CN110923145A - Mycoplasma freeze-drying protective agent, freeze-dried mycoplasma and preparation method of freeze-dried mycoplasma - Google Patents
Mycoplasma freeze-drying protective agent, freeze-dried mycoplasma and preparation method of freeze-dried mycoplasma Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly provides a mycoplasma freeze-drying protective agent, a mycoplasma freeze-drying protective agent and a preparation method of the mycoplasma freeze-drying protective agent. The freeze-drying protective solution comprises 1-3 w/v% dextran, 2-4 w/v% sorbitol, 4-6 w/v% hydrolyzed milk protein, 0.5-2 w/v% transferrin, 0.5-3 w/v% gelatin, 1-5 w/v% trehalose, 1-3 w/v% skim milk, 0.5-1.5 w/v% Na2HPO4.12H2O and 0.05-0.15 w/v% NaH2PO4.2H2O and the balance of water. The freeze-drying protective solution is designed aiming at freeze-drying preservation of mycoplasma strains, and the components have synergistic effect, so that the mycoplasma death in the freeze-drying process and the preservation period is obviously reduced, the mycoplasma is effectively protected, and the freeze-drying preservation time of the mycoplasma is prolonged.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a mycoplasma freeze-drying protective agent, a mycoplasma freeze-drying protective agent and a preparation method of the mycoplasma freeze-drying protective agent.
Background
Mycoplasma (mycoplasma) is a group of the smallest prokaryotic cell type microorganisms that have no cell wall, are highly polymorphic, can pass through a bacterial filter, and can be propagated by culturing in artificial media, and has a size of 0.1-0.3 microns. It is called mycoplasma because of its ability to form filamentous and branched forms. Mycoplasma is widely present in humans and animals, mostly non-pathogenic, but also a few pathogenic mycoplasma such as mycoplasma hyopneumoniae, mycoplasma genitalium, mycoplasma bovis, etc. Mycoplasma bovis is an important pathogenic pathogen which can cause pneumonia, arthritis, mastitis, keratoconjunctivitis, otitis, genital tract inflammation, even abortion, infertility and other diseases of cattle, and can cause secondary infection of the diseases under the action of other stress factors such as environment.
The freeze-drying preservation of mycoplasma strains is generally involved in the actual mycoplasma research process, but due to the basic characteristics of mycoplasma, the freeze-drying of mycoplasma strains has certain difficulty, and the freeze-drying often causes massive death of mycoplasma. Most of mycoplasma freeze-drying protective agents reported at present mainly comprise serum, trehalose, skim milk and the like which are single or combined components, but the freeze-drying effect is not satisfactory, and the viable bacteria rate and the storage life of mycoplasma freeze-drying are required to be further improved.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a mycoplasma freeze-drying protective agent and a preparation method thereof, so as to solve the problems that the mycoplasma freeze-drying protective agent in the prior art is not ideal in effect, low in viable bacteria rate of freeze-dried mycoplasma and short in storage life.
The second objective of the present invention is to provide a preparation method of a freeze-dried mycoplasma and the obtained freeze-dried mycoplasma, so as to alleviate the lack of a freeze-dried mycoplasma with high viable bacteria rate and short expiration date in the prior art.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
mycoplasma freeze-drying methodA protectant, the lyoprotectant comprising: 1-3 w/v% dextran, 2-4 w/v% sorbitol, 4-6 w/v% hydrolyzed milk protein, 0.5-2 w/v% transferrin, 0.5-3 w/v% gelatin, 1-5 w/v% trehalose, 1-3 w/v% skim milk, 0.5-1.5 w/v% Na2HPO4.12H2O and 0.05-0.15 w/v% NaH2PO4.2H2O and the balance of water.
Further, the lyoprotectant includes: 1.2-2.8 w/v% dextran, 2.2-3.8 w/v% sorbitol, 4.2-5.8 w/v% hydrolyzed milk protein, 0.7-1.8 w/v% transferrin, 0.7-2.8 w/v% gelatin, 1.5-4.5 w/v% trehalose, 1.2-2.8 w/v% skim milk, 0.7-1.3 w/v% Na2HPO4.12H2O and 0.07-0.13 w/v% NaH2PO4.2H2O and the balance of water.
