CN103299986A - Parasitic protozoon lyophilized powder and preparation method thereof - Google Patents
Parasitic protozoon lyophilized powder and preparation method thereof Download PDFInfo
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- CN103299986A CN103299986A CN201310190462XA CN201310190462A CN103299986A CN 103299986 A CN103299986 A CN 103299986A CN 201310190462X A CN201310190462X A CN 201310190462XA CN 201310190462 A CN201310190462 A CN 201310190462A CN 103299986 A CN103299986 A CN 103299986A
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- 230000003071 parasitic effect Effects 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000008176 lyophilized powder Substances 0.000 title abstract 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 15
- 238000001291 vacuum drying Methods 0.000 claims abstract description 15
- 239000000725 suspension Substances 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 238000004108 freeze drying Methods 0.000 claims abstract description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 9
- 239000000843 powder Substances 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 12
- 239000003223 protective agent Substances 0.000 claims description 10
- 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 description 8
- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 claims description 8
- 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 description 8
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 7
- 235000013824 polyphenols Nutrition 0.000 claims description 7
- 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 description 6
- 241000224016 Plasmodium Species 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 230000012447 hatching Effects 0.000 claims description 4
- 241000224489 Amoeba Species 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 125000000647 trehalose group Chemical group 0.000 claims description 2
- 230000006378 damage Effects 0.000 abstract description 10
- 238000007710 freezing Methods 0.000 abstract description 7
- 230000008014 freezing Effects 0.000 abstract description 7
- 229960005486 vaccine Drugs 0.000 abstract description 6
- 208000027418 Wounds and injury Diseases 0.000 abstract description 5
- 208000014674 injury Diseases 0.000 abstract description 5
- 238000003860 storage Methods 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 3
- 230000007774 longterm Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 2
- 229930182555 Penicillin Natural products 0.000 abstract 2
- 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 abstract 2
- 229940049954 penicillin Drugs 0.000 abstract 2
- 239000000654 additive Substances 0.000 abstract 1
- 230000000996 additive effect Effects 0.000 abstract 1
- 230000001681 protective effect Effects 0.000 abstract 1
- 238000004321 preservation Methods 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 238000004821 distillation Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 241000223104 Trypanosoma Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002389 environmental scanning electron microscopy Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000004017 vitrification Methods 0.000 description 1
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- Medicinal Preparation (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a preparation method of a parasitic protozoon lyophilized powder. The preparation method comprises the following steps: (1) uniformly dispersing parasitic protozoa into a freeze-drying protective additive to form a parasitic protozoon suspension liquid, separately filling the parasitic protozoon suspension liquid into a penicillin bottle, incubating at room temperature, and putting the penicillin bottle into liquid nitrogen to be prefrozen; and (2) transferring the prefrozen parasitic protozoon suspension liquid into a freeze dryer for vacuum drying so as to obtain the parasitic protozoon lyophilized powder. A direct liquid nitrogen input mode is adopted to quickly vitrify and prefreeze the parasitic protozoa, so that freezing injury and drying stress injury can be effectively relieved, and the rehydration recovery of the parasitic protozoon lyophilized powder is greatly increased; series optimized technological parameters are adopted for vacuum drying, the drying time is short and the operation is simple; the parasitic protozoon lyophilized powder has the advantages of low water content, stable performance, convenience in long-term storage at room temperature and remote transportation. The obtained parasitic protozoon lyophilized powder can be applied to scientific research or biological vaccine development.
Description
Technical field
The present invention relates to a kind of freeze-dried powder, particularly a kind of parasitic protozoa freeze-dried powder and preparation method thereof belongs to cryobiology and biological medicine technology field.
Background technology
Biovaccine concerns health care and the life security of organism, and its preservation problem is the focus that receives much concern always.Because biovaccine belongs to heat-sensitive substance, to heat (high temperature) sensitivity, very easily inactivation all adopts the deepfreeze method to the preservation of biovaccine both at home and abroad at present usually.Yet this method complex operation, cost is higher, and it is preserved the transportation cold chain and relates to a plurality of links again, and each link needs strict operation, and there have slightly to be improper, all will have a strong impact on the preservation quality of vaccine.For example, once caused " the Shanxi vaccine event " of government department and various circles of society's extensive concern in 2010, it is dead or disabled to cause nearly hundred children to inoculate behind the problem vaccine.Through investigation, the basic reason that causes this event is that vaccine sex change takes place and polluted the serious toxic and side effect of generation after human body uses because being exposed to hot environment in cold storage procedure.
