CN111235086A - Antibacterial composition for cell culture and application thereof - Google Patents
Antibacterial composition for cell culture and application thereof Download PDFInfo
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- CN111235086A CN111235086A CN202010090761.6A CN202010090761A CN111235086A CN 111235086 A CN111235086 A CN 111235086A CN 202010090761 A CN202010090761 A CN 202010090761A CN 111235086 A CN111235086 A CN 111235086A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2500/80—Undefined extracts from animals
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to an antibacterial composition for cell culture and application thereof. The antibacterial composition for cell culture is characterized in that the effective component of the antibacterial composition is catfish body surface mucus, and the body surface mucus comprises catfish body surface antibacterial polypeptide and active polysaccharide. The catfish body surface antibacterial polypeptide has the effects of sterilizing and preventing bacterial infection, the peptide substance and the active polysaccharide have the effect of providing necessary nutrition for cell culture, and the two components play a role in synergy.
Description
This application will likely serve as a priority basis for subsequent patent applications (including, but not limited to, chinese invention patent applications, chinese utility model applications, PCT applications, foreign applications based on the paris convention).
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to an antibacterial composition and application thereof in cell culture and antibiosis.
Background
At present, the common cell culture modes comprise three types of adherent culture, suspension culture and immobilized culture, wherein the suspension culture and the immobilized culture are easy to produce contamination due to long culture time, and the operation is difficult, and the time and money are wasted. The most common method for preventing bacterial contamination during cell culture is to add antibiotics to the culture medium-for example, the currently used cell culture antibiotics are penicillin, streptomycin, gentamicin, tetracycline, erythromycin, etc., but each antibiotic usually can only inhibit bacteria against a certain bacteria, such as penicillin and streptomycin, in combination, so-called "double-resistance", mainly against gram-positive bacteria and gram-negative bacteria. For bacterial infections, gentamicin or kanamycin may also be used. If the fungus pollution is serious, amphotericin/nystatin can be used for controlling; if the mycoplasma contamination is caused, a combined antibiotic of gentamicin/tetracycline/erythromycin can be used
However, there is a certain potential hazard with the use of antibiotics in cell culture: frequent use of antibiotics may cause bacterial tolerance and even induce new types of bacteria. And the antibiotics in the waste can cause serious pollution if being discarded in the environment at will. Meanwhile, the development progress of antibiotics is far shorter than the evolution speed of new bacteria, and bacterial infection is likely to be caused in the future without medicine. Therefore, there is a need to research and apply a novel antibacterial agent that is non-toxic, non-hazardous, and non-resistant to drugs instead of antibiotics.
Disclosure of Invention
In view of the above, the present invention aims to provide a novel antibacterial composition for cell culture.
In order to achieve the above object, the technical solution of the present invention is as follows.
An antibacterial composition for cell culture, the effective component of the antibacterial composition is catfish body surface mucus; the catfish body surface mucus comprises catfish body surface antibacterial polypeptide and active polysaccharide; the molecular weight of the catfish body surface antibacterial polypeptide is 2000-30000, and the molecular weight of the active polysaccharide is 2000-30000.
Further, the catfish species include one or more of silurus meridionalis, clarias fuscus, or clarias fuscus.
The antibacterial composition is a broad-spectrum antibacterial substance, has an inhibiting effect on infection caused by gram-positive bacteria and gram-negative bacteria, and can inhibit bacteria including but not limited to staphylococcus aureus, escherichia coli and bacillus subtilis.
Further, the antibacterial composition also comprises ethanol, glycerol and water, wherein the ethanol accounts for 400 parts, the glycerol accounts for 10-50 parts, and the water accounts for 1000 parts.
Preferably, the ethanol accounts for 100-200 parts, the glycerol accounts for 10-30 parts, and the water accounts for 600-900 parts.
The ethanol and the water are solvents, and the glycerol is a thickening agent. Through the specific proportion, the catfish body surface antibacterial polypeptide and the active polysaccharide molecules can be fully dissolved, and meanwhile, the consistency is proper, so that the whole antibacterial composition has higher stability and is convenient to be subsequently used for cell culture.