Further, the lyoprotectant includes: 1.5-2.5 w/v% dextran, 2.5-3.5 w/v% sorbitol, 4.5-5.5 w/v% hydrolyzed milk protein, 0.8-1.5 w/v% transferrin, 1-2.5 w/v% gelatin, 2-4 w/v% trehalose, 1.5-2.5 w/v% skim milk, 0.8-1.2 w/v% Na2HPO4.12H2O and 0.08-0.12 w/v% NaH2PO4.2H2O and the balance of water.
Further, the lyoprotectant includes: 2 w/v% dextran, 3 w/v% sorbitol, 5 w/v% hydrolyzed milk protein, 1 w/v% transferrin, 1.5 w/v% gelatin, 3 w/v% trehalose, 2 w/v% skim milk, 1 w/v% Na2HPO4.12H2O and 0.1 w/v% NaH2PO4.2H2O and the balance of water.
Further, the mycoplasma includes mycoplasma bovis, mycoplasma hyopneumoniae, mycoplasma capricolum pleuropneumoniae, or mycoplasma ovipneumoniae.
The preparation method of the freeze-drying protective agent comprises the steps of mixing dextran, sorbitol, hydrolyzed lactoprotein, transferrin, gelatin, trehalose, skim milk and Na2HPO4.12H2O and NaH2PO4.2H2And O, uniformly mixing with water to obtain the freeze-drying protective agent.
Further, uniformly mixing the mixture with water, and sterilizing to obtain the freeze-drying protective agent;
preferably, the sterilization is performed by filtration through a 0.22 micron filter.
A method for preparing lyophilized mycoplasma comprises mixing mycoplasma with lyophilized protectant, and lyophilizing.
Further, the volume ratio of the mycoplasma to the lyoprotectant is (0.1-1.5): 1.
The freeze-dried mycoplasma is obtained by the preparation method of the freeze-dried mycoplasma.
Compared with the prior art, the invention has the beneficial effects that:
the freeze-drying protective solution for mycoplasma provided by the invention comprises dextran, sorbitol, hydrolyzed lactoprotein, transferrin, gelatin, trehalose, skim milk and Na2HPO4.12H2O and NaH2PO4.2H2And O. Wherein, Na2HPO4.12H2O and NaH2PO4.2H2O plays the effect of buffering stable system, and dextran plays the effect that prevents the crystallization of saccharide in freeze-drying process, and hydrolyzed milk protein, skimmed milk, transferrin play the effect that prevents the oxidation reaction of save in-process, and gelatin plays and prevents that effective substance from sublimating and scattering and making the effect of taking shape together with vapor, and sorbitol, trehalose play the effect of low temperature protection and dehydration protection effect. The freeze-drying protective solution is designed aiming at freeze-drying preservation of mycoplasma strains, the technical problems that the freeze-drying effect of the conventional freeze-drying protective solution is unsatisfactory, the viable bacteria rate is too low, and the storage period is short are solved, and the components have synergistic effect, so that the death of mycoplasma in the freeze-drying process and the preservation period is obviously reduced, the mycoplasma is effectively protected, and the freeze-drying preservation time of the mycoplasma is prolonged.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
A lyoprotectant for mycoplasma comprising: 1-3 w/v% dextran, 2-4 w/v% sorbitol, 4-6 w/v% hydrolyzed milk protein, 0.5-2 w/v% transferrin, 0.5-3 w/v% gelatin, 1-5 w/v% trehalose, 1-3 w/v% skim milk, 0.5-1.5 w/v% Na2HPO4.12H2O and 0.05-0.15 w/v% NaH2PO4.2H2O and the balance of water.
Na2HPO4.12H2O and NaH2PO4.2H2O plays the effect of buffering stable system, and dextran plays the effect that prevents the crystallization of saccharide in freeze-drying process, and hydrolyzed milk protein, skimmed milk, transferrin play the effect that prevents the oxidation reaction of save in-process, and gelatin plays and prevents that effective substance from sublimating and scattering and making the effect of taking shape together with vapor, and sorbitol, trehalose play the effect of low temperature protection and dehydration protection effect. The freeze-drying protective solution is designed aiming at freeze-drying preservation of mycoplasma strains, the technical problems that the freeze-drying effect of the conventional freeze-drying protective solution is unsatisfactory, the viable bacteria rate is too low, and the storage period is short are solved, and the components have synergistic effect, so that the death of mycoplasma in the freeze-drying process and the preservation period is obviously reduced, the mycoplasma is effectively protected, and the freeze-drying preservation time of the mycoplasma is prolonged.
