CN106676039B - Rhodooomycete thiophilic microbial inoculum, extracellular protein, preparation method of microbial inoculum and extracellular protein and application of microbial inoculum - Google Patents
Rhodooomycete thiophilic microbial inoculum, extracellular protein, preparation method of microbial inoculum and extracellular protein and application of microbial inoculum Download PDFInfo
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Abstract
The invention discloses a small sulfur-tolerant rhodobacter strain, a microbial inoculum, an extracellular protein, a preparation method of the microbial inoculum and the extracellular protein, and an application of the microbial inoculum and the extracellular protein, wherein the small sulfur-tolerant rhodobacter strain is small sulfur-tolerant rhodobacter IV-1(Rhodovulum Sulfidophilum IV-1), is preserved in China center for type culture collection with the preservation number of CCTCC M2012369, the preservation unit address is located at Wuhan university in China, and the preservation date is 2012, 9 and 26 days. According to the invention, extracellular protein with antagonistic action on pepper anthracnose bacteria can be secreted from the rhodobacter sulfidophilus IV-1 microbial inoculum, so that the inhibition effect on pepper anthracnose is achieved; the invention extracts extracellular protein from extracellular products of the rhodobacter sulfidophilum IV-1 microbial inoculum for the first time, the extraction process is simple, the flow is short, the cost is low, and the extraction process has no pollution to the environment; the protein component extracted from the rhodobacter sulfidophilum IV-1 microbial inoculum has the molecular weight of about 50kD, high thermal stability, storage resistance and good inhibition effect on pepper anthracnose.
Description
Technical Field
The invention relates to the technical field of biological pesticides, in particular to a rhodobacter sulfidophilus microbial inoculum, an extracellular protein, a preparation method of the microbial inoculum and the extracellular protein and application of the microbial inoculum and the extracellular protein.
Background
The Pepper Anthracnose (Pepper Anthracnose) is an important disease on Pepper and pimento. In China, the pepper anthracnose is mainly caused by red anthracnose bacterium ((R))C.gloeosporioides=Gloeosporiumpiperatum) Colletotrichum nigrescens (C.coccodes=C.nigrum) Colletotrichum gloeosporioides (A), (B), (C)C.capsici) And anthrax (B) B.oxysporum: (C.acutatum （Simmonds）)4 pathogenic bacteria. If the climate is high temperature and high humidity, the pepper anthracnose is rapidly spread and has long harm time, the pepper seedling death and rotten fruit can be caused, the pepper yield is reduced by 30-50 percent, and even up to 80 percent in severe cases, the pepper anthracnose has great influence on the pepper quality, the pepper yield and the processing and production of mature fruits, and the pepper anthracnose is one of the main obstacles for pepper production.
Controlling and reducing the harm of anthracnose to pepper production, and generally adopting measures such as chemical prevention and control in production. But with the long-term use of chemical bactericides in large quantities, pathogens are caused to generate drug resistance and drug resistance, and beneficial microorganisms are killed; meanwhile, chemical pesticides diffuse into the environment along with atmospheric circulation and water circulation, so that environmental pollution is caused; furthermore, the pepper has special requirements on the appearance and the internal quality of the pepper as an edible economic crop, pesticide residues and the influence on food sanitation, and the large-scale utilization of the chemical bactericide is limited. With the continuous improvement of the demand of the quantity and the quality of crops in the world, the increasing importance of various countries on the protection of the living environment of human beings, the continuous deepening of continuous agriculture and continuous plant protection concept, and the increasing production demand of environmental problems and pollution-free food, the biological control of pepper anthracnose is imperative.