The invention also aims to provide application of the antibacterial composition.
The application of the antibacterial composition in preparing cell culture antibacterial agents.
Further, the antibacterial composition can be used for preparing antibacterial agents for cell suspension culture and/or cell immobilization culture.
In daily experimental operations, cell suspension culture and cell immobilization culture are easy to be infected with bacteria, so antibiotics are often added, but the use of antibiotics has certain potential risks. The antibacterial composition is particularly suitable for the two cell culture modes and is safer compared with the addition of antibiotics. The research team of the invention finds that the addition of 1-10% of the dosage range has the best bacteriostatic effect by searching in a laboratory.
The invention also aims to provide a preparation method of the antibacterial agent containing the antibacterial composition.
A preparation method of a cell culture antibacterial agent comprises the following steps:
1) collecting catfish body surface mucus;
2) extracting components with molecular weight of 2000-30000 in the catfish body surface mucus to obtain a catfish body surface macromolecule mixture containing catfish body surface antibacterial polypeptide and active polysaccharide, drying to obtain catfish body surface macromolecule mixture powder, and freezing and storing; the invention adopts gel chromatography to extract the components with the molecular weight between 2000-30000 in the mucus;
3) and dissolving the catfish body surface macromolecule mixture powder in deionized water containing ethanol and glycerol according to the weight percentage of not less than 1% to obtain the cell culture antibacterial agent.
Further, the weight percentage is 1% -10%.
Further, in the step 1), clear water for feeding the fresh and alive catfish is filtered every 12-24 hours.
The invention preferably uses the filtering of the water body for feeding the catfish to obtain the catfish body surface mucus, but other methods, such as towel wiping collection, also belong to the protection scope of the invention.
Further, the method for drying and preparing powder in the step 2) comprises a freeze drying method and a spray drying method, wherein the drying temperature is-20 to-50 ℃.
Further, in the step 3), the ethanol accounts for 400 parts, the glycerol accounts for 10-50 parts, and the water accounts for 1000 parts.
The invention has the beneficial effects
The cell culture antibacterial composition provided by the invention comprises the active ingredients of catfish body surface mucus macromolecule components, wherein the body surface mucus macromolecule components comprise catfish body surface antibacterial polypeptide and active polysaccharide, the catfish body surface antibacterial polypeptide has the effects of sterilizing and preventing bacterial infection, the peptide substances and the active polysaccharide have the effect of providing necessary nutrition for cell culture, and the two components play a role in synergy.
The catfish body surface antibacterial polypeptide provided by the invention is a natural antibacterial substance, does not bring drug resistance to bacteria, is safer than antibiotics, avoids abuse of antibiotics and has no side effect. The active polysaccharide and the antibacterial polypeptide are both from feed water for feeding the fresh and live catfish, belong to waste utilization, save water resources and are environment-friendly. In addition, the two substances of the antibacterial polypeptide and the active polysaccharide are concomitantly generated in the obtaining process, do not need to be respectively mixed, have simple preparation process and are suitable for industrial scale-up production.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
Extracting a mixture of catfish body surface antibacterial polypeptide and active polysaccharide:
the experiment adopts 5 fresh healthy silurus meridionalis fish, the silurus meridionalis fish is fed in a water tank for 7 days by using clean water, the clean water for feeding is filtered by using a plurality of layers of gauzes every 12 to 24 hours, filtrate is collected by using a test tube, and the filtrate is stored at the temperature of 0 to 4 ℃.
Extracting components with molecular weight of 2000-30000 in the filtrate by gel chromatography, freeze drying at-20-50 deg.C to obtain powder or spray drying to obtain block, pulverizing the block into powder of about 80 meshes, and storing at-80 deg.C in a clean brown bottle. The optimal separation condition of gel chromatography is 0.02mol/L Tris-HCl buffer solution with pH8.0, and the elution speed is 0.3 ml/min.