Adopt Freeze Drying Technique that biovaccine is lyophilized into powder at low temperatures, long preservation and consistency can come into operation after being dissolved in water at room temperature after packing.If this technology can be succeedd, the store method of biovaccine will greatly be simplified, convenient transportation, saving cost.At present, it is ripe that freeze drying is used for the technology of long preservation aspect of biovaccine is that all right, also has many difficult problems.
Freeze-drying is called for short freeze-drying, will be rich in the material of moisture exactly, be frozen into by cooling in advance solid-state, then under the condition of vacuum and low temperature, by to freeze material slowly heating make water sublimed wherein conciliate sucking-off, to obtain the technology of dry matter.Freeze drying mainly is divided into pre-freeze and two megastages of vacuum drying, and the purpose of pre-freeze is for water congeals into ice with free state in the material at low temperatures, is beneficial to the distillation of moisture.Vacuum drying is exactly to make the ice distillation and remove the unfrozen water that adsorbs in the material by the method that heats, to reach dry purpose.Freezing dry process is because of freezing and drying stress that violent environmental change produces, can cause in various degree damage to cell, finally causes it dead or lose activity.Freezing injury mainly is to be formed by ice crystal to cause, and when freezing rate was too fast, moisture had little time to exosmose in the born of the same parents, form ice crystal in cell, thereby cell membrane causes mechanical destruction, i.e. mechanical damage.Cross when slow when freezing rate, preferentially form ice crystal in the extracellular, moisture constantly exosmoses in the born of the same parents, thereby causes cell because of dead in the solution that is exposed to high concentration, and namely solute damages.Lyophilization also can cause cells injury, overdrying can cause cell dead because of dehydration, finishes drying in advance and can cause too much moisture as plasticizer again, makes the vitrification point of freeze-drying prods be lower than storage temperature, thereby the acceleration cellular metabolism descends cytoactive.
Parasitic protozoa is unicellular eucaryon animal, and volume is small and can independently finish whole physiological functions of vital movement, and is extensive in distributed in nature, seeks parasitic life mostly, and part can be caused a disease.Parasitic protozoa is the important source material of development parasitosis vaccine, it is protected and plants also is to carry out the requisite important step of correlative study, therefore long term storage at room temperature after the parasitic protozoa freeze-drying is had very important significance, yet do not see as yet both at home and abroad that at present the cryodesiccated report of parasitic protozoa success is arranged.
Summary of the invention
In order to overcome the shortcoming and defect of prior art, primary and foremost purpose of the present invention is to provide a kind of preparation method of parasitic protozoa freeze-dried powder.
Another object of the present invention is to obtain the parasitic protozoa freeze-dried powder by above-mentioned preparation method.
For achieving the above object, the present invention is achieved through the following technical solutions:
A kind of preparation method of parasitic protozoa freeze-dried powder comprises following steps:
(1) parasitic protozoa is dispersed in formation parasitic protozoa suspension in the frozen-dried protective agent solution, is sub-packed in the cillin bottle, after at room temperature hatching, drop into pre-freeze in the liquid nitrogen;
(2) the parasitic protozoa suspension with above-mentioned pre-freeze is transferred in the freeze dryer, carries out vacuum drying, obtains the parasitic protozoa freeze-dried powder.
In the preparation method of above-mentioned parasitic protozoa freeze-dried powder, described parasitic protozoa comprises the protozoon of all parasitic lives such as plasmodium, Amoeba, arc worm, trypanosome.
In the preparation method of above-mentioned parasitic protozoa freeze-dried powder, in the described frozen-dried protective agent solution, freeze drying protectant is trehalose and Tea Polyphenols, and the concentration of trehalose is 0.05 ~ 0.5mol/L, and the concentration of Tea Polyphenols is 0 ~ 0.5 mol/L, and solvent is the PBS buffer solution.
In the preparation method of above-mentioned parasitic protozoa freeze-dried powder, the concentration before the described parasitic protozoa pre-freeze in the frozen-dried protective agent solution is 1 * 10
7~ 10
8Individual/ml.