With the intensive research on biocontrol bacteria and the increasing renewal of biocontrol products, efforts are being made to excavate antibacterial active substances. The routes for biocontrol bacteria to produce antibacterial active substances are generally classified into two routes, ribosome synthesis and non-ribosome synthesis. Antibacterial substances produced by the ribosome synthesis pathway include bacteriocins, enzymes and other active proteins; the antibacterial substances produced by the non-ribosome synthetic pathway include lipopeptides, antibacterial peptides and antibacterial substances produced by other substances such as macrolides, polyenes, phenols and the like, and include non-ribosome synthetic lipopeptide antibiotics, ribosome synthetic bacteriocins and the like. Among a plurality of active substances, the antibacterial protein is an important natural substance for resisting the invasion of pathogenic bacteria by organisms and maintaining the continuation and operation of life activities, is an important disease-resistant resource and can be widely applied to the prevention and treatment of pepper anthracnose.
Disclosure of Invention
The invention aims to provide a rhodobacter sulfidophilum microbial agent, an extracellular protein, a preparation method of the microbial agent and the extracellular protein and application of the microbial agent and the extracellular protein, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a rhodobacter sulfidophilus microbial inoculum is prepared by using rhodobacter sulfidophilus strain IV-1, storing in China center for type culture collection with the preservation number of CCTCC NO: M2012369, and the preservation unit address is located at the university of Wuhan, China with the preservation date of 2012 9 months and 20 days, and performing activation, seed culture, production culture and centrifugation on the rhodobacter sulfidophilus strain.
The preparation method of the rhodobacter sulfidophilum microbial inoculum comprises the following steps:
the first step is as follows: and (3) activation: culturing the preserved strain of the rhodobacter sulfidophilus strain by a double-layer plate culture method until a red single colony appears;
the second step is that: seed culture: inoculating the red single colony cultured in the first step into a serum bottle, and culturing the red single colony in a photosynthetic bacteria liquid culture medium to a logarithmic phase under the conditions of the temperature of 30-32 ℃, the illumination condition of 2500 Lx-4000 Lx and the pH value of 7.0 to obtain a bacterial liquid;
the third step: production and culture: inoculating the bacterial liquid obtained after the seed culture in the second step into a production bottle, and carrying out production culture by using a photosynthetic bacteria production culture medium, wherein the inoculation amount of the bacterial liquid is 5 percent of the total volume of the photosynthetic bacteria strain production culture medium, and the bacterial liquid is cultured to a logarithmic phase under the conditions that the temperature is 30-32 ℃, the illumination condition is 2500 Lx-4000 Lx and the pH = 7.0;
the fourth step: centrifuging: centrifuging the bacterial liquid obtained by the production culture in the third step at 8000rpm, 4 ℃ for 15min, collecting supernatant, and performing suction filtration by using a 0.22-micron filter membrane to obtain filtrate, namely the rhodobacter sulfidophilum microbial inoculum is prepared.
An extracellular protein, wherein the extracellular protein is extracted from the rhodobacter sulfidophilus microbial inoculum or the rhodobacter sulfidophilus microbial inoculum prepared by the preparation method.
The preparation method of the extracellular protein specifically comprises the following steps:
the first step is as follows: ammonium sulfate precipitation-salting out: adding ammonium sulfate into the rhodobacter sulfidophilus to 85% saturation, and separating out protein in the rhodobacter sulfidophilus to obtain a mixed solution;
the second step is that: centrifuging: centrifuging the mixed solution obtained in the first step at the rotating speed of 11000rpm for 30min to obtain a precipitate, and re-suspending the precipitate in a PBS buffer solution to obtain a protein re-suspension solution;
the third step: ATKA protein purification system HiTrap desaling 5mL Desalting column desalination: freezing and concentrating the protein heavy suspension prepared in the second step at a low temperature, and Desalting by using a HiTrap desaling 5mL Desalting column to obtain a protein crude product;
the fourth step: the ATKA protein purification system Superdex 200 Increate 10/300GL chromatographic column separation: and (4) carrying out chromatographic separation on the crude protein product prepared in the third step by using a Superdex 200 Increate 10/300GL chromatographic column to obtain extracellular protein.
As a still further scheme of the invention: the concentration of PBS buffer in the second step was 0.01M and the pH was 7.0.
As a still further scheme of the invention: and in the third step, carrying out HiTrap desaling 5mL column desalination on the protein resuspension at the loading ratio of 15% to obtain a crude protein, and diluting the crude protein to 10 mg/mL.