In this example, the temperature conditions used for freeze drying and the temperature conditions for storage were optimum conditions, but not exclusive conditions. Experiments prove that the temperature is controlled to be at-4 ℃ during freeze drying and low-temperature storage, so that the bacteriostatic activity of the freeze drying and low-temperature storage agent is kept, but the bacteriostatic activity is gradually weakened along with the prolonging of time in the temperature environment.
Example 2
Test for bacteriostatic Activity
The antibacterial activity of the catfish body surface mucus extract on three bacteria is inspected in a bacteriostatic circle mode.
The experiment adopts an Oxford cup method for detection, and comprises the following steps: spreading an agar culture medium as bottom layer in a culture dish, cooling, and adding a thin layer of agar culture medium containing test bacteria (Staphylococcus aureus, Escherichia coli, and Bacillus subtilis) (50 μ L of agar culture medium with bacteria content of about 9 × 10)8Cell culture medium of about 10 cells/mL6one/mL). Dissolving the catfish body surface mucus extract powder in pure water by a certain multiple to prepare a concentration gradient antibacterial sample (as shown in table 1, in the experiment, the concentration of the catfish body surface mucus extract is 1%, 2.5%, 5%, 7.5% and 10%), punching a hole on a flat plate containing test bacteria by using an Oxford cup (the diameter is 9mm), adding 15-20 mu L of the antibacterial sample into each hole, using physiological saline as negative control, placing the flat plate into a refrigerator at 4 ℃ for standing for 12h after sample addition, fully diffusing the antibacterial sample into agar, then placing the flat plate upside down into an incubator at 37 ℃ for incubation for 24h, and observing the antibacterial effect.
Finally, as shown in table 1, it can be seen from the table that the catfish body surface mucus extract has stronger bacteriostatic activity on staphylococcus aureus, escherichia coli and bacillus subtilis, and the bacteriostatic effect is more obvious with the increase of the concentration of the catfish body surface mucus extract. Wherein, the diameter of the inhibition zone is more than 0, which indicates that the catfish body surface mucus extract has the inhibition function on staphylococcus aureus, escherichia coli, bacillus subtilis, gram-positive bacteria and gram-negative bacteria; the larger the diameter of the inhibition zone is, the stronger the antibacterial activity is.
TABLE 1 bacteriostatic Activity of catfish body surface mucus extracts on different bacteria
Here, the concentration is 10% at 100mg/mL and 1% at 10 mg/mL.
Example 3
The mixture of the catfish body surface antibacterial peptide and the active polysaccharide in the first embodiment is as follows: cell culture fluid 1:9 the cell culture solution added with catfish body surface mucus is prepared according to the proportion.
The cells were taken out from the incubator and observed under a microscope for growth state and density. The amount of medium in the well of the plate was first observed. Sucking out the cells in the two holes together with the culture solution and PBS by using a suction pipe, putting the cells in the holes of the culture plate, slightly blowing the hole walls, centrifuging, setting a centrifuge at 1000 rpm for 4-5 minutes. Taking out the centrifuge tube, slightly sucking out the supernatant, and pouring into a waste liquid tank. Sucking about 7mL of culture solution, placing the culture solution into a centrifuge tube, sucking 1mL of cell suspension, transferring the cell suspension into an EP tube, and preparing for counting. The remaining cells were placed in six wells, approximately 1mL per well. Aspirate medium into the wells of the plate to 1/3 well capacity. The cells were found to be evenly distributed by microscopic observation, and no several bacteria appeared.
Example 4
A method for preparing cell culture antibacterial agent containing catfish body surface mucus is provided.
S1 collecting mucus secreted by the body surface of the catfish: filtering to obtain clear water for feeding catfish, collecting filtrate, and freezing for storage.
S2 separation and drying: extracting components with molecular weight of 2000-30000 from the filtrate by gel chromatography to obtain a mixture of catfish body surface antibacterial peptide and active polysaccharide, drying to obtain powder, and storing at-80-100 deg.C.
