CN114085878A - Preparation method of biological foaming agent based on microbial fermentation - Google Patents
Preparation method of biological foaming agent based on microbial fermentation Download PDFInfo
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Abstract
The invention discloses a method for preparing a biological foaming agent synthesized by adopting a biological fermentation technology, wherein the main component of the foaming agent contains 0.01-0.05 thousandth of active substances, a specific strain is subjected to amplification culture and fermentation synthesis in a proper environment, the product solubility is changed by adjusting the pH value of a fermented solution to precipitate the product, and a target product with higher purity is obtained by high-speed centrifugation, organic solvent extraction and rotary evaporation through a centrifuge. The biological foaming agent has strong foaming capacity, can quickly generate fine and dense foam after being mixed by taking gas as power, has excellent foaming performance, is non-toxic and degradable, has the advantages of green and environment-friendly production process, good foaming effect, convenient use and low cost, and can provide a high-quality foaming material for the technologies of fire-fighting foam extinguishment, mine foam dust fall, foam flotation and the like.
Description
Technical Field
The invention belongs to the technical field of foam, and particularly relates to a biological foaming agent and a preparation method thereof.
Background
Foam generally refers to an aggregate of gas bubbles separated by a thin film of liquid, and can be used in fire fighting foam extinguishing, mine foam dust settling, foam flotation, flooding technologies and the like. Generally, water is difficult to directly form foam, and it is necessary to generate bubbles by adding a foaming agent to water and dispersing a gas phase in a liquid phase by means of air blowing, stirring, etc., wherein an active material is capable of effectively reducing the surface tension of the liquid, and hydrophilic and hydrophobic groups in molecules are affected by hydrophobic effect after being dissolved in water, hydrophilic ends extend into water and hydrophobic ends extend into air, and double electron layers are arranged on the surface of the liquid film to surround the air, thereby forming foam. Therefore, the foaming agent becomes a key factor influencing the application effect of the foam technology, but the traditional foaming agent is generally a chemical foaming agent, has the problems of insufficient foaming efficiency, poor biodegradability, certain toxicity and the like, increases the use amount and the cost in application, has certain corrosivity to personnel, instruments and equipment, and easily pollutes the surrounding environment.
Disclosure of Invention
Aiming at the defects of the traditional chemical foaming agent, the invention provides a preparation scheme of a biological foaming agent, which consists of a bioactive substance and water, and comprises the following specific steps:
a. obtaining an engineering strain: respectively adding an activation culture solution into standard bacillus subtilis strains and mixing to enable the strains to be dispersed in the activation culture solution, extracting a strain-activation solution mixture by using a liquid transfer device, respectively inoculating the strain-activation solution mixture into a liquid activation culture medium and a solid activation culture medium, uniformly coating a bacterial solution in the solid activation culture medium by using a triangular rod, and culturing under a constant temperature condition, wherein if bacterial colonies in the liquid activation culture medium are light pink, round, irregular in edge, capable of folding and opaque, and change from wet to dry flat bacterial colonies, and the solid activation culture medium is obvious in microscopic bacterial colonies, the bacterial strains can be used as primary engineering strains for synthesizing the biological foaming agent.
b. Screening high-yield strains: the method comprises the steps of adding a small amount of engineering strain liquid in a liquid activated culture medium into a blood agar plate, uniformly coating the liquid by using a triangular rod, culturing under a constant temperature condition, obtaining the largest strain by calculating the specific area of a hemolysis ring, inoculating the strain into a solid growth culture medium and a liquid growth culture medium, and culturing under the constant temperature condition, wherein the solid culture medium is used for storage, and the liquid culture medium is used for enrichment culture.
c. Strain preservation and rejuvenation: the strain to be preserved is diluted fully with SCP culture medium, spread to solid growth culture medium, cultured at constant temperature and preserved in low temperature for short period. Fully diluting the strain to be preserved by using an SCP culture medium, inoculating the strain to a liquid growth culture medium, culturing at a constant temperature, fully mixing a bacterium solution and glycerol, placing the prepared solution in a freezing tube, quickly freezing, and refrigerating in a low-temperature refrigerator. Can be used for long-term storage. The deposited strain needs to be revived before use. The strain stored for a short period to a solid growth medium was cultured in a constant temperature incubator, and the colony morphology was observed. The strains, which were cryopreserved in glycerol tubes for a long period of time, were thawed before being retested. And uniformly mixing the bacterial liquid and the liquid growth culture medium according to a certain proportion. Then inoculating bacterial liquid and uniformly coating the bacterial liquid on a solid activation culture medium. The bacterial solution and the plate were incubated at constant temperature and observed for morphological changes. If the colony characteristics of the solid activated culture medium are obvious, and the colony is in a flat colony shape which is light pink, round, irregular in edge, capable of being folded and opaque and is changed from wetting to drying, the strain is proved to be successfully rejuvenated.