It is understood that "w/v%" in the present invention means the volume to mass ratio of the components, for example, dextran content of 1-3 w/v% means that the freeze-drying protection solution contains 1-3g dextran in 1L volume. Dextran is typically but not limited to 1 w/v%, 1.2 w/v%, 1.4 w/v%, 1.6 w/v%, 1.8 w/v%, 2 w/v%, 2.2 w/v%, 2.4 w/v%, 2.6 w/v%, 2.8 w/v%, or 3 w/v%; sorbitol is typically, but not limited to, 2 w/v%, 2.2 w/v%, 2.4 w/v%, 2.6 w/v%, 2.8 w/v%, 3 w/v%, 3.2 w/v%, 3.4 w/v%, 3.6 w/v%, 3.8 w/v%, or 4 w/v%; the hydrolyzed milk protein is typically, but not limited to, 4 w/v%, 4.2 w/v%, 4.5 w/v%, 4.8 w/v%, 5 w/v%, 5.3 w/v%, 5.5 w/v%v%, 5.7 w/v% or 6 w/v%; transferrin is typically, but not limited to, 0.5 w/v%, 0.7 w/v%, 0.9 w/v%, 1.1 w/v%, 1.3 w/v%, 1.5 w/v%, 1.7 w/v%, 1.9 w/v%, or 2 w/v%; gelatin is typically, but not limited to, 0.5 w/v%, 0.8 w/v%, 1.2 w/v%, 1.5 w/v%, 1.8 w/v%, 2.1 w/v%, 2.4 w/v%, 2.7 w/v%, or 3 w/v%; trehalose is typically, but not limited to, 1 w/v%, 1.5 w/v%, 2 w/v%, 2.5 w/v%, 3 w/v%, 3.5 w/v%, 4 w/v%, 4.5 w/v%, or 5 w/v%; skim milk is typically, but not limited to, 1 w/v%, 1.5 w/v%, 2 w/v%, 2.5 w/v%, or 3 w/v%; na (Na)2HPO4.12H2O is typically but not limited to 0.5 w/v%, 0.7 w/v%, 0.9 w/v%, 1.1 w/v%, 1.3 w/v%, or 1.5 w/v%; NaH2PO4.2H2O is typically, but not limited to, 0.05 w/v%, 0.07 w/v%, 0.09 w/v%, 0.11 w/v%, 0.13 w/v%, or 0.15 w/v%.
In a preferred embodiment, the lyoprotectant includes: 1.2-2.8 w/v% dextran, 2.2-3.8 w/v% sorbitol, 4.2-5.8 w/v% hydrolyzed milk protein, 0.7-1.8 w/v% transferrin, 0.7-2.8 w/v% gelatin, 1.5-4.5 w/v% trehalose, 1.2-2.8 w/v% skim milk, 0.7-1.3 w/v% Na2HPO4.12H2O and 0.07-0.13 w/v% NaH2PO4.2H2O and the balance of water.
Further preferably, the lyoprotectant includes: 1.5-2.5 w/v% dextran, 2.5-3.5 w/v% sorbitol, 4.5-5.5 w/v% hydrolyzed milk protein, 0.8-1.5 w/v% transferrin, 1-2.5 w/v% gelatin, 2-4 w/v% trehalose, 1.5-2.5 w/v% skim milk, 0.8-1.2 w/v% Na2HPO4.12H2O and 0.08-0.12 w/v% NaH2PO4.2H2O and the balance of water.
Even more preferably, the lyoprotectant comprises: 2 w/v% dextran, 3 w/v% sorbitol, 5 w/v% hydrolyzed milk protein, 1 w/v% transferrin, 1.5 w/v% gelatin, 3 w/v% trehalose, 2 w/v% skim milk, 1 w/v% Na2HPO4.12H2O and 0.1 w/v% NaH2PO4.2H2O and the balance of water.
By gradually optimizing the content of each component in the freeze-drying protective agent, the test of the invention finds that the freeze-drying viable bacteria rate and the storage time of mycoplasma are obviously improved.
In a preferred embodiment, the lyoprotectant may be used for lyophilised mycoplasma including mycoplasma bovis, mycoplasma hyopneumoniae, mycoplasma capricolum pleuropneumoniae or mycoplasma ovipneumoniae, preferably mycoplasma bovis. The freeze-drying protective agent provided by the invention is designed and developed aiming at the mycoplasma, which is a special flora, and the freeze-drying protective agent which can obviously improve the freeze-drying effect of the mycoplasma is provided aiming at the self characteristics of no cell wall, high polymorphism and the like of the mycoplasma, and can meet the freeze-drying requirement in the research and production of various mycoplasma.