S3 preparation of antibacterial agent: dissolving the antibacterial peptide and active polysaccharide mixture powder in deionized water containing ethanol and glycerol at a ratio of 0.5% -5% (preferably 1: 100, i.e. 1%), and stirring to obtain medicinal solution; dipping the medicinal solution in medicinal cotton, and drying in dark to obtain the antibacterial agent. The antibacterial agent has high stability, can be kept in a clear state after being placed for a long time in a normal temperature environment, and does not generate precipitates and the like. In addition, water, ethanol and glycerol are highly safe solvents and do not cause cytotoxicity, and thus are also suitable as antibacterial agents for cell culture.
And in the step S1, filtering clear water for feeding the fresh and live catfish by using multiple layers of gauze every 12-24 hours.
Wherein the drying step of S2 comprises freeze drying and spray drying, and the drying temperature is-20 to-50 ℃; the block obtained by spray drying method is crushed and then treated by sieve mesh to obtain granular powder.
In the drying step of S3, the deionized water containing ethanol and glycerol has a volume ratio of ethanol: glycerol: 160 parts of water: 20: 820.
example 5
The mixture of the catfish body surface antibacterial peptide and the active polysaccharide, penicillin (100 mug/mL) and deionized water without adding the bacteriostatic agent are respectively prepared into solutions with cell culture solution in the first embodiment to be subjected to cell culture, and the solutions are specifically prepared into the cell culture solution by taking the ratio of various bacteriostatic agents to the cell culture solution of 1: 9.
The cells were taken out from the incubator and observed under a microscope for growth state and density. The cell culture antibacterial agent is suitable for various common cell strains for experiments, including but not limited to Hela cells, Vero cells, CHO-K1 cells or 293 cells and the like. The amount of medium in the well of the plate was first observed. Sucking out the cells in the two holes together with the culture solution and PBS by using a suction pipe, putting the cells in the holes of the culture plate, slightly blowing the hole walls, centrifuging, setting a centrifuge at 1000 rpm for 4-5 minutes. Taking out the centrifuge tube, slightly sucking out the supernatant, and pouring into a waste liquid tank. Sucking about 7mL of culture solution, placing the culture solution into a centrifuge tube, sucking 1mL of cell suspension, transferring the cell suspension into an EP tube, and preparing for counting. The remaining cells were placed in six wells, approximately 1mL per well. The medium was aspirated into the wells of the plate to a volume of 1/3 wells and cultured for 24 hours. Taking out, observing the cell culture condition under a microscope, and finding that the cells added with the catfish body surface macromolecule antibacterial agent have good growth, normal growth speed, no abnormal cell morphology and no visible bacteria generation, and the culture solution becomes viscous; the cells added with penicillin grow well, the growth speed is normal, the cell morphology is not abnormal, no visible bacteria is generated, and the culture solution becomes viscous; cells without adding the bacteriostatic agent are dead and broken, the culture solution turns yellow and turbid, and obvious black fine sand-shaped bacteria are generated. To test the stability of the antimicrobial formulations of the present invention, the cell suspension was cultured in larger culture tubes (e.g., 36 hours, 48 hours) and still no visible bacteria were produced in the tubes containing the macromolecular antimicrobial formulation from the surface of catfish.
While embodiments of the present invention have been described, the present invention is not limited to the above-described embodiments, which are intended to be illustrative rather than limiting, and many modifications may be made by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims. For example, in addition to the catfish body surface mucus, the present invention also uses other catfish (clarias fuscus, etc.) body surface mucus to make antibacterial compositions, which also have stable antibacterial effect and can be used for cell culture.
Claims (11)
1. An antibacterial composition for cell culture, characterized in that the effective component of the antibacterial composition is catfish body surface mucus; the catfish body surface mucus comprises catfish body surface antibacterial polypeptide and active polysaccharide; the molecular weight of the catfish body surface antibacterial polypeptide is 2000-30000, and the molecular weight of the active polysaccharide is 2000-30000.
2. The antimicrobial composition of claim 1, wherein the catfish species comprises one or more of silurus meridionalis, or clarias fuscus.
3. The antimicrobial composition of claim 1, further comprising ethanol, glycerol, and water; the ethanol accounts for 400 portions, the glycerol accounts for 10-50 portions, and the water accounts for 1000 portions.
