CN102430125A - Heat-resistant stabilizer for chicken embryos fluid lyophilization, as well as preparation method and application thereof - Google Patents
Heat-resistant stabilizer for chicken embryos fluid lyophilization, as well as preparation method and application thereof Download PDFInfo
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- CN102430125A CN102430125A CN2011104554205A CN201110455420A CN102430125A CN 102430125 A CN102430125 A CN 102430125A CN 2011104554205 A CN2011104554205 A CN 2011104554205A CN 201110455420 A CN201110455420 A CN 201110455420A CN 102430125 A CN102430125 A CN 102430125A
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- liquid
- pvp
- gallus domesticus
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- purified water
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- 241000287828 Gallus gallus Species 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 238000004108 freeze drying Methods 0.000 title abstract description 3
- 239000012530 fluid Substances 0.000 title abstract 7
- 239000003381 stabilizer Substances 0.000 title abstract 6
- 210000002257 embryonic structure Anatomy 0.000 title abstract 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims abstract description 34
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims abstract description 34
- 230000001681 protective effect Effects 0.000 claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims abstract description 22
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 claims abstract description 14
- 229960002401 calcium lactate Drugs 0.000 claims abstract description 14
- 235000011086 calcium lactate Nutrition 0.000 claims abstract description 14
- 239000001527 calcium lactate Substances 0.000 claims abstract description 14
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 11
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 11
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 11
- 229930006000 Sucrose Natural products 0.000 claims abstract description 11
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 11
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 11
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 11
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims abstract description 11
- 235000013923 monosodium glutamate Nutrition 0.000 claims abstract description 11
- 229940073490 sodium glutamate Drugs 0.000 claims abstract description 11
- 239000000600 sorbitol Substances 0.000 claims abstract description 11
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims description 67
- 210000001161 mammalian embryo Anatomy 0.000 claims description 32
- 239000008213 purified water Substances 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 13
- 239000005018 casein Substances 0.000 claims description 10
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 10
- 235000021240 caseins Nutrition 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- 238000009835 boiling Methods 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000009993 protective function Effects 0.000 abstract 1
- 229960004793 sucrose Drugs 0.000 abstract 1
- 229960005486 vaccine Drugs 0.000 description 11
- 239000003223 protective agent Substances 0.000 description 7
- 238000004321 preservation Methods 0.000 description 6
- 208000010359 Newcastle Disease Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 206010006451 bronchitis Diseases 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005267 amalgamation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
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- Medicinal Preparation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a heat-resistant stabilizer for chicken embryos fluid lyophilization, as well as a preparation method and application thereof, and aims at providing a heat-resistant stabilizer with simple preparation method, low cost and excellent protective function. The invention adopts a technical scheme that: the heat-resistant stabilizer for chicken embryos fluid consists of a basic fluid and a PVP (polyvinyl pyrrolidone) fluid in a volume ratio of 4 to 1, wherein the basic fluid consists of the following components in each 800ml by volume, 38 to 42g of calcium lactate, 9 to 11g of thiourea, 48 to 52g of cane sugar, 4 to 6g of trehalose, 48 to 52g of sodium glutamate, 36 to 39g of NZ-AmineAS, and the balance of water; and the PVP fluid consists of the following components in each 200ml by volume, 28 to 32g of PVP, 9 to 11g of sorbitol, and the balance of water. The heat-resistant stabilizer and the preparation method thereof provided by the invention have the beneficial effects that: the heat-resistant stabilizer has excellent protective performance; and the preparation method is simple and low in cost.
Description
Technical field
The present invention relates to heat-resisting lyophilized protecting agent, be specially a kind of the be used for freeze dried heat resisting protective of Embryo Gallus domesticus liquid, its preparation method and application thereof.
Background technology
Freeze drying protectant can influence biological product quality, tire and stability, change the production technology of biological product, increase stability among product batches.Heat-resisting lyophilized protecting agent has immunocompetence but parmacodynamics-less activity, can when lyophilizing and preservation, keep the stability of vaccine.Heat-resisting lyophilized protecting agent and preparation method thereof is a key technology in the production of vaccine.