As a still further scheme of the invention: and in the fourth step, the protein crude product is subjected to Superdex 200 Increate 10/300GL chromatographic column separation according to the loading proportion of 2%.
As a still further scheme of the invention: and in the fourth step, each protein component obtained after chromatographic separation is subjected to activity determination, the active components are combined, and the extracellular protein is obtained by low-temperature freezing concentration.
The application of the extracellular protein extracted according to the extracellular protein or the extraction method is to prevent and treat pepper anthracnose.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention can secrete extracellular protein with antagonistic action on pepper anthracnose pathogen from the rhodobacter sulfidophilus IV-1 microbial inoculum, thereby playing the role of inhibiting pepper anthracnose.
2. The invention extracts extracellular protein from extracellular products of the rhodobacter sulfidophilum IV-1 microbial inoculum for the first time, has simple extraction process, short flow, low cost and no environmental pollution in the extraction process.
3. The molecular weight of a protein component extracted from the rhodobacter sulfidophilum IV-1 microbial inoculum is about 50kD, and the protein component has high thermal stability, storage resistance and obvious inhibiting effect on pepper anthracnose.
Drawings
FIG. 1 is a photograph showing a colony of R.thiophilus IV-1 strain in example 1 of the present invention.
FIG. 2 is a drawing showing the plate measurement test of anthrax hyphae of pepper in example 4 of the present invention.
FIG. 3 is a diagram of a test for measuring the amount of produced spores of anthracnose of hot pepper in example 4 of the present invention.
FIG. 4 is a fruit set test chart of the pepper anthracnose pathogen of example 4 of the present invention.
FIG. 5 is a greenhouse disease test chart of the pepper anthracnose in example 4 of the present invention.
Detailed Description
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Referring to fig. 1-5, the Rhodovulum thiophanate strain is Rhodovulum thiophanate (Rhodovulum Sulfidophilum) IV-1, which is preserved in the China center for type culture Collection with the preservation number of CCTCC NO: M2012369, the preservation unit address is located in Wuhan university, China, and the preservation date is 9/20 days 2012.
The rhodobacter sulfidophilus microbial inoculum is prepared by the rhodobacter sulfidophilus strain through activation, seed culture, production culture and centrifugation.
The preparation method of the rhodobacter sulfidophilum microbial inoculum comprises the following steps:
the first step is as follows: and (3) activation: culturing the preserved strain of the rhodobacter sulfidophilus strain by a double-layer plate culture method until a red single colony appears;
the second step is that: seed culture: inoculating the red single colony cultured in the first step into a serum bottle, and culturing the red single colony in a photosynthetic bacteria liquid culture medium to a logarithmic phase under the conditions of the temperature of 30-32 ℃, the illumination condition of 2500 Lx-4000 Lx and the pH value of 7.0 to obtain a bacterial liquid;
the third step: production and culture: inoculating the bacterial liquid obtained after the seed culture in the second step into a production bottle, and carrying out production culture by using a photosynthetic bacteria production culture medium, wherein the inoculation amount of the bacterial liquid is 5 percent of the total volume of the photosynthetic bacteria strain production culture medium, and the bacterial liquid is cultured to a logarithmic phase under the conditions that the temperature is 30-32 ℃, the illumination condition is 2500 Lx-4000 Lx and the pH = 7.0;
the fourth step: centrifuging: centrifuging the bacterial liquid obtained by the production culture in the third step at 8000rpm, 4 ℃ for 15min, collecting supernatant, and performing suction filtration by using a 0.22-micron filter membrane to obtain filtrate, namely the rhodobacter sulfidophilum microbial inoculum is prepared.
An extracellular protein, wherein the extracellular protein is extracted from the rhodobacter sulfidophilus microbial inoculum or the rhodobacter sulfidophilus microbial inoculum prepared by the preparation method.