d. And (3) microbial fermentation: inoculating a small amount of liquid growth culture medium to a certain amount of liquid fermentation culture medium, placing the liquid culture medium in a water bath shaking table, and culturing for more than 24h under the conditions of a certain temperature, pH value and shaking table rotation speed by adopting different shaking modes. After fermentation, the secondary metabolite can be obtained.
e. Active substance extraction: extracting active substances in the fermentation liquor by using an acidification fractional precipitation method, placing a conical flask filled with the fermentation liquor in a low-temperature water bath kettle to ensure that microorganisms do not continue to ferment, pouring the fermentation liquor into a high-speed centrifuge for centrifugation, taking supernate, adding hydrochloric acid to adjust the pH value to acidity, and standing overnight under a low-temperature condition; repeatedly centrifuging and acidifying for several times, and collecting precipitate to obtain crude product of surface active peptide. Adding anhydrous methanol into the crude product of the surface active peptide, fully dissolving the crude product by using ultrasonic waves, centrifuging the solution by using a high-speed centrifuge, taking supernatant, evaporating the supernatant to dryness by using a rotary evaporator under reduced pressure, adding ultrapure water into the evaporated material, adjusting the pH value to be alkaline by using NaOH, and filtering by using a filter membrane; adjusting the pH of the filtrate to acidity with concentrated hydrochloric acid, centrifuging in a high-speed centrifuge, collecting precipitate, and drying. The precipitate is dried to obtain the pure product of the surface active peptide.
f. The biological foaming agent consists of the following components: active substance, water.
A preparation method of a biological foaming agent comprises the following steps:
a. obtaining an engineering strain: preparing a 10ml test tube filled with 5ml of liquid activated medium and two solid activated media, sucking 0.5ml of liquid activated medium by a pipette gun, pumping into a freeze-drying tube, fully mixing, pouring bacterial liquid back into the solid activated medium, uniformly mixing, sucking the bacterial liquid, uniformly coating the bacterial liquid on solid activated medium plates, coating 0.1-0.3ml of bacterial liquid on each plate, and finishing the whole process on a sterile operating platform. Placing the liquid activation culture medium and the solid activation culture medium in a constant temperature incubator at 30-40 ℃ for culturing for 12-36 h; if the bacterial colony in the liquid activated culture medium is in a flat bacterial colony shape which is light pink, round, irregular in edge, capable of being folded and opaque and is changed from wetting to drying, and the bacterial colony detected by a solid activated culture medium through a mirror is obvious, the bacterial colony can be used as a primary engineering bacterial strain for synthesizing the biological foaming agent.
b. Screening high-yield strains: the surface active peptide of the secondary metabolite of the bacillus subtilis has hemolytic property, and the higher the concentration of the substance, the stronger the hemolytic ability. According to the characteristics, bacteria with stronger surface active peptide synthesis capacity can be preliminarily screened. Taking a certain amount of culture solution in the liquid activation culture medium, and diluting with SCP culture medium by a certain multiple in a grading way, wherein the dilution multiple is determined by an OD600 value, and the OD value is the absorbance measured at 600nm by adopting a spectrophotometer. Uniformly coating the diluted strain on a blood agar plate, culturing at 30-40 ℃ for 6-18h, screening out strains with large specific area of a hemolysis cycle, respectively inoculating the strains to a solid growth culture medium and a liquid growth culture medium, culturing at 30-40 ℃ for 6-18h, storing and marking the solid culture medium in a refrigerator at 2-5 ℃, and enriching and culturing the screened strains in the liquid culture medium.