In the preparation method of above-mentioned parasitic protozoa freeze-dried powder, the described vacuum drying condition of step (2) is: drying pressure 0 ~ 30Pa, baffle temperature-40 ~-10 ℃, condenser temperature-196 ~-57 ℃, 30 ~ 120 minutes drying times.
In the preparation method of above-mentioned parasitic protozoa freeze-dried powder, the described time of at room temperature hatching is 5-10 minute, and the time of pre-freeze is 24 hours in liquid nitrogen.
Compared with prior art, the present invention has following beneficial effect:
(1) adopts the mode that directly drops into liquid nitrogen that parasitic protozoa is carried out rapid glass pre-freeze, can effectively alleviate the damage of freezing injury and drying stress, improve the rehydration rate of recovery of parasitic protozoa freeze-dried powder greatly;
(2) process of vacuum drying adopts serial optimized parameters, and drying time is short, and is simple to operate;
(3) low, the stable in properties of parasitic protozoa freeze-dried powder moisture content, be convenient to long term storage and remote transportation under the room temperature.
(4) gained parasitic protozoa freeze-dried powder can be applied to the development of scientific research or biovaccine.
Description of drawings
Fig. 1 is trypanosome freeze-dried powder SEM photo (5 μ m).
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail:
Embodiment 1
Take by weighing trehalose and Tea Polyphenols, be dissolved in the PBS buffer solution, be mixed with the frozen-dried protective agent solution, trehalose concentration is 0.4 mol/L, and Tea Polyphenols concentration is 0.2 mol/L, and is standby in 4 ℃ of refrigerators preservations, wherein.From the heart extracting arterial blood, collect stand-by the mouse that infects trypanosoma bocagei.To contain the mouse blood of trypanosome and frozen-dried protective agent solution by volume the abundant mixing of 1:1 make trypanosome suspension, being sub-packed in volume is in the 2.5ml cillin bottle, every bottle of 0.2ml trypanosome suspension, at room temperature hatch 5 minutes after, directly drop into pre-freeze in the liquid nitrogen.After 24 hours, the trypanosome suspension that pre-freeze is good is transferred in the freeze dryer together with cillin bottle, carries out vacuum drying, obtains the parasitic protozoa freeze-dried powder at last.The vacuum drying condition is: drying pressure 10Pa, baffle temperature-15 ℃, condenser temperature-57 ℃, 60 minutes drying times.Utilize the survival rate of light microscope detection polypide before and after the vacuum drying, calculate recovery rate.The parasitic protozoa freeze-dried powder can be directly with after the cillin bottle gland seal under room temperature long preservation.Fig. 1 is for adopting ESEM (SEM) photo of the resulting trypanosome freeze-dried powder of present embodiment, and photo shows intact red cells and trypanosome in the trypanosome freeze-dried powder, and stay after the ice crystal distillation enrich hole.
Embodiment 2
Take by weighing trehalose and Tea Polyphenols, be dissolved in the PBS buffer solution, be mixed with the frozen-dried protective agent solution, trehalose concentration is 0.1 mol/L, and is standby in 4 ℃ of refrigerators preservations, wherein.To infect plasmodial mouse from the heart extracting arterial blood, collect stand-by.To contain plasmodial mouse blood and frozen-dried protective agent solution by volume the abundant mixing of 1:1 make trypanosome suspension; being sub-packed in volume is in the 2.5ml cillin bottle; every bottle of 0.2ml plasmodium suspension, at room temperature hatch 10 minutes after, directly drop into pre-freeze in the liquid nitrogen.After 24 hours, the plasmodium suspension that pre-freeze is good is transferred in the freeze dryer together with cillin bottle, carries out vacuum drying, obtains the parasitic protozoa freeze-dried powder at last.The vacuum drying condition is: drying pressure 20Pa, baffle temperature-30 ℃, condenser temperature-180 ℃, 100 minutes drying times.Utilize the survival rate of light microscope detection polypide before and after the vacuum drying, calculate recovery rate.The parasitic protozoa freeze-dried powder can be directly with after the cillin bottle gland seal under room temperature long preservation.