4. The antimicrobial composition of claim 3, wherein the ethanol is 100-200 parts, the glycerol is 10-30 parts, and the water is 600-900 parts.
5. Use of the antimicrobial composition of any one of claims 1 to 4 for the preparation of cell culture antimicrobial agents.
6. The use according to claim 5, wherein the antimicrobial composition is used for the preparation of antimicrobial agents for cell suspension and/or cell immobilization culture.
7. A preparation method of a cell culture antibacterial agent is characterized by comprising the following steps:
1) collecting catfish body surface mucus;
2) extracting components with molecular weight of 2000-30000 in the catfish body surface mucus to obtain a catfish body surface macromolecule mixture containing catfish body surface antibacterial polypeptide and active polysaccharide, drying to obtain catfish body surface macromolecule mixture powder, and freezing and storing;
3) and dissolving the catfish body surface macromolecule mixture powder in deionized water containing ethanol and glycerol according to the weight percentage of not less than 1% to obtain the cell culture antibacterial agent.
8. The method of claim 7, wherein the weight percentage is 1% to 10%.
9. The process according to claim 7, wherein the clear water used for feeding live catfish in step 1) is filtered every 12 to 24 hours.
10. The preparation method of claim 7, wherein the drying method for preparing the catfish body surface macromolecule mixture powder in the step 2) comprises a freeze drying method and a spray drying method, and the drying temperature is-20 to-50 ℃.
11. The method as claimed in claim 7, wherein the ethanol content in step 3) is 400 portions, the glycerol content is 10-50 portions, and the water content is 1000 portions.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010090761.6A CN111235086A (en) | 2020-02-13 | 2020-02-13 | Antibacterial composition for cell culture and application thereof |
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| CN202010090761.6A CN111235086A (en) | 2020-02-13 | 2020-02-13 | Antibacterial composition for cell culture and application thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990077430A (en) * | 1998-03-25 | 1999-10-25 | 윤덕용 | Novel peptide isolated from catfish and the use thereof |
| US6316594B1 (en) * | 1998-03-25 | 2001-11-13 | Korea Advanced Institute Of Science And Technology | Antimicrobial peptide isolated from parasilurus asotus and its uses |
| CN106047796A (en) * | 2016-08-15 | 2016-10-26 | 江苏吉锐生物技术有限公司 | Reagent capable of preventing and eliminating pollution of mammalian cells |
| CN107594187A (en) * | 2017-09-25 | 2018-01-19 | 中国标准化研究院 | The application of catfish body surface antibacterial peptide and active polysaccharide mixture in feed is prepared |
| CN107693771A (en) * | 2017-09-25 | 2018-02-16 | 中国标准化研究院 | Promotion Wound healing composition containing catfish body surface antibacterial peptide and active polysaccharide |
-
2020
- 2020-02-13 CN CN202010090761.6A patent/CN111235086A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990077430A (en) * | 1998-03-25 | 1999-10-25 | 윤덕용 | Novel peptide isolated from catfish and the use thereof |
| US6316594B1 (en) * | 1998-03-25 | 2001-11-13 | Korea Advanced Institute Of Science And Technology | Antimicrobial peptide isolated from parasilurus asotus and its uses |
| CN106047796A (en) * | 2016-08-15 | 2016-10-26 | 江苏吉锐生物技术有限公司 | Reagent capable of preventing and eliminating pollution of mammalian cells |
| CN107594187A (en) * | 2017-09-25 | 2018-01-19 | 中国标准化研究院 | The application of catfish body surface antibacterial peptide and active polysaccharide mixture in feed is prepared |
| CN107693771A (en) * | 2017-09-25 | 2018-02-16 | 中国标准化研究院 | Promotion Wound healing composition containing catfish body surface antibacterial peptide and active polysaccharide |
Non-Patent Citations (1)
| Title |
|---|
| 韩庆等: "洞庭湖鲶鱼体表黏液和肌肉营养组成对比分析", 《食品科学》 * |
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