c. Strain preservation and rejuvenation: fully diluting the strain to be preserved with SCP culture medium, coating onto solid growth culture medium slant, culturing at 30-40 deg.C for 6-18h, and storing in 2-5 deg.C refrigerator for a short period of no more than 20 days; fully diluting a strain to be preserved by using an SCP culture medium, inoculating the strain to a liquid growth culture medium, culturing for 6-18h at 30-40 ℃, fully mixing a bacterium solution with 40% glycerol to prepare 1ml of solution in a freezing tube, freezing quickly, and refrigerating in a low-temperature refrigerator, wherein the solution can be used for long-term preservation, but the preservation time is not longer than 400 days; the deposited strain needs to be revived before use. The strain preserved for a short period to a solid growth medium was cultured in a constant temperature incubator at 30-40 ℃ for 6-18 hours, and the colony morphology was observed. The strains, which were cryopreserved in glycerol tubes for a long period of time, were thawed before being retested. Then 0.5ml of bacterial liquid is taken to be uniformly mixed with the liquid growth culture medium according to the proportion of 1:1 (v/v). Then inoculating 50-200 mul of bacterial liquid and uniformly coating the bacterial liquid on a solid activation culture medium. Placing the bacterial liquid and the flat plate in a constant temperature incubator at 30-40 ℃, culturing for 12-24h, and observing morphological change. If the colony characteristics of the solid activation medium plate are obvious, and the plate colony is light pink, round, irregular in edge, capable of being folded and opaque, and is in a flat colony form which is changed from wetting to drying, the strain is successfully rejuvenated.
d. 50-70mL of liquid fermentation medium is filled in a 250mL triangular flask, a liquid transfer device is used for accurately taking bacterial liquid in the liquid growth medium to be injected into the triangular flask, the inoculation amount is ensured to be 1.5-3%, the inoculated liquid fermentation medium is placed into a rotary water bath shaking table, and the liquid fermentation medium is cultured for more than 48h under the conditions that the temperature is 30-40 ℃, the pH value is 7-8, the rotation speed of the shaking table is 200-. After fermentation, the secondary metabolite can be obtained.
e. Extracting active substances from the fermentation liquor by an acidification, grading and precipitation method, and carrying out thermostatic water bath on the fermentation liquor for 25-35min at low temperature (4 ℃); placing the fermentation liquor in a high-speed centrifuge, and centrifuging for 15-25min under the condition that the relative centrifugal force is 6000-8000 g; adding 6mol/L concentrated hydrochloric acid into the supernatant to adjust the pH to 2.0, and standing overnight at the temperature of 2-5 ℃; centrifuging for 15-25min under the condition that the relative centrifugal force is 10000-; resuspending the precipitate with ultra-clean water, adjusting pH to 2.0, and acid-washing again; and centrifuging, and collecting precipitate to obtain the crude product of the surfactin. Adding 20-40mL of anhydrous methanol into the crude product of the surfactin, performing 20-60KHZ ultrasonic extraction for 25-35min to fully dissolve the crude product in a methanol reagent, centrifuging for 15-25min under the condition that the relative centrifugal force is 16000-; adding ultra-clean water into the steamed dry matter, adjusting the pH to 12.0 by using 1mol/LNaOH, and filtering by using a filter membrane; adjusting the pH of the filtrate to 2.0 by using 6mol/L concentrated hydrochloric acid; centrifuging for 15-30min under the condition that the relative centrifugal force is 8000-11000g, collecting the precipitate and drying. The precipitate is dried to obtain the pure active substance.
f. Adding 0.01-0.05 per mill of active substance into water according to weight, heating and controlling the temperature at about 37 ℃; stirring with magnetic stirrer for 40-50min to mix active substances in water; stopping stirring, and standing the foaming agent solution to room temperature to form a white, transparent, uniform and stable biological foaming agent.
Compared with the traditional chemical foaming agent, the invention has the following advantages:
firstly, the environmental protection is good. The main component of the biological foaming agent is an active substance generated by microbial metabolism, a microbial fermentation method is adopted to obtain a microbial secondary metabolite, and no toxic substances are generated in the process, wherein bacillus subtilis and bacillus licheniformis which are required by the microorganisms are ideal biocontrol microorganisms, widely exist in soil and are harmless to people.
Secondly, the preparation process is simple. The biological foaming agent disclosed by the invention is excellent in foaming performance and good in stability, the preparation process is simple to operate, and the foaming agents with different concentrations can be obtained only by adding active substances into water according to the weight and fully stirring and uniformly mixing the active substances.
Thirdly, the surface activity is high and the foaming ability is strong. The active substance in the biological foaming agent is a microbial secondary metabolite, the critical micelle concentration of the biological foaming agent is about 0.03 per thousand, and the using amount is one thirtieth of that of the traditional chemical foaming agent at most; the active substance has excellent foaming performance, hydrophilic and hydrophobic groups contained in the active substance can be orderly arranged in a gas-liquid interface and a liquid phase, and the cyclic/long-chain active molecules can well reduce the surface tension of water and increase the foam forming rate.