A lyophilized protectant is prepared from dextran, sorbitol, hydrolyzed lactoprotein, transferrin, gelatin, trehalose, skimmed milk, and Na2HPO4.12H2O and NaH2PO4.2H2And O, uniformly mixing with water to obtain the freeze-drying protective agent. The preparation of the freeze-drying protective agent provided by the invention has no special requirements, and all the components are uniformly mixed in water according to the raw material proportion, so that the cost is low, and the operation is simple and convenient.
In a preferred embodiment, the lyoprotectant is obtained by sterilization after mixing with water, preferably by filtration through a 0.22 micron filter. The filtration sterilization can effectively sterilize, avoid polluting mycoplasma strains, and simultaneously avoid the damage to hydrolyzed lactoprotein and the like in the components to influence the effect of the freeze-drying protective agent.
A method for preparing lyophilized mycoplasma comprises mixing mycoplasma with lyophilized protectant, and lyophilizing.
In a preferred embodiment, the ratio of mycoplasma to lyoprotectant is (0.1-1.5):1 by volume. It is understood that mycoplasma refers to mycoplasma that have been cultured to yield 1.0-40.0X 108The volume ratio of the CFU/ml bacterial liquid, the mycoplasma bacterial liquid and the freeze-drying protective agent is (0.1-1.5):1, and the volume ratio is, for example but not limited to, 0.1:1, 0.5:1, 1:1 or 1.5: 1.
The invention finally provides the freeze-dried mycoplasma obtained by the preparation method of the freeze-dried mycoplasma.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1
A lyoprotectant for mycoplasma comprising: 1 w/v% dextran, 4 w/v% sorbitol, 4 w/v% hydrolyzed milk protein, 2 w/v% transferrin, 0.5 w/v% gelatin, 5 w/v% trehalose, 1 w/v% skim milk, 1.5 w/v% Na2HPO4.12H2O and 0.05 w/v% NaH2PO4.2H2O and the balance of water.
Example 2
A lyoprotectant for mycoplasma comprising: 3 w/v% dextran, 2 w/v% sorbitol, 6 w/v% hydrolyzed milk protein, 0.5 w/v% transferrin, 3 w/v% gelatin, 1 w/v% trehalose, 3 w/v% skim milk, 0.5 w/v% Na2HPO4.12H2O and 0.15 w/v% NaH2PO4.2H2O and the balance of water.
Example 3
A lyoprotectant for mycoplasma comprising: 1.5 w/v% dextran, 3.5 w/v% sorbitol, 4.5 w/v% hydrolyzed milk protein, 1.5 w/v% transferrin, 1 w/v% gelatin, 4 w/v% trehalose, 1.5 w/v% skim milk, 1.2 w/v% Na2HPO4.12H2O and 0.08 w/v% NaH2PO4.2H2O and the balance of water.
Example 4
A lyoprotectant for mycoplasma comprising: 2.5 w/v% dextran, 2.5 w/v% sorbitol, 5.5 w/v% hydrolyzed milk protein, 0.8 w/v% transferrin, 2.5 w/v% gelatin, 2 w/v% trehalose, 2.5 w/v% skim milk, 0.8 w/v% Na2HPO4.12H2O and 0.12 w/v% NaH2PO4.2H2O and the balance of water.
Example 5
A lyoprotectant for mycoplasma comprising: 2 w/v% dextran, 3 w/v% sorbitol, 5 w/v% hydrolyzed milk protein1 w/v% transferrin, 1.5 w/v% gelatin, 3 w/v% trehalose, 2 w/v% skim milk, 1 w/v% Na2HPO4.12H2O and 0.1 w/v% NaH2PO4.2H2O and the balance of water.
Comparative example 1
A lyoprotectant for mycoplasma comprising: 0.1 w/v% dextran, 10 w/v% sorbitol, 1 w/v% hydrolyzed milk protein, 6 w/v% transferrin, 0.05 w/v% gelatin, 10 w/v% trehalose, 0.5 w/v% skim milk, 5 w/v% Na2HPO4.12H2O and 0.01 w/v% NaH2PO4.2H2O and the balance of water.
