Marine crustacean mycoplasma culture medium and separation and purification method

Abstract

The invention relates to the technical field of culture medium preparation, in particular to a marine crustacean mycoplasma culture medium and a separation and purification method. The culture medium comprises the following components in parts by weight: 1 to 3 parts of a compound growth promoting agent, 6 to 8 parts of peptone, 3 to 5 parts of a yeast extract, 1 to 5 parts of glucose, 10 to 20 parts of bovine serum, 12 to 15 parts of a phosphate buffer solution, 0.2 to 0.4 part of beta-mercaptoethanol, 0.12 to 0.42 part of a rhodiola rosea extract and 1.2 to 2.5 parts of a DTPA chelating agent, wherein the composite growth promoting agent is prepared by mixing spirulina and sea urchin according to a mass ratio of 1: (0.05-0.07), performing enzymolysis, and compounding with phosphatidic acid; a composite growth promoting agent is adopted, a nutrient source needed by growth is provided for mycoplasma through spirulina, growth of gram-positive and gram-negative bacteria is inhibited through sea urchin, the influence of adverse compounds is overcome through phosphatidic acid, growth of the mycoplasma is promoted, meanwhile, the mycoplasma is separated and purified through a multi-time plate streaking method, and operation is easy, convenient and fast.

Description

Technical Field

[0001] The invention relates to the technical field of culture medium preparation, in particular to a culture medium for mycoplasma of marine crustaceans and a separation and purification method. Background Art

[0002] Marine crustaceans, as an indispensable part of the marine ecosystem, play an important role in the food chain with their diverse species and unique physiological structure. However, beneath these seemingly indestructible shells, there is also a lurking microbial world, including the tiny but not negligible biological group of mycoplasmas. Mycoplasmas, as a type of prokaryotic microorganism, are known for their unique biological characteristics and wide host range. In marine crustaceans, mycoplasmas can use the nutrition and environmental conditions provided by the host to grow and reproduce, while affecting the health of the host and causing infection symptoms in the host, such as growth retardation and increased mortality.

[0003] However, the existing mycoplasma culture medium has a single component, which is prone to contamination by other microorganisms, limiting the growth of mycoplasmas, and has a low titer of viable bacteria. In addition, there are few studies on the mycoplasma culture medium for marine crustaceans. In view of this, we propose a marine crustacean mycoplasma culture medium and a separation and purification method. Summary of the invention

[0004] The object of the present invention is to provide a culture medium and a separation and purification method for mycoplasma of marine crustaceans to solve the problems raised in the above background technology.

[0005] To achieve the above-mentioned purpose, the present invention provides a marine crustacean mycoplasma culture medium, comprising the following components: 1-3 parts by weight of a composite growth promoter, 6-8 parts by weight of peptone, 3-5 parts by weight of yeast extract, 1-5 parts by weight of glucose, 10-20 parts by weight of bovine serum, 12-15 parts by weight of phosphate buffer, 0.2-0.4 parts by weight of β-mercaptoethanol, 0.12-0.42 parts by weight of rhodiola rosea extract and 1.2-2.5 parts by weight of DTPA chelating agent; wherein the composite growth promoter is prepared by mixing and enzymolyzing spirulina and sea urchin in a mass ratio of 1:0.05-0.07, and then compounding with phosphatidic acid.

[0006] Preferably, the preparation method of the composite growth promoter is as follows: Use a homogenizer to crush the pretreated sea urchin and spirulina into slurry respectively, then mix the slurries of the two raw materials in proportion, crush and sterilize, and add them to a solution containing a composite enzyme inhibitor, prepare a composite enzymatic solution with phosphate buffer, transfer the pretreated mixture to a reaction vessel, add the composite enzymatic solution, and adjust the pH value. The reaction temperature is set to 37-40°C, the reaction time is 5-7h, and a magnetic stirrer is used to keep stirring to ensure that the reaction is uniform. Place the reaction mixture in a water bath and heat it to 80-100°C for 10-15min, cool it naturally to room temperature, and then centrifuge the reaction mixture at 4000-6000rpm / min for 10-15min, collect the supernatant, and then filter the supernatant with filter paper to remove residual solid particles to obtain a mixed enzymatic solution; weigh phosphatidic acid powder and dissolve it in dimethyl sulfoxide to prepare a solution with a concentration of 5-10μg / mL, and add the evenly mixed phosphatidic acid solution to the mixed enzymatic solution while stirring to obtain a composite growth promoter.