Fourth, the foam is uniform and fine. The air blowing method can be used for generating uniform and dense foam, the size of the bubbles is uniform, the distribution is fine, the pressure difference between the bubbles is small, the gas diffusion rate is reduced, the foam can be maintained for a certain time, the instability of the bubbles caused by diffusion can be reduced, and the stability of the bubbles is improved.
Detailed Description
Example 1: foaming base material for mine dust suppression foam technology
Firstly, preparing a 10ml test tube filled with 5ml of liquid activated culture medium and two solid activated culture mediums, sucking 0.5ml of liquid activated culture medium by a liquid transfer gun, pumping the liquid activated culture medium into a freeze-drying tube, fully mixing, pouring bacterial liquid back to the solid activated culture medium, uniformly mixing, sucking the bacterial liquid, uniformly coating the bacterial liquid on solid activated culture medium plates, coating 0.2ml of bacterial liquid on each plate, and finishing the whole process on a sterile operating platform. The liquid activation culture medium and the solid activation culture medium are placed in a constant temperature incubator at 37 ℃ and cultured for 24 hours; if the bacterial colony in the liquid activated culture medium is in a flat bacterial colony shape which is light pink, round, irregular in edge, capable of being folded and opaque and is changed from wetting to drying, and the bacterial colony detected by a solid activated culture medium through a mirror is obvious, the bacterial colony can be used as a primary engineering bacterial strain for synthesizing the biological foaming agent.
Secondly, taking a certain amount of culture solution in the liquid activation culture medium, and diluting the culture solution by a certain multiple by SCP culture medium in a grading way, wherein the dilution multiple is determined by an OD600 value, and the OD value is the absorbance measured by a spectrophotometer at 600 nm. After dilution, the mixture is evenly spread on a blood agar plate, and is cultured for 12 hours at 37 ℃, strains with large specific area of hemolysis cycle are screened out, and are respectively inoculated with a liquid growth culture medium and cultured for 12 hours at 37 ℃.
Then, 59.89mL of liquid fermentation medium is filled in a 250mL triangular flask, a liquid transfer device is used for accurately sucking bacterial liquid in the liquid growth medium and injecting the bacterial liquid into the triangular flask, the inoculation amount is ensured to be 2.17%, the inoculated liquid fermentation medium is placed into a rotary water bath shaking table and is cultured for more than 48 hours under the conditions that the temperature is 37.56 ℃, the pH value is 7.99, the rotation speed of the shaking table is 220r/min and the oxygen introduction amount is 80 mL/min. After fermentation, the secondary metabolite can be obtained.
Thirdly, extracting active substances in the fermentation liquor by adopting an acidification grading precipitation method, and carrying out constant-temperature water bath on the fermentation liquor for 45min at low temperature (4 ℃); placing the fermentation liquor in a high-speed centrifuge, and centrifuging for 25min under the condition that the relative centrifugal force is 7500 g; adding 6mol/L concentrated hydrochloric acid into the supernatant to adjust the pH to 2.0, and standing overnight at 4 ℃; centrifuging for 30min under the condition that the relative centrifugal force is 12000g, and removing supernatant; resuspending the precipitate with ultra-clean water, adjusting pH to 2.0, and acid-washing again; and centrifuging, and collecting precipitate to obtain the crude product of the surfactin. Adding 40mL of anhydrous methanol into the crude product of the surfactin, performing 40KHZ ultrasonic extraction for 30min, centrifuging for 20min under the condition that the relative centrifugal force is 18000g, and evaporating the supernatant to dryness in a rotary evaporator at 55 ℃ under vacuum of 0.6 under reduced pressure; adding ultra-clean water into the steamed dry matter, adjusting the pH to 12.0 by using 1mol/LNaOH, and filtering by using a filter membrane; adjusting the pH of the filtrate to 2.0 by using 6mol/L concentrated hydrochloric acid; centrifuging for 20min under the condition that the relative centrifugal force is 10000g, collecting the precipitate and drying. The precipitate is dried to obtain the pure active substance.
Finally, 0.003kg of active substance by weight is added to 99.997kg of water, heated and controlled at about 37 ℃; stirring with magnetic stirrer for 40-50min to mix active substances in water; stopping stirring, and standing the foaming agent solution to room temperature to form a white, transparent, uniform and stable biological foaming agent.
Example 2: foaming base material for mine fire foam technology
Firstly, preparing a 