The preparation method of the extracellular protein specifically comprises the following steps:
the first step is as follows: ammonium sulfate precipitation-salting out: adding ammonium sulfate into the rhodobacter sulfidophilus to 85% saturation, and separating out protein in the rhodobacter sulfidophilus to obtain a mixed solution;
the second step is that: centrifuging: centrifuging the mixed solution obtained in the first step at the rotating speed of 11000rpm for 30min to obtain a precipitate, and re-suspending the precipitate in a PBS buffer solution to obtain a protein re-suspension solution;
the third step: ATKA protein purification system HiTrap desaling 5mL Desalting column desalination: freezing and concentrating the protein heavy suspension prepared in the second step at a low temperature, and Desalting by using a HiTrap desaling 5mL Desalting column to obtain a protein crude product;
the fourth step: the ATKA protein purification system Superdex 200 Increate 10/300GL chromatographic column separation: and (4) carrying out chromatographic separation on the crude protein product prepared in the third step by using a Superdex 200 Increate 10/300GL chromatographic column to obtain extracellular protein.
As a still further scheme of the invention: the concentration of PBS buffer in the second step was 0.01M and the pH was 7.0.
As a still further scheme of the invention: and in the third step, carrying out HiTrap desaling 5mL column desalination on the protein resuspension at the loading ratio of 15% to obtain a crude protein, and diluting the crude protein to 10 mg/mL.
As a still further scheme of the invention: and in the fourth step, the protein crude product is subjected to Superdex 200 Increate 10/300GL chromatographic column separation according to the loading proportion of 2%.
As a still further scheme of the invention: and in the fourth step, each protein component obtained after chromatographic separation is subjected to activity determination, the active components are combined, and the extracellular protein is obtained by low-temperature freezing concentration.
The application of the extracellular protein extracted according to the extracellular protein or the extraction method is to prevent and treat pepper anthracnose.
According to the invention, extracellular protein with antagonistic action on pepper anthracnose bacteria can be secreted from the rhodobacter sulfidophilus IV-1 microbial inoculum, so that the inhibition effect on pepper anthracnose is achieved; the invention extracts extracellular protein from extracellular products of the rhodobacter sulfidophilum IV-1 microbial inoculum for the first time, the extraction process is simple, the flow is short, the cost is low, and the extraction process has no pollution to the environment; the protein component extracted from the rhodobacter sulfidophilum IV-1 microbial inoculum has the molecular weight of about 50kD, high thermal stability, storage resistance and good inhibition effect on pepper anthracnose.
Example 1
A Rhodooomycete thiophila strain is Rhodooomycete thiophila strain (Rhodooomycete thiophila) (R)Rhodovulum sulfidophilum) IV-1, preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2012369, the preservation unit address is located at the university of Wuhan, China, the preservation date is 9 months and 20 days in 2012, the strain is named as Haematococcus thiophilic (C)Rhodovulum sulfidophilum）IV-1。
Referring to fig. 1: the rhodobacter sulfidophilus strain IV-1 is a gram-negative bacterium, is identified as rhodobacter sulfidophilus through physiological and biochemical identification and molecular biology, and has the following main biological characteristics: culturing for 4-8 days on a double-layer solid plate culture medium to form red round colonies, wherein the edges of the colonies are neat and smooth, and the diameter of the colonies is 0.2-0.60 mm; culturing in liquid culture medium for 7d to obtain deep red; good growth under anaerobic and micro-aerobic conditions; the physiochemical characteristics are V-P reaction and methyl red reaction negative, H2The S reaction, the gelatin liquefaction, the urease test and the indole test are all positive, the optimal growth temperature is 30-32 ℃, and the pH =7.