[0007] Spirulina is a cyanobacteria that has attracted attention for its high nutritional value. It is rich in protein, vitamins, minerals and antioxidants. When Spirulina is treated with a complex enzyme, its internal cell walls and proteins will be broken down into polysaccharides, polypeptides and amino acids, which can provide the nutrient source needed for the growth of mycoplasma and enrich the nutrition of the culture medium. Sea urchin is a marine invertebrate that contains edible parts, including gonads (called "yolk" or "sea urchin yolk"), muscle tissue and other internal organs. Their tissues and internal organs contain a variety of biological macromolecules such as proteins, polysaccharides, and lipids. The lipase in the complex enzyme can process the lipid components in sea urchins to obtain products such as glycerol and free fatty acids. Sea urchins also contain a variety of biologically active compounds. These active compounds can inhibit Gram-positive and Gram-negative bacteria, and can therefore act as antibiotics to inhibit the growth of Gram-positive and Gram-negative bacteria, prevent them from over-reproduction in the culture medium, and create an environment that is conducive to the growth of mycoplasma but not conducive to the growth of other microorganisms.

[0008] However, the antimicrobial peptides in Spirulina can bind to the bacterial cell membrane, destroy the integrity of the cell membrane, cause the leakage of cell contents, and ultimately cause bacterial death, which will affect the growth of mycoplasma cells. For this reason, through enzymatic hydrolysis, the antimicrobial peptides and phycocyanin are decomposed by a composite enzyme preparation to destroy their antimicrobial components, thereby affecting their activity. When the enzymatic hydrolysis reaction is incomplete, certain antimicrobial components still exist, so phosphatidic acid is added to improve its comprehensive performance. Since phosphatidic acid is one of the main components of the cell membrane, it can provide a physical barrier for the cell membrane, participate in various biochemical processes such as cell signal transmission and material transport, and help maintain the integrity and fluidity of the cell membrane, thereby improving the quality of the cell membrane and compensating for the adverse effects of the antimicrobial components in Spirulina.

[0009] Preferably, the composite enzyme preparation contains cellulase, lipase, pectinase and protease, and the mass ratio of the components is 5:2-4:0.5-0.9:1-3.

[0010] Preferably, the concentration of the composite enzymatic hydrolysate is 0.5-1% (w / v), and the ratio of the composite enzymatic hydrolysate to the two raw materials is 1:8-10 (v / w).

[0011] Preferably, the mass ratio of the mixed enzymatic hydrolysate to the phosphatidic acid solution is 1:0.8-1.2.

[0012] Preferably, the pH value is adjusted to 6.5-7.5.

[0013] In another aspect, the present invention provides a method for separating and purifying a marine crustacean mycoplasma culture medium, which is used for any one of the marine crustacean mycoplasma culture mediums described above, comprising the following steps: Adding a composite growth promoter, peptone, yeast extract, glucose, bovine serum, β-mercaptoethanol, rhodiola rosea extract and DTPA chelating agent to deionized water in sequence, stirring to dissolve solid components, adjusting the pH value of the mixed solution to a range suitable for mycoplasma growth with a phosphate buffer, filtering the mixed solution with a 0.22-0.45 μm sterile filter to remove impurities, filling the filtered culture medium into sterile bottles, sterilizing at 121-125° C. for 10-15 minutes in a high-pressure steam sterilizer, and refrigerating and storing at 2-4° C. to obtain a marine crustacean mycoplasma culture medium; Select marine crustaceans infected with mycoplasma as sample sources, collect samples from animal gill tissues using aseptic techniques, homogenize the collected samples using a grinding rod under sterile conditions, use a centrifuge to remove larger particles and cell debris from the homogenized samples, inoculate the treated samples onto the prepared culture medium, and culture them anaerobically at 37-40°C for 2-4 days; When colony formation is observed, single colonies are selected with an inoculation loop and streaked on new culture medium in parallel. Culture is continued for 1-3 days, and the above plate streaking steps are repeated 4-5 times until colonies of a single morphology are observed. The purified strain can then be frozen for storage.

[0014] Rhodiola rosea extract contains a variety of biologically active ingredients, mainly including salidroside and other phenolic compounds, which have antioxidant effects and can be used to help mycoplasma cells reduce oxidative stress, enhance the effect of the culture medium, protect cells from damage by free radicals, and promote the proliferation of mycoplasma; in addition, the additionally added DTPA chelating agent can also be used to offset the effects of adverse compounds in Spirulina on mycoplasma cells, and can reduce the catalytic effect of metal ions by chelating with metal ions, further optimizing the combination effect of the compound growth promoter and the mycoplasma culture medium components, ensuring that the culture medium can not only provide sufficient nutrition, but also overcome possible inhibitory factors, thereby promoting the growth of mycoplasma.

[0015] Preferably, the phosphate buffer adjusts the pH value of the mixed solution to 7.0-7.5.

[0016] Preferably, the centrifugation temperature is 2-4°C, the centrifugation speed is 2000-3000 rpm / min, and the centrifugation time is 5-10 min.