For the application of heat resisting protective in Embryo Gallus domesticus liquid protection, the selection of raw and auxiliary material, preparation, sterilization, preservation and use in the heat resisting protective play crucial effects for the protection quality of heat-resisting lyophilized protecting agent.
Summary of the invention
The object of the invention is to provide that a kind of compound method is simple, cost is low, have a good protection performance be used for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid.
Concrete technical scheme of the present invention is: be used for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, be made up of according to the volume ratio of 4:1 basal liquid and polyvinylpyrrolidone PVP liquid; Wherein, Said basal liquid is grouped into by following one-tenth: contain calcium lactate 38～42g, thiourea 9～11g, sucrose 48～52g, trehalose 4～6g, sodium glutamate 48～52g, enzymolysis casein NZ-Amine AS 36～39g in every 800ml volume, all the other are purified water; Said PVP liquid is grouped into by following one-tenth: contain PVP 28～32g, sorbitol 9～11g in every 200ml volume, all the other are purified water.
Said basal liquid is grouped into by following one-tenth: contain calcium lactate 40g, thiourea 10g, sucrose 50g, trehalose 5g, sodium glutamate 50g, enzymolysis casein NZ-Amine AS37.5g in every 800ml volume, all the other are purified water.
Said PVP liquid is grouped into by following one-tenth: contain PVP 30g, sorbitol 10g in every 200ml volume, all the other are purified water.
Another technical problem to be solved by this invention provides the method for preparing of above-mentioned heat resisting protective; Its technical scheme is: preparation is used for the method for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid; The preparation of basal liquid: (1) heating purified water is to boiling; Slowly add calcium lactate in said ratio, stir transparently, filter with qualitative filter paper until dissolving fully; (2) successively thiourea, sucrose, trehalose, sodium glutamate, enzymolysis casein NZ-Amine AS are dissolved in the calcium lactate solution in said ratio, add purified water to said ratio; (3) filtration sterilization; (4) room temperature is preserved; Can not put 2-8 ℃ of preservation, separate out and precipitate otherwise have; The preparation of PVP liquid: (1) heating purified water adds PVP to boiling in said ratio, adds sorbitol again, stirs transparent until dissolving fully; (2) autoclaving; Basal liquid is mixed according to the volume ratio of 4:1 with PVP liquid, promptly get.It is available that room temperature is preserved 1-2 week.
Said filtration sterilization adopts the mode of microporous filter membrane to carry out.
Said microporous filter membrane aperture is 0.22um.
Said autoclaving is under 15 pounds of pressure, to sterilize 15 minutes.
The present invention is used for the application of the freeze dried heat resisting protective of Embryo Gallus domesticus liquid at protection Embryo Gallus domesticus liquid, and heat resisting protective and Embryo Gallus domesticus liquid are according to the volume ratio preparation of 1:1.
Beneficial effect of the present invention is: be applicable to chicken Newcastle disease live vaccine, infectious bronchitis of chicken live vaccine, newcastle disease infectious bronchitis of chicken bigeminal live vaccine; But heat resisting protective room temperature is preserved; Embryo Gallus domesticus liquid there is the excellent protection performance; Compound method is simple, cost is low.
The specific embodiment
Embodiment 1:
Be used for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, form according to the volume ratio of 4:1 by basal liquid and polyvinylpyrrolidone PVP liquid; Wherein, said basal liquid is grouped into by following one-tenth: contain calcium lactate 40g, thiourea 10g, sucrose 50g, trehalose 5g, sodium glutamate 50g, enzymolysis casein NZ-Amine AS 37.5g in every 800ml volume, all the other are purified water; Said PVP liquid is grouped into by following one-tenth: contain PVP 30g, sorbitol 10g in every 200ml volume, all the other are purified water.
Preparation is used for the method for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, the preparation of basal liquid: (1) heating purified water slowly adds calcium lactate to boiling in said ratio, stirs transparently until dissolving fully, filters with qualitative filter paper; (2) successively thiourea, sucrose, trehalose, sodium glutamate, enzymolysis casein NZ-Amine AS (available from Beijing Baeyer enlightening biotech firm) are dissolved in the calcium lactate solution in said ratio, add purified water to said ratio; (3) adopting the aperture is the filtering with microporous membrane degerming of 0.22um; (4) room temperature is preserved; The preparation of PVP liquid: (1) heating purified water adds PVPK30 (available from east, Beijing ring amalgamation factory) to boiling in said ratio, adds sorbitol again, stirs transparent until dissolving fully; (2) autoclaving 15 minutes under 15 pounds of pressure; Basal liquid is mixed according to the volume ratio of 4:1 with PVPK30 liquid, promptly get.It is available that room temperature is preserved 1-2 week.