Example 2
A rhodobacter sulfidophilus microbial inoculum is prepared by the rhodobacter sulfidophilus strain of example 1 through activation, seed culture, production culture and centrifugation, and the specific preparation method comprises the following steps:
the first step is as follows: and (3) activation: culturing the preserved strain of the rhodobacter sulfidophilus strain by a double-layer plate culture method until a red single colony appears;
the second step is that: seed culture: inoculating the red single colony cultured in the first step into a serum bottle, and culturing the red single colony in a photosynthetic bacteria liquid culture medium to a logarithmic phase under the conditions of the temperature of 30-32 ℃, the illumination condition of 2500 Lx-4000 Lx and the pH value of 7.0 to obtain a bacterial liquid;
the third step: production and culture: inoculating the bacterial liquid obtained after the seed culture in the second step into a production bottle, and carrying out production culture by using a photosynthetic bacteria production culture medium, wherein the inoculation amount of the bacterial liquid is 5 percent of the total volume of the photosynthetic bacteria strain production culture medium, and the bacterial liquid is cultured to a logarithmic phase under the conditions that the temperature is 30-32 ℃, the illumination condition is 2500 Lx-4000 Lx and the pH = 7.0;
the fourth step: centrifuging: centrifuging the bacterial liquid obtained by the production culture in the third step at 8000rpm, 4 ℃ for 15min, collecting supernatant, and performing suction filtration by using a 0.22-micron filter membrane to obtain filtrate, namely the rhodobacter sulfidophilum microbial inoculum is prepared.
Example 3
An extracellular protein extracted from the Rhodooomycete thiophila microbial inoculum of example 2, the specific preparation method comprising the steps of:
the first step is as follows: ammonium sulfate precipitation-salting out: adding ammonium sulfate into the rhodobacter sulfidophilus to 85% saturation, and separating out protein in the rhodobacter sulfidophilus to obtain a mixed solution;
the second step is that: centrifuging: centrifuging the mixed solution obtained in the first step at the rotating speed of 11000rpm for 30min to obtain a precipitate, and re-suspending the precipitate in a PBS buffer solution to obtain a protein re-suspension solution; the concentration of PBS buffer solution is 0.01M, and the pH value is 7.0;
the third step: ATKA protein purification system HiTrap desaling 5mL Desalting column desalination: freezing and concentrating the protein heavy suspension prepared in the second step at a low temperature, and Desalting by using a HiTrap desaling 5mL Desalting column to obtain a protein crude product; carrying out HiTrap desaling 5mL column desalination on the protein resuspension solution at the loading proportion of 15% to obtain a protein crude product, and diluting the protein crude product to 10 mg/mL;
the fourth step: the ATKA protein purification system Superdex 200 Increate 10/300GL chromatographic column separation: carrying out chromatographic separation on the crude protein product prepared in the third step by using a Superdex 200 Increate 10/300GL chromatographic column to obtain extracellular protein; carrying out Superdex 200 Increate 10/300GL chromatographic column separation on the crude protein product according to the loading proportion of 2%; and carrying out activity determination on each protein component obtained after chromatographic separation, combining the active components, and carrying out low-temperature freezing concentration to obtain the extracellular protein.
Example 4:
an application of the extracellular protein of the embodiment 3 in prevention and treatment of pepper anthracnose disease is as follows:
hot pepper anthrax hypha plate determination test: the method takes colletotrichum gloeosporioides as a target and adopts a plate method to carry out an antibacterial test. Adding the extracellular protein into a 10mLPDA culture medium to prepare a protein plate with final concentrations of 200 mug/mL, 100 mug/mL, 50 mug/mL and 25 mug/mL, inactivating 200 mug/mL at 100 ℃ for 15min as a control, inoculating and culturing pepper anthracnose pathogen for 7 days, culturing at 28 ℃, repeating for 5 times, and calculating the inhibition rate of protein liquid on the anthracnose pathogen after culturing at 28 ℃ for 3 days.
Inhibition rate = (colony diameter)Control coloniesDiameter of coloniesProtein processing) Colony diameterControl colonies×100%。
Referring to FIG. 2, numbers 1, 2, 3, 4, 5 and 6 in FIG. 2 are inactivated protein, bovine serum albumin 200. mu.g/mL, blank control and extracellular protein fraction treatments of 50. mu.g/mL, 100. mu.g/mL and 200. mu.g/mL, respectively, and it can be known from FIG. 2 that: the extracellular protein liquid still has certain activity at the concentration of 50 mug/mL; the prevention and treatment effect of the protein after high-temperature treatment on pepper anthracnose is lost. When the concentration reaches more than 200 mu g/mL, the control effect on pepper anthracnose reaches 72.53%. PBS buffer 0.01M, pH =7.0 was blank control.