[0017] Preferably, the gas mixture ratio under the anaerobic condition is 80-85% N2 and 15-20% CO2.

[0018] Compared with the prior art, the present invention has the following beneficial effects: In the marine crustacean mycoplasma culture medium and separation and purification method, a composite growth promoter is used to prepare a marine crustacean mycoplasma culture medium; spirulina is rich in nutrients, which are hydrolyzed to release the nutrients, providing a nutrient source required for the growth of mycoplasma; sea urchins also contain a variety of bioactive compounds, which inhibit the growth of Gram-positive and Gram-negative bacteria, prevent them from over-reproduction in the culture medium, and provide an excellent growth environment for mycoplasma; phosphatidic acid provides a physical barrier for the cell membrane, which helps maintain the integrity and fluidity of the cell membrane, overcomes the influence of adverse compounds in spirulina, and further promotes the growth of mycoplasma; and the mycoplasma is separated and purified by a multiple plate streaking method, which is simple and convenient to operate. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The marine crustacean mycoplasma culture medium of the present invention comprises the following components: 1-3 weight parts of a composite growth promoter, 6-8 weight parts of peptone, 3-5 weight parts of yeast extract, 1-5 weight parts of glucose, 10-20 weight parts of bovine serum, 12-15 weight parts of phosphate buffer, 0.2-0.4 weight parts of beta-mercaptoethanol, 0.12-0.42 weight parts of rhodiola rosea extract and 1.2-2.5 weight parts of a DTPA chelating agent; wherein the composite growth promoter is prepared by mixing and enzymolyzing spirulina and sea urchin at a mass ratio of 1:0.05-0.07, and then compounding with phosphatidic acid.

[0021] Example 1: A culture medium and a method for separating and purifying mycoplasma of marine crustaceans, comprising the following steps: Prepare components: 3 parts by weight of a composite growth promoter, 6 parts by weight of peptone, 3 parts by weight of yeast extract, 1 part by weight of glucose, 10 parts by weight of bovine serum, 12 parts by weight of phosphate buffer, 0.2 parts by weight of β-mercaptoethanol, 0.12 parts by weight of rhodiola rosea extract and 1.2 parts by weight of a DTPA chelating agent; wherein the composite growth promoter is prepared by mixing spirulina and sea urchin with a mass ratio of 1:0.05 and then compounding with phosphatidic acid, the composite enzyme preparation contains cellulase, lipase, pectinase and protease, the mass ratio of the components is 5:2:0.5:1, the concentration of the composite enzymatic solution is 0.5% (w / v), the ratio of the composite enzymatic solution to the two raw materials is 1:8 (v / w); the mass ratio of the mixed enzymatic solution to the phosphatidic acid solution is 1:0.8; Use a homogenizer to crush the pretreated sea urchin and spirulina into slurries respectively, then mix the slurries of the two raw materials in proportion, crush and sterilize, and add them to a solution containing a composite enzyme inhibitor, prepare a composite enzymatic solution with phosphate buffer, transfer the pretreated mixture to a reaction vessel, add the composite enzymatic solution, and adjust the pH value to 7.0, set the reaction temperature to 37°C, and the reaction time to 5h. Use a magnetic stirrer to keep stirring to ensure that the reaction is uniform, place the reaction mixture in a water bath and heat it to 100°C for 10min, cool it naturally to room temperature, and then centrifuge the reaction mixture at 4000rpm / min for 10min, collect the supernatant, and then filter the supernatant with filter paper to remove residual solid particles to obtain a mixed enzymatic solution; weigh phosphatidic acid powder and dissolve it in dimethyl sulfoxide to prepare a solution with a concentration of 5-10μg / mL, add the evenly mixed phosphatidic acid solution to the mixed enzymatic solution while stirring to obtain a composite growth promoter; Adding a composite growth promoter, peptone, yeast extract, glucose, bovine serum, β-mercaptoethanol, rhodiola rosea extract and DTPA chelating agent to deionized water in sequence, stirring to dissolve solid components, adjusting the pH value of the mixed solution to 7.5 with a phosphate buffer, filtering the mixed solution with a 0.22 μm sterile filter to remove impurities, and filling the filtered culture medium into sterile bottles, sterilizing at 121° C. for 10 min in a high-pressure steam sterilizer, and refrigerating and storing at 4° C. to obtain a marine crustacean mycoplasma culture medium; Marine crustaceans infected with mycoplasma were selected as sample sources. Samples were collected from the gill tissues of animals using aseptic techniques. The collected samples were homogenized using a grinding rod under aseptic conditions. The homogenized samples were centrifuged to remove larger particles and cell debris. The centrifugal temperature was 4°C, the centrifugal speed was 3000 rpm / min, and the centrifugal time was 5 min. The treated samples were inoculated into the prepared culture medium and cultured anaerobically at 37°C for 2 days. The gas mixture ratio under anaerobic conditions was 80% N2 and 20% CO2. When colony formation is observed, single colonies are selected with an inoculation loop and streaked in parallel on new culture medium. Culture is continued for 1 day, and the above plate streaking steps are repeated 5 times until colonies of a single morphology are observed. The purified strain can then be frozen for storage.