Be used for the application of the freeze dried heat resisting protective of Embryo Gallus domesticus liquid at protection Embryo Gallus domesticus liquid, heat resisting protective and Embryo Gallus domesticus liquid are according to the volume ratio preparation of 1:1.
Embodiment 2:
Be used for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, form according to the volume ratio of 4:1 by basal liquid and polyvinylpyrrolidone PVP liquid; Wherein, said basal liquid is grouped into by following one-tenth: contain calcium lactate 42g, thiourea 9g, sucrose 52g, trehalose 4g, sodium glutamate 52g, enzymolysis casein NZ-Amine AS 36g in every 800ml volume, all the other are purified water; Said PVP liquid is grouped into by following one-tenth: contain PVP 28g, sorbitol 11g in every 200ml volume, all the other are purified water.
Preparation is used for the method for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, with embodiment 1.
Be used for of the application of the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, with embodiment 1 at protection Embryo Gallus domesticus liquid.
Embodiment 3:
Be used for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, it is characterized in that, form according to the volume ratio of 4:1 by basal liquid and polyvinylpyrrolidone PVP liquid; Wherein, said basal liquid is grouped into by following one-tenth: contain calcium lactate 38g, thiourea 11g, sucrose 48g, trehalose 6g, sodium glutamate 48g, enzymolysis casein NZ-Amine AS 39g in every 800ml volume, all the other are purified water; Said PVP liquid is grouped into by following one-tenth: contain PVP 32g, sorbitol 9g in every 200ml volume, all the other are purified water.
Preparation is used for the method for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, with embodiment 1.
Be used for of the application of the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, with embodiment 1 at protection Embryo Gallus domesticus liquid.
Test Example: protection potency test.
1, test material: strain: newcastle hangs down virulence live vaccine (La Sota strain), available from China Veterinery Drug Inspection Office; The SPF Embryo Gallus domesticus is available from academy of agricultural sciences, Shandong Province poultry institute;
2, experimental vaccine:,, after lyophilizing is handled, get vaccine with of the volume ratio preparation of Embryo Gallus domesticus liquid according to 1:1 according to the inventive method preparation heat-resisting lyophilized protecting agent;
3, the preservation of experimental vaccine and titration: place 2～8 ℃ and room temperature preservation respectively, measure the different time viral level by version " Chinese veterinary drug allusion quotation " method in 2010, result of the test sees the following form, and (unit is EID
50/ plumage part):
| Preservation condition | After the lyophilizing | Preserved 3 months | Preserved 6 months | Preserved 12 months | Preserved 24 months |
| 2～8℃ | 10 6.5 | 10 6.5 | 10 6.5 | 10 6.5 | 10 6.5 |
| Room temperature | 10 6.5 | 10 6.5 | 10 6.5 | 10 6.5 | 10 6.375 |
Result of the test shows that the chicken Newcastle disease live vaccine heat resistance that heat-resisting lyophilized protecting agent of the present invention is prepared is good, and heat-resisting lyophilized protecting agent of the present invention has the excellent protection effect to the newcastle live vaccine.
Claims (8)
1. be used for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid, it is characterized in that, form according to the volume ratio of 4:1 by basal liquid and polyvinylpyrrolidone PVP liquid; Wherein, Said basal liquid is grouped into by following one-tenth: contain calcium lactate 38～42g, thiourea 9～11g, sucrose 48～52g, trehalose 4～6g, sodium glutamate 48～52g, enzymolysis casein NZ-Amine AS 36～39g in every 800ml volume, all the other are purified water; Said PVP liquid is grouped into by following one-tenth: contain PVP28～32g, sorbitol 9～11g in every 200ml volume, all the other are purified water.