Comparative example 2
A lyoprotectant for mycoplasma comprising: 5 w/v% dextran, 0.5 w/v% sorbitol, 10 w/v% hydrolyzed milk protein, 0.1 w/v% transferrin, 6 w/v% gelatin, 0.1 w/v% trehalose, 6 w/v% skim milk, 0.1 w/v% Na2HPO4.12H2O and 3 w/v% NaH2PO4.2H2O and the balance of water.
Comparative example 3
A lyoprotectant for mycoplasma comprising: 2 w/v% dextran, 3 w/v% sorbitol, 5 w/v% hydrolyzed milk protein, 1.5 w/v% gelatin, 3 w/v% trehalose, 2 w/v% skim milk, 1 w/v% Na2HPO4.12H2O and 0.1 w/v% NaH2PO4.2H2O and the balance of water.
Comparative example 4
A lyoprotectant for mycoplasma comprising: 2 w/v% dextran, 3 w/v% sorbitol, 1 w/v% transferrin, 1.5 w/v% gelatin, 3 w/v% trehalose, 2 w/v% skim milk, 1 w/v% Na2HPO4.12H2O and 0.1 w/v% NaH2PO4.2H2O and the balance of water.
Comparative example 5
A lyoprotectant for mycoplasma comprising: 2 w/v% dextran, 3 w/v% sorbitol, 5 w/v% hydrolyzed milk protein, 1 w/v% transferrin, 1.5 w/v% gelatin, 3 w/v% trehalose, 1 w/v% Na2HPO4.12H2O and 0.1 w/v% NaH2PO4.2H2O and the balance of water.
Comparative example 6
A lyoprotectant for mycoplasma comprising: 2 w/v% dextran, 3 w/v% sorbitol, 5 w/v% hydrolyzed milk protein, 1 w/v% transferrin, 3 w/v% trehalose, 2 w/v% skim milk, 1 w/v% Na2HPO4.12H2O and 0.1 w/v% NaH2PO4.2H2O and the balance of water.
Comparative example 7
A lyoprotectant for mycoplasma comprising: 7.5 w/v% skimmed milk and 2.5 w/v% trehalose, the balance being water.
Comparative example 8
A lyoprotectant for mycoplasma comprising: 7.5 w/v% skim milk, 2.5 w/v% trehalose and 40 w/v% serum, the balance being water.
Test examples
1. Preparing bacterial liquid: taking out mycoplasma bovis strain from-80 deg.C, thawing at room temperature, inoculating mycoplasma bovis special culture medium according to 15% inoculation ratio, culturing in 37 deg.C carbon dioxide constant temperature incubator for 4 days, collecting bacterial liquid, and counting viable bacteria.
2. Preparing a freeze-drying protective agent: lyoprotectants (100 ml content) were prepared according to the formulations of the lyoprotectants in examples 1-5 and comparative examples 1-8, respectively.
3. Freeze-drying
3.1 mixing: the lyoprotectants of examples 1-5 and comparative examples 1-8 were mixed with Mycoplasma bovis bacteria at a ratio of 1:1 (by volume), and the viable bacteria were counted. The mixed solution is respectively subpackaged into 7 ml sterile bottles, 2ml sterile bottles and covered with a T-shaped plug.
3.2 freeze-drying: the samples were lyophilized according to the lyophilization procedure of a lyophilizer.
Three batches of the above lyophilized samples were prepared in succession according to the above procedure.
4. Examination of Freeze drying Effect
4.1 Property and solubility observations: for each lyoprotectant, 10 vials of lyophilized samples were randomly selected and observed for lyophilizate properties and solubility. The shape of the product is spongy loose lumps which are easy to separate from the bottle wall, and the product can be quickly dissolved after being added with diluent. And judging the product to be qualified.
4.2 vacuum degree detection: for each lyoprotectant, 10 vials of the lyophilized sample were randomly selected and the vacuum was checked. The bottle is judged to be qualified when white, pink or purple glow appears in the bottle.
4.3 residual moisture: for each lyoprotectant machine, 10 vials of the lyophilized sample were selected and the remaining water was measured. The residual water content is not more than 4.0 percent, and the product is judged to be qualified.
4.4 determination of viable cell ratio: and randomly selecting 3 bottles of freeze-dried samples for each freeze-drying protective agent, counting viable bacteria on the day after freeze-drying is finished, and calculating the viable bacteria rate.
4.5 shelf life test: for each lyoprotectant, 3 vials of lyophilized samples were randomly selected and viable count was performed on the day of lyophilization, at-20 ℃ (12, 24, and 36 months).