[0022] Example 2: Marine crustacean mycoplasma culture medium and separation and purification method, comprising the following steps: Prepare components: 3 parts by weight of a composite growth promoter, 6 parts by weight of peptone, 3 parts by weight of yeast extract, 1 part by weight of glucose, 10 parts by weight of bovine serum, 12 parts by weight of phosphate buffer, 0.2 parts by weight of β-mercaptoethanol, 0.12 parts by weight of rhodiola rosea extract and 1.2 parts by weight of a DTPA chelating agent; wherein the composite growth promoter is prepared by mixing spirulina and sea urchin with a mass ratio of 1:0.06 and then compounding with phosphatidic acid, the composite enzyme preparation contains cellulase, lipase, pectinase and protease, the mass ratio of the components is 5:2:0.5:1, the concentration of the composite enzymatic solution is 0.5% (w / v), the ratio of the composite enzymatic solution to the two raw materials is 1:8 (v / w); the mass ratio of the mixed enzymatic solution to the phosphatidic acid solution is 1:1.0; Use a homogenizer to crush the pretreated sea urchin and spirulina into slurries respectively, then mix the slurries of the two raw materials in proportion, crush and sterilize, and add them to a solution containing a composite enzyme inhibitor, prepare a composite enzymatic solution with phosphate buffer, transfer the pretreated mixture to a reaction vessel, add the composite enzymatic solution, and adjust the pH value to 7.0, set the reaction temperature to 37°C, and the reaction time to 5h. Use a magnetic stirrer to keep stirring to ensure that the reaction is uniform, place the reaction mixture in a water bath and heat it to 100°C for 10min, cool it naturally to room temperature, and then centrifuge the reaction mixture at 4000rpm / min for 10min, collect the supernatant, and then filter the supernatant with filter paper to remove residual solid particles to obtain a mixed enzymatic solution; weigh phosphatidic acid powder and dissolve it in dimethyl sulfoxide to prepare a solution with a concentration of 5-10μg / mL, add the evenly mixed phosphatidic acid solution to the mixed enzymatic solution while stirring to obtain a composite growth promoter; Adding a composite growth promoter, peptone, yeast extract, glucose, bovine serum, β-mercaptoethanol, rhodiola rosea extract and DTPA chelating agent to deionized water in sequence, stirring to dissolve solid components, adjusting the pH value of the mixed solution to 7.5 with a phosphate buffer, filtering the mixed solution with a 0.22 μm sterile filter to remove impurities, and filling the filtered culture medium into sterile bottles, sterilizing at 121° C. for 10 min in a high-pressure steam sterilizer, and refrigerating and storing at 4° C. to obtain a marine crustacean mycoplasma culture medium; Marine crustaceans infected with mycoplasma were selected as sample sources. Samples were collected from the gill tissues of animals using aseptic techniques. The collected samples were homogenized using a grinding rod under aseptic conditions. The homogenized samples were centrifuged to remove larger particles and cell debris. The centrifugal temperature was 4°C, the centrifugal speed was 3000 rpm / min, and the centrifugal time was 5 min. The treated samples were inoculated into the prepared culture medium and cultured anaerobically at 37°C for 2 days. The gas mixture ratio under anaerobic conditions was 80% N2 and 20% CO2. When colony formation is observed, single colonies are selected with an inoculation loop and streaked in parallel on new culture medium. Culture is continued for 1 day, and the above plate streaking steps are repeated 5 times until colonies of a single morphology are observed. The purified strain can then be frozen for storage.