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spiritual essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1. the preparation method of a parasitic protozoa freeze-dried powder is characterized in that may further comprise the steps:
(1) parasitic protozoa is dispersed in formation parasitic protozoa suspension in the frozen-dried protective agent solution, is sub-packed in the cillin bottle, after at room temperature hatching, drop into pre-freeze in the liquid nitrogen;
(2) the parasitic protozoa suspension with above-mentioned pre-freeze is transferred in the freeze dryer, carries out vacuum drying, obtains the parasitic protozoa freeze-dried powder.
2. preparation method according to claim 1, it is characterized in that: described parasitic protozoa comprises plasmodium, Amoeba, arc worm or trypanosome.
3. according to the described preparation method of claim 1; it is characterized in that: in the described frozen-dried protective agent solution, freeze drying protectant is trehalose and Tea Polyphenols, and the concentration of trehalose is 0.05 ~ 0.5mol/L; the concentration of Tea Polyphenols is 0 ~ 0.5 mol/L, and solvent is the PBS buffer solution.
4. according to the described preparation method of claim 1, it is characterized in that: the concentration before the described parasitic protozoa pre-freeze in the frozen-dried protective agent solution is 1 * 10
7~ 10
8Individual/ml.
5. according to the described preparation method of claim 1, it is characterized in that: the described vacuum drying condition of step (2) is: drying pressure 0 ~ 30Pa, baffle temperature-40 ~-10 ℃, condenser temperature-196 ~-57 ℃, 30 ~ 120 minutes drying times.
6. preparation method according to claim 1, it is characterized in that: the described time of at room temperature hatching is 5-10 minute, the time of pre-freeze is 24 hours in liquid nitrogen.
7. parasitic protozoa freeze-dried powder, each described preparation method obtains by claim 1 ~ 5.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310190462XA CN103299986A (en) | 2013-07-03 | 2013-07-03 | Parasitic protozoon lyophilized powder and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310190462XA CN103299986A (en) | 2013-07-03 | 2013-07-03 | Parasitic protozoon lyophilized powder and preparation method thereof |
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| CN103299986A true CN103299986A (en) | 2013-09-18 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018067647A1 (en) * | 2016-10-05 | 2018-04-12 | Zoetis Services Llc | Lyophilization methods that provide stably dehydrated protozoans for use as potent live vaccines |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102145020A (en) * | 2011-04-15 | 2011-08-10 | 常州市炉奇虫草行 | Chinese caterpillar fungus freeze-dried fresh-keeping processing technique |
-
2013
- 2013-07-03 CN CN201310190462XA patent/CN103299986A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102145020A (en) * | 2011-04-15 | 2011-08-10 | 常州市炉奇虫草行 | Chinese caterpillar fungus freeze-dried fresh-keeping processing technique |
Non-Patent Citations (2)
| Title |
|---|
| 王海燕等: "连用DCS与显微方法的冷冻干燥系统及实验研究", 《制冷学报》, vol. 32, no. 3, 16 June 2011 (2011-06-16), pages 70 - 73 * |
| 王海燕等: "锥虫保护剂冻结及冻干特性研究", 《工程热物理学报》, vol. 32, no. 8, 31 August 2011 (2011-08-31), pages 1365 - 1367 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018067647A1 (en) * | 2016-10-05 | 2018-04-12 | Zoetis Services Llc | Lyophilization methods that provide stably dehydrated protozoans for use as potent live vaccines |
| CN109789097A (en) * | 2016-10-05 | 2019-05-21 | 硕腾服务有限责任公司 | The protozoic freeze drying process for stablizing dehydration for being used as potent live vaccine is provided |
| CN109789097B (en) * | 2016-10-05 | 2021-05-18 | 硕腾服务有限责任公司 | Lyophilization process to provide stably dehydrated protozoa for use as potent live vaccines |
| EP3522871B1 (en) * | 2016-10-05 | 2021-11-17 | Zoetis Services LLC | Lyophilization methods that provide stably dehydrated protozoans for use as potent live vaccines |
| US11453854B2 (en) | 2016-10-05 | 2022-09-27 | Zoetis Services Llc | Lyophilization methods that provide stably dehydrated protozoans for use as potent live vaccines |
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Application publication date: 20130918 |