The samples which are qualified in the detection of the properties, the solubility, the vacuum degree and the residual moisture of 3 batches of freeze-dried mycoplasma bovis prepared by the freeze-drying protective agents in the examples 1-5 and the comparative examples 1-8 respectively enter the test detection of the viable bacteria rate and the storage life.
Effect example 1 viable cell ratio
Viable bacteria count was performed before and after freeze-drying of each sample, and viable bacteria rate was calculated, and the average results of 3 samples are shown in the following table:
remarking: the 'Y' represents a spongy loose block with the character of being easy to separate from the bottle wall and quickly dissolved after being added with diluent. The ' N ' represents ' the character of compact block, not easy to be separated from the bottle wall or slow to be dissolved after adding diluent.
Effect example 2 shelf life
Viable bacteria count was performed on the day of freeze-drying completion for each sample, storage at-20 ℃ (12, 24, and 36 months), and viable bacteria ratio was calculated, and the results are shown in the following table:
while particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. A lyoprotectant for a mycoplasma comprising: 1-3 w/v% dextran, 2-4 w/v% sorbitol, 4-6 w/v% hydrolyzed milk protein, 0.5-2 w/v% transferrin, 0.5-3 w/v% gelatin, 1-5 w/v% trehalose, 1-3 w/v% skim milk, 0.5-1.5 w/v% Na2HPO4.12H2O and 0.05-0.15 w/v% NaH2PO4.2H2O and the balance of water.
2. The lyoprotectant of claim 1, wherein said lyoprotectant comprises: 1.2-2.8 w/v% dextran, 2.2-3.8 w/v% sorbitol, 4.2-5.8 w/v% hydrolyzed milk protein, 0.7-1.8 w/v% transferrin, 0.7-2.8 w/v% gelatin, 1.5-4.5 w/v% trehalose, 1.2-2.8 w/v% skim milk, 0.7-1.3 w/v% Na2HPO4.12H2O and 0.07-0.13 w/v% NaH2PO4.2H2O and the balance of water.
3. The lyoprotectant of claim 2, wherein said lyoprotectant comprises: 1.5-2.5 w/v% dextran, 2.5-3.5 w/v% sorbitol, 4.5-5.5 w/v% hydrolyzed milk protein, 0.8-1.5 w/v% transferrin, 1-2.5 w/v% gelatin, 2-4 w/v% trehalose, 1.5-2.5 w/v% skim milk, 0.8-1.2 w/v% Na2HPO4.12H2O and 0.08-0.12 w/v% NaH2PO4.2H2O and the balance of water.
4. The lyoprotectant of claim 3, wherein said lyoprotectant comprises: 2 w/v% dextran, 3 w/v% sorbitol, 5 w/v% hydrolyzed milk protein, 1 w/v% transferrin, 1.5 w/v% gelatin, 3 w/v% trehalose, 2 w/v% skim milk, 1 w/v% Na2HPO4.12H2O and 0.1 w/v% NaH2PO4.2H2O and the balance of water.
5. The lyoprotectant of any of claims 1-4, wherein said mycoplasma comprises Mycoplasma bovis, Mycoplasma hyopneumoniae, contagious caprine Mycoplasma pleuropneumoniae, or Mycoplasma ovipneumoniae.
6. A process for the preparation of a lyoprotectant according to any of claims 1 to 5, characterized in that formulated amounts of dextran, sorbitol, hydrolysed milk protein, transferrin, gelatin, trehalose, skim milk, Na2HPO4.12H2O and NaH2PO4.2H2And O, uniformly mixing with water to obtain the freeze-drying protective agent.
7. The preparation method according to claim 6, characterized in that the cryoprotectant is obtained by sterilizing after mixing with water;
preferably, the sterilization is performed by filtration through a 0.22 micron filter.
8. A method for preparing a freeze-dried mycoplasma, which is characterized in that the mycoplasma and the freeze-drying protective agent of any one of claims 1 to 5 are mixed and freeze-dried.
9. The method according to claim 8, wherein the ratio of the volume of the mycoplasma to the lyoprotectant is (0.1-1.5): 1.
10. A lyophilized Mycoplasma obtained by the method according to claim 8 or 9.
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| CN201911300057.2A CN110923145B (en) | 2019-12-16 | 2019-12-16 | Mycoplasma freeze-drying protective agent, freeze-dried mycoplasma and preparation method of freeze-dried mycoplasma |
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