[0023] Example 3: Marine crustacean mycoplasma culture medium and separation and purification method, comprising the following steps: Prepare components: 3 parts by weight of a composite growth promoter, 6 parts by weight of peptone, 3 parts by weight of yeast extract, 1 part by weight of glucose, 10 parts by weight of bovine serum, 12 parts by weight of phosphate buffer, 0.2 parts by weight of β-mercaptoethanol, 0.12 parts by weight of rhodiola rosea extract and 1.2 parts by weight of a DTPA chelating agent; wherein the composite growth promoter is prepared by mixing spirulina and sea urchin with a mass ratio of 1:0.07 and then compounding with phosphatidic acid, the composite enzyme preparation contains cellulase, lipase, pectinase and protease, the mass ratio of the components is 5:2:0.5:1, the concentration of the composite enzymatic solution is 0.5% (w / v), the ratio of the composite enzymatic solution to the two raw materials is 1:8 (v / w); the mass ratio of the mixed enzymatic solution to the phosphatidic acid solution is 1:1.2; Use a homogenizer to crush the pretreated sea urchin and spirulina into slurry respectively, then mix the slurries of the two raw materials in proportion, crush and sterilize, and add them to a solution containing a composite enzyme inhibitor, prepare a composite enzymatic solution with a phosphate buffer, transfer the pretreated mixture to a reaction vessel, add the composite enzymatic solution, and adjust the pH value to 7.0, set the reaction temperature to 37°C, the reaction time to 5h, and use a magnetic stirrer to keep stirring to ensure that the reaction is uniform, place the reaction mixture in a water bath and heat it to 100°C for 10min, cool it naturally to room temperature, then centrifuge the reaction mixture at 4000rpm / min for 10min, collect the supernatant, and then filter the supernatant with filter paper to remove residual solid particles to obtain a mixed enzymatic solution; weigh phosphatidic acid powder and dissolve it in dimethyl sulfoxide to prepare a solution with a concentration of 5-10μg / mL, add the evenly mixed phosphatidic acid solution to the mixed enzymatic solution while stirring to obtain a composite growth promoter; Adding a composite growth promoter, peptone, yeast extract, glucose, bovine serum, β-mercaptoethanol, rhodiola rosea extract and DTPA chelating agent to deionized water in sequence, stirring to dissolve solid components, adjusting the pH value of the mixed solution to 7.5 with a phosphate buffer, filtering the mixed solution with a 0.22 μm sterile filter to remove impurities, and filling the filtered culture medium into sterile bottles, sterilizing at 121° C. for 10 min in a high-pressure steam sterilizer, and refrigerating and storing at 4° C. to obtain a marine crustacean mycoplasma culture medium; Marine crustaceans infected with mycoplasma were selected as sample sources. Samples were collected from the gill tissues of animals using aseptic techniques. The collected samples were homogenized using a grinding rod under aseptic conditions. The homogenized samples were centrifuged to remove larger particles and cell debris. The centrifugal temperature was 4°C, the centrifugal speed was 3000 rpm / min, and the centrifugal time was 5 min. The treated samples were inoculated into the prepared culture medium and cultured anaerobically at 37°C for 2 days. The gas mixture ratio under anaerobic conditions was 80% N2 and 20% CO2. When colony formation is observed, single colonies are selected with an inoculation loop and streaked in parallel on new culture medium. Culture is continued for 1 day, and the above plate streaking steps are repeated 5 times until colonies of a single morphology are observed. The purified strain can then be frozen for storage.

[0024] Example 4: Marine crustacean mycoplasma culture medium and separation and purification method, comprising the following steps: Prepare components: 1 weight part of composite growth promoter, 8 weight parts of peptone, 5 weight parts of yeast extract, 5 weight parts of glucose, 20 weight parts of bovine serum, 15 weight parts of phosphate buffer, 0.4 weight parts of β-mercaptoethanol, 0.42 weight parts of rhodiola rosea extract and 2.5 weight parts of DTPA chelating agent; wherein the composite growth promoter is prepared by mixed enzymatic hydrolysis of spirulina and sea urchin at a mass ratio of 1:0.07, and then compounded with phosphatidic acid, the composite enzyme preparation contains cellulase, lipase, pectinase and protease, the mass ratio of the components is 5:4:0.9:3, the concentration of the composite enzymatic hydrolysate is 1% (w / v), the ratio of the composite enzymatic hydrolysate to the two raw materials is 1:10 (v / w); the mass ratio of the mixed enzymatic hydrolysate to the phosphatidic acid solution is 1:1.2; Use a homogenizer to crush the pretreated sea urchin and spirulina into slurry respectively, then mix the slurries of the two raw materials in proportion, crush and sterilize, and add them to a solution containing a composite enzyme inhibitor, prepare a composite enzymatic solution with a phosphate buffer, transfer the pretreated mixture to a reaction vessel, add the composite enzymatic solution, and adjust the pH value to 7.0, set the reaction temperature to 37°C, the reaction time to 5h, and use a magnetic stirrer to keep stirring to ensure that the reaction is uniform, place the reaction mixture in a water bath and heat it to 100°C for 10min, cool it naturally to room temperature, then centrifuge the reaction mixture at 4000rpm / min for 10min, collect the supernatant, and then filter the supernatant with filter paper to remove residual solid particles to obtain a mixed enzymatic solution; weigh phosphatidic acid powder and dissolve it in dimethyl sulfoxide to prepare a solution with a concentration of 5-10μg / mL, add the evenly mixed phosphatidic acid solution to the mixed enzymatic solution while stirring to obtain a composite growth promoter; Adding a composite growth promoter, peptone, yeast extract, glucose, bovine serum, β-mercaptoethanol, rhodiola rosea extract and DTPA chelating agent to deionized water in sequence, stirring to dissolve solid components, adjusting the pH value of the mixed solution to 7.5 with a phosphate buffer, filtering the mixed solution with a 0.22 μm sterile filter to remove impurities, and filling the filtered culture medium into sterile bottles, sterilizing at 121° C. for 10 min in a high-pressure steam sterilizer, and refrigerating and storing at 4° C. to obtain a marine crustacean mycoplasma culture medium; Marine crustaceans infected with mycoplasma were selected as sample sources. Samples were collected from the gill tissues of animals using aseptic techniques. The collected samples were homogenized using a grinding rod under aseptic conditions. The homogenized samples were centrifuged to remove larger particles and cell debris. The centrifugal temperature was 4°C, the centrifugal speed was 3000 rpm / min, and the centrifugal time was 5 min. The treated samples were inoculated into the prepared culture medium and cultured anaerobically at 37°C for 2 days. The gas mixture ratio under anaerobic conditions was 80% N2 and 20% CO2. When colony formation is observed, single colonies are selected with an inoculation loop and streaked in parallel on new culture medium. Culture is continued for 1 day, and the above plate streaking steps are repeated 5 times until colonies of a single morphology are observed. The purified strain can then be frozen for storage.