And (3) a pepper anthracnose sporulation amount determination test: taking colletotrichum gloeosporioides as a target, uniformly mixing extracellular protein into 20mL of potato liquid culture medium, adjusting the final concentration of the protein to be 200 mu g/mL, inoculating a pepper colletotrichum gloeosporioides bacterial cake (6 mm) cultured for 5 days, shaking the bacterial cake at 28 ℃ and 180rpm, culturing for 8 days, and counting the sporulation amount.
Referring to FIG. 3, numbers 1, 2 and 3 in FIG. 3 are 100mg/mL azoxystrobin, extracellular protein 200. mu.g/mL and blank control treatment, respectively, and it can be known from FIG. 3 that: the spore yield of extracellular protein treatment is 4.1 multiplied by 104The spore yield per ml is equivalent to that of azoxystrobin after treatment, while the spore yield of a blank control reaches 3.6 multiplied by 106One/ml. PBS buffer 0.01M, pH =7.0 was blank control.
Fruit determination test for pepper anthracnose pathogen: collecting mature fruit, sterilizing surface, pricking 2 inoculation points on fruit surface with needle of HAMILTON microsyringe, and mixing the decoction of Capsici fructus leaves: extracellular proteins: spore = 1: 1: spore suspension (concentration 1X 10) formulated at 1 ratio6one/mL), 10 μ L of the extract was added to each of the samples, and the samples were cultured at 25-28 ℃ under 12h illumination/day and RH ≥ 95%, and the disease incidence was investigated on day 7. The disease with the lesion diameter being more than or equal to 4mm is regarded as the disease incidence, and the disease incidence is counted, namely the percentage of the number of the lesion spots in the total inoculation points. There were 4 replicates of at least 5 fruits per replicate.
Referring to FIG. 4, numbers 1, 2 and 3 in FIG. 4 represent azoxystrobin at a concentration of 100mg/mL, extracellular protein at a concentration of 200. mu.g/mL, respectively, and blank control treatment, and it can be seen from FIG. 4 that: the morbidity of pepper anthracnose pathogen treated by contrast is 90%, and the pepper treated by the extracellular protein liquid has no morbidity and has the effect equivalent to that of azoxystrobin. PBS buffer 0.01M, pH =7.0 was blank control.
Greenhouse bioassay test for pepper anthracnose: preparing spore suspension (concentration is 1 × 10) from 10% pepper leaf decoction6one/mL) is sprayed and inoculated to seedlings with the seedling age of 4-5 leaf stages, preferably dripping water on leaf surfaces. Moisture preservation is carried out for 24h immediately after inoculation, and water spraying is carried out for 24h after 7 d. During the identification period, the seedlings are cultured under the conditions of 26-28 ℃ and 10h of illumination/day, and investigation is carried out after 14 days. Grading the disease condition standard: grade 0, no symptomatic plaques; grade 1, the diameter of the disease spot is 1mm, and 1-2 leaves are attacked; grade 2, the diameter of the disease spot is larger than 1mm, and 2 true leaves are attacked; grade 3, many large spots, sporulation orMore than 2 true leaves are diseased, and plants are malformed and dwarfed; grade 4, there are many large spots, a large number of spores are produced, and diseased strains are dying or have died.
Referring to FIG. 5, numbers 1, 2 and 3 in FIG. 5 represent azoxystrobin at a concentration of 100mg/mL, extracellular protein at a concentration of 200. mu.g/mL, respectively, and blank control treatment, as can be seen in FIG. 4: the pepper treated by the blank control is completely withered, and the control effect of pepper anthracnose after being treated by the extracellular protein solution reaches 75.63%. PBS buffer 0.01M, pH =7.0 was blank control.
Although the preferred embodiments of the present patent have been described in detail, the present patent is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present patent within the knowledge of those skilled in the art.