2. the freeze dried heat resisting protective of Embryo Gallus domesticus liquid that is used for according to claim 1; It is characterized in that; Said basal liquid is grouped into by following one-tenth: contain calcium lactate 40g, thiourea 10g, sucrose 50g, trehalose 5g, sodium glutamate 50g, enzymolysis casein NZ-Amine AS 37.5g in every 800ml volume, all the other are purified water.
3. the freeze dried heat resisting protective of Embryo Gallus domesticus liquid that is used for according to claim 1 is characterized in that said PVP liquid is grouped into by following one-tenth: contain PVP 30g, sorbitol 10g in every 200ml volume, all the other are purified water.
4. prepare any one said method that is used for the freeze dried heat resisting protective of Embryo Gallus domesticus liquid of claim 1-3, it is characterized in that:
The preparation of basal liquid: (1) heating purified water slowly adds calcium lactate to boiling in said ratio, stirs transparently until dissolving fully, filters with qualitative filter paper; (2) successively thiourea, sucrose, trehalose, sodium glutamate, enzymolysis casein NZ-Amine AS are dissolved in the calcium lactate solution in said ratio, add purified water to said ratio; (3) filtration sterilization; (4) room temperature is preserved;
The preparation of PVP liquid: (1) heating purified water adds PVP to boiling in said ratio, adds sorbitol again, stirs transparent until dissolving fully; (2) autoclaving;
Basal liquid is mixed according to the volume ratio of 4:1 with PVP liquid, promptly get.
5. method according to claim 4 is characterized in that: said filtration sterilization adopts the mode of microporous filter membrane to carry out.
6. method according to claim 5 is characterized in that: said microporous filter membrane aperture is 0.22um.
7. method according to claim 4 is characterized in that: said autoclaving is sterilization under 15 pounds of pressure 15 minutes.
8. claim 1 is used for the application of the freeze dried heat resisting protective of Embryo Gallus domesticus liquid at protection Embryo Gallus domesticus liquid, it is characterized in that, heat resisting protective and Embryo Gallus domesticus liquid are according to the volume ratio preparation of 1:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104554205A CN102430125B (en) | 2011-12-31 | 2011-12-31 | Heat-resistant stabilizer for chicken embryos fluid lyophilization, as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104554205A CN102430125B (en) | 2011-12-31 | 2011-12-31 | Heat-resistant stabilizer for chicken embryos fluid lyophilization, as well as preparation method and application thereof |
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| Publication Number | Publication Date |
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| CN102430125A true CN102430125A (en) | 2012-05-02 |
| CN102430125B CN102430125B (en) | 2013-04-17 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111184871A (en) * | 2020-03-23 | 2020-05-22 | 辽宁益康生物股份有限公司 | Heat-resistant protective agent for newcastle disease and bronchitis combined live vaccine and preparation process thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102166362A (en) * | 2011-04-13 | 2011-08-31 | 武汉中博生物股份有限公司 | Porcine reproductive and respiratory syndrome live vaccine heat-resistant freeze-drying protective agent and preparation method thereof |
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2011
- 2011-12-31 CN CN2011104554205A patent/CN102430125B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102166362A (en) * | 2011-04-13 | 2011-08-31 | 武汉中博生物股份有限公司 | Porcine reproductive and respiratory syndrome live vaccine heat-resistant freeze-drying protective agent and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《江苏农业科学》 20041231 侯继波等 "不同基质对鸡新城疫病毒冻干保护作用的研究" 第80－82页 1-8 , 第5期 * |
| 侯继波等: ""不同基质对鸡新城疫病毒冻干保护作用的研究"", 《江苏农业科学》, no. 5, 31 December 2004 (2004-12-31), pages 80 - 82 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111184871A (en) * | 2020-03-23 | 2020-05-22 | 辽宁益康生物股份有限公司 | Heat-resistant protective agent for newcastle disease and bronchitis combined live vaccine and preparation process thereof |
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Denomination of invention: Heat resistant protective agent for freeze-drying chicken embryo liquid, its preparation method and application Effective date of registration: 20231011 Granted publication date: 20130417 Pledgee: Weihai Commercial Bank Co.,Ltd. Binzhou Branch Pledgor: SHANDONG BINZHOU BOLAIWEI BIOTECH Co.,Ltd. Registration number: Y2023980060827 |
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