[0025] Comparative Example 1: The method of Example 3 was adopted, and the amount of the composite growth promoter added to the culture medium was 0.05 parts by weight.

[0026] Comparative Example 2: The method of Example 3 was adopted, and the composite growth promoter was obtained by enzymatic hydrolysis of Spirulina and then compounded with phosphatidic acid, but no sea urchin was added.

[0027] Comparative Example 3: The method of Example 3 was adopted, the composite growth promoter was obtained by enzymatic hydrolysis of sea urchin and then compounded with phosphatidic acid, but Spirulina was not added.

[0028] Comparative Example 4: The method of Example 3 was adopted, and the composite growth promoter was prepared by enzymatic hydrolysis of Spirulina and sea urchin, but no phosphatidic acid was added.

[0029] The performance index test items and test standards of the marine crustacean mycoplasma culture medium prepared by the composite growth promoter of the present invention are as follows: The viable bacterial titer is used to characterize the number of viable bacteria in a sample. It is an important indicator for measuring the viable bacterial concentration in a microbial population and can evaluate the growth ability of microorganisms on a given culture medium. A 48-well plate was prepared. 500 μL / well of the culture medium of Example 1 was added to the first row of wells, 500 μL / well of the culture medium of Example 2 was added to the second row of wells, and so on. The culture medium of Comparative Examples 1-3 was added. The marine crustacean mycoplasma was inoculated at an initial concentration of 5×10 8 100 μL of activated marine crustacean mycoplasma was taken and diluted step by step by 10 times until the required dilution reached 10 -5, inoculate it into the first row of wells of the culture medium of Example 1 and mix evenly, and perform the same operation on the remaining culture media. After inoculation, all are placed in an incubator at 37°C and containing 20% ​​CO2. After culturing for 3 days, continue to blindly transfer to a new 24-well plate at a volume percentage ratio of 10%, and repeat 5 times. Before the 5th repetition, 0.03g / L color indicator phenol red is added to each culture medium respectively. The growth speed of mycoplasma is judged by observing the time of color change under the same conditions of each group of culture media; and the mycoplasma antigen detection card is used for detection. After 15 minutes of reaction, observe whether there are bands on the standard single card and the depth of the bands. The depth of the bands is positively correlated with the virus titer. The interpretation method is: 0 color value is a negative result; 1-2 color values ​​are weak positive results; 3-6 color values ​​are medium-intensity positive results; 7-10 color values ​​are strong positive results.

[0030] According to the above standards, the marine crustacean mycoplasma culture medium and separation and purification method prepared in the above Examples 1-4 and Comparative Examples 1-4 were tested, and the obtained data are shown in Table 1: Table 1 Performance data of Examples 1-4 and Comparative Examples 1-4 The above data fully show that compared with Comparative Examples 1-4, Examples 1-4 can fully show the effect of the composite growth promoter on the mycoplasma growth performance of marine crustacean mycoplasma culture medium.

[0031] Since the present invention adopts a composite growth promoter to prepare a marine crustacean mycoplasma culture medium, the performance of the marine crustacean mycoplasma culture medium is effectively improved by the composite growth promoter, as follows: It can be seen from Examples 1-3 that as the proportion of spirulina and sea urchin in the composite growth promoter continues to increase, the performance of the marine crustacean mycoplasma culture medium in promoting mycoplasma growth is significantly improved. This is because spirulina, as a high-protein microalgae, is rich in amino acids, vitamins and minerals, and can be used as a nutrient source for mycoplasma growth. Sea urchins also contain a variety of proteins, lipids and trace elements, which can also provide necessary nutrients for mycoplasma. At the same time, sea urchins also contain a variety of biologically active compounds, which can inhibit Gram-positive and Gram-negative bacteria, and thus can act as antibiotics to inhibit the growth of Gram-positive and Gram-negative bacteria, prevent them from over-reproduction in the culture medium, and provide a favorable growth environment for mycoplasma. Phosphatidic acid is the main component of the cell membrane, which can provide a physical barrier for the cell membrane and improve the quality of the cell membrane. As the content of spirulina, sea urchin and phosphatidic acid increases, the composite growth promoter provides more nutrients and protective effects, changes the culture environment, and thus promotes the growth of marine crustacean mycoplasma.

[0032] It can be seen from Examples 3 and 4 that, with the continuous change of the content of the overall components in the culture medium, the marine crustacean mycoplasma culture medium has no obvious change in the performance of promoting the growth of mycoplasma. This shows that the change of the overall components in the culture medium within a certain range is not enough to cause a significant change in the performance of the culture medium in promoting the growth of mycoplasma. Therefore, under the premise of ensuring sufficient growth needs of mycoplasma, the change of the overall component content has no obvious change in the performance of promoting the growth of mycoplasma.

[0033] According to the above test experiments, the marine crustacean mycoplasma culture medium prepared according to Example 3 has the best performance, so Example 3 is taken as the best example; By comparing Example 3 with Comparative Examples 1-4, it can be seen that: In Comparative Example 1, the amount of the composite growth promoter added to the culture medium is 0.05 parts by weight. The marine crustacean mycoplasma culture medium has a worse performance in promoting the growth of mycoplasma. This is because the composite growth promoter contains a variety of nutrients required for the growth of mycoplasma, which can selectively inhibit the growth of other microorganisms. When the amount of the composite growth promoter added is small, these nutrients are not enough to meet the growth needs of mycoplasma, resulting in slow growth or stagnation, and are not enough to provide effective inhibition of foreign bacteria and protection of mycoplasma cell membranes, thereby affecting the growth of mycoplasma. Therefore, the performance of promoting the growth of mycoplasma is poor.

[0034] In Comparative Example 2, the composite growth promoter is obtained by enzymatic hydrolysis of Spirulina and then compounded with phosphatidic acid, but no sea urchin is added. The marine crustacean mycoplasma culture medium has poor performance in promoting the growth of mycoplasma. This is because sea urchin contains certain bioactive molecules that can effectively inhibit the growth of other bacteria. The lack of these ingredients will cause the bacteria to compete for nutrients, thereby affecting the growth performance of mycoplasma, and thus has poor performance in promoting the growth of mycoplasma.

[0035] In comparative example 3, the composite growth promoter is obtained by enzymatic hydrolysis of sea urchin and then compounded with phosphatidic acid, but Spirulina is not added. The marine crustacean mycoplasma culture medium has poor performance in promoting the growth of mycoplasma. This is because the enzymatic hydrolysis product of Spirulina can provide rich nutrients such as amino acids, vitamins and minerals, and the lack of Spirulina will result in these ingredients being insufficient to meet the growth needs of mycoplasma, which in turn leads to slow or unstable growth of mycoplasma, and therefore has poor performance in promoting the growth of mycoplasma.

[0036] In Comparative Example 4, the composite growth promoter is made of spirulina and sea urchin enzymatic hydrolysis, but no phosphatidic acid is added. The marine crustacean mycoplasma culture medium has poor performance in promoting the growth of mycoplasma. This is because phosphatidic acid is the main component of the cell membrane, which can help maintain the integrity and fluidity of the cell membrane, thereby improving the quality of the cell membrane. Without phosphatidic acid, it is impossible to compensate for the adverse effects of the antibacterial components in Spirulina, so it has poor performance in promoting the growth of mycoplasma.

[0037] In summary, a marine crustacean mycoplasma culture medium was prepared using a composite growth promoter. The rich nutrients of Spirulina were used to provide the nutrient source required for the growth of mycoplasma. The bioactive compounds in sea urchins provided an excellent growth environment for mycoplasma. Phosphatidic acid was used to improve the quality of cell membranes, offset the adverse effects of Spirulina, and thus promote the growth of mycoplasma.

[0038] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only preferred examples of the present invention, and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A marine crustacean mycoplasma culture medium, characterized in that: The invention comprises the following components: 1-3 parts by weight of a composite growth promoter, 6-8 parts by weight of peptone, 3-5 parts by weight of a yeast extract, 1-5 parts by weight of glucose, 10-20 parts by weight of bovine serum, 12-15 parts by weight of a phosphate buffer, 0.2-0.4 parts by weight of beta-mercaptoethanol, 0.12-0.42 parts by weight of a rhodiola rosea extract and 1.2-2.5 parts by weight of a DTPA chelating agent; Among them, the compound growth promoter is prepared by enzymatically hydrolyzing spirulina and sea urchin in a mass ratio of 1:0.05-0.07 and then compounding with phosphatidic acid.

2. The marine crustacean mycoplasma culture medium according to claim 1, characterized in that: The preparation method of the composite growth promoter is as follows: Use a homogenizer to crush the pretreated sea urchin and spirulina into slurry respectively, then mix the slurries of the two raw materials in proportion, crush and sterilize, and add them to a solution containing a composite enzyme inhibitor, prepare a composite enzymatic solution with phosphate buffer, transfer the pretreated mixture to a reaction vessel, add the composite enzymatic solution, and adjust the pH value. The reaction temperature is set to 37-40°C, the reaction time is 5-7h, and a magnetic stirrer is used to keep stirring to ensure that the reaction is uniform. Place the reaction mixture in a water bath and heat it to 80-100°C for 10-15min, cool it naturally to room temperature, and then centrifuge the reaction mixture at 4000-6000rpm / min for 10-15min, collect the supernatant, and then filter the supernatant with filter paper to remove residual solid particles to obtain a mixed enzymatic solution; weigh phosphatidic acid powder and dissolve it in dimethyl sulfoxide to prepare a solution with a concentration of 5-10μg / mL, and add the evenly mixed phosphatidic acid solution to the mixed enzymatic solution while stirring to obtain a composite growth promoter.

3. The marine crustacean mycoplasma culture medium according to claim 2, characterized in that: The composite enzyme preparation contains cellulase, lipase, pectinase and protease, and the mass ratio of the components is 5:2-4:0.5-0.9:1-3.

4. The marine crustacean mycoplasma culture medium according to claim 2, characterized in that: The concentration of the composite enzymatic hydrolysate is 0.5-1% (w / v), and the ratio of the composite enzymatic hydrolysate to the two raw materials is 1:8-10 (v / w).

5. The marine crustacean mycoplasma culture medium according to claim 2, characterized in that: The mass ratio of the mixed enzymatic hydrolysate to the phosphatidic acid solution is 1:0.8-1.

2.

6. The marine crustacean mycoplasma culture medium according to claim 2, characterized in that: The pH value is adjusted to 6.5-7.

5.

7. A method for separating and purifying a marine crustacean mycoplasma culture medium, used for preparing the marine crustacean mycoplasma culture medium according to any one of claims 1 to 6, characterized in that: The steps include: Adding a composite growth promoter, peptone, yeast extract, glucose, bovine serum, β-mercaptoethanol, rhodiola rosea extract and DTPA chelating agent to deionized water in sequence, stirring to dissolve solid components, adjusting the pH value of the mixed solution to a range suitable for mycoplasma growth with a phosphate buffer, filtering the mixed solution with a 0.22-0.45 μm sterile filter to remove impurities, filling the filtered culture medium into sterile bottles, sterilizing at 121-125° C. for 10-15 minutes in a high-pressure steam sterilizer, and refrigerating and storing at 2-4° C. to obtain a marine crustacean mycoplasma culture medium; Select marine crustaceans infected with mycoplasma as sample sources, collect samples from animal gill tissues using aseptic techniques, homogenize the collected samples using a grinding rod under sterile conditions, use a centrifuge to remove larger particles and cell debris from the homogenized samples, inoculate the treated samples onto the prepared culture medium, and culture them anaerobically at 37-40°C for 2-4 days; When colony formation is observed, single colonies are selected with an inoculation loop and streaked on new culture medium in parallel. Culture is continued for 1-3 days, and the above plate streaking steps are repeated 4-5 times until colonies of a single morphology are observed. The purified strain can then be frozen for storage.

8. The method for separating and purifying the marine crustacean mycoplasma culture medium according to claim 7, characterized in that: The phosphate buffer adjusts the pH value of the mixed solution to 7.0-7.

5.

9. The method for separating and purifying the marine crustacean mycoplasma culture medium according to claim 7, characterized in that: The centrifugation temperature is 2-4°C, the centrifugation speed is 2000-3000 rpm / min, and the centrifugation time is 5-10 min.

10. The method for separating and purifying the marine crustacean mycoplasma culture medium according to claim 7, characterized in that: The gas mixture ratio under the anaerobic condition is 80-85% N2 and 15-20% CO2.

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