Claims (5)
1. The application of the extracellular protein is characterized in that the extracellular protein is applied to control of pepper anthracnose;
the extracellular protein is extracted from a rhodobacter sulfidophilus microbial agent;
the rhodobacter sulfidophilus microbial inoculum is prepared by adopting a rhodobacter sulfidophilus strain through activation, seed culture, production culture and centrifugation; the Rhodooomycete thiophila strain is Rhodooomycete thiophila (Rhodovulum sulfidophilum) IV-1, preserving in China center for type culture Collection with a preservation number of CCTCC NO of M2012369, wherein the preservation unit address is located in Wuhan university, China, and the preservation date is 9 months and 20 days in 2012;
the preparation method of the rhodobacter sulfidophilum microbial inoculum comprises the following steps: the first step is as follows: and (3) activation: culturing the preserved strain of the rhodobacter sulfidophilus strain by a double-layer plate culture method until a red single colony appears; the second step is that: seed culture: inoculating the red single colony cultured in the first step into a serum bottle, and culturing the red single colony in a photosynthetic bacteria liquid culture medium to a logarithmic phase under the conditions of the temperature of 30-32 ℃, the illumination condition of 2500 Lx-4000 Lx and the pH value of 7.0 to obtain a bacterial liquid; the third step: production and culture: inoculating the bacterial liquid obtained after the seed culture in the second step into a production bottle, and carrying out production culture by using a photosynthetic bacteria production culture medium, wherein the inoculation amount of the bacterial liquid is 5 percent of the total volume of the photosynthetic bacteria strain production culture medium, and the bacterial liquid is cultured to a logarithmic phase under the conditions that the temperature is 30-32 ℃, the illumination condition is 2500 Lx-4000 Lx and the pH = 7.0; the fourth step: centrifuging: centrifuging the bacterial solution obtained by the production culture in the third step at 8000rpm, 4 ℃ for 15min, collecting supernatant, and performing suction filtration by using a 0.22-micron filter membrane to obtain filtrate, namely the rhodobacter sulfidophilus microbial inoculum is prepared;
the preparation method of the extracellular protein specifically comprises the following steps: the first step is as follows: ammonium sulfate precipitation-salting out: adding ammonium sulfate into the rhodobacter sulfidophilus to 85% saturation, and separating out protein in the rhodobacter sulfidophilus to obtain a mixed solution; the second step is that: centrifuging: centrifuging the mixed solution obtained in the first step at the rotating speed of 11000rpm for 30min to obtain a precipitate, and re-suspending the precipitate in a PBS buffer solution to obtain a protein re-suspension solution; the third step: ATKA protein purification system HiTrap desaling 5mL Desalting column: freezing and concentrating the protein heavy suspension prepared in the second step at a low temperature, and desalting by adopting a HiTrapDesalting 5mL desalting column to obtain a protein crude product; the fourth step: the ATKA protein purification system Superdex 200 Increate 10/300GL chromatographic column separation: and (4) carrying out chromatographic separation on the crude protein product prepared in the third step by using a Superdex 200 Increate 10/300GL chromatographic column to obtain extracellular protein.
2. The use of extracellular protein according to claim 1, wherein the concentration of PBS buffer in the second step of the preparation method of extracellular protein is 0.01M and pH is 7.0.
3. The use of extracellular proteins, according to claim 1, wherein in the third step of the preparation method of extracellular proteins, the protein resuspension is desalted by HiTrap desaling 5mL column at a loading ratio of 15% to obtain crude protein, and the crude protein is diluted to 10 mg/mL.
4. The application of the extracellular proteins as claimed in claim 1, wherein in the fourth step of the preparation method of the extracellular proteins, the crude proteins are subjected to Superdex 200 Increate 10/300GL chromatographic column separation according to the loading proportion of 2%.
5. The use of extracellular protein according to claim 1, wherein in the fourth step of the preparation method of extracellular protein, each protein component obtained after chromatography separation is subjected to activity measurement, the active components are combined, and the extracellular protein is obtained by low-temperature freeze concentration.
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