Amniotic fluid protein powder and preparation method and application thereof |
| CN114569636A (en) * | 2020-12-02 | 2022-06-03 | 西南交通大学 | Processed chicken embryo, and its preparation method and application |
| CN115025124A (en) * | 2022-06-17 | 2022-09-09 | 济南万泉生物技术有限公司 | Hematopoietic anti-aging active component extracted from chick embryo tissue and application thereof |
| CN115025124B (en) * | 2022-06-17 | 2024-04-30 | 济南万泉生物技术有限公司 | Hematopoietic anti-aging active component extracted from chick embryo tissue and application thereof |
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Application publication date: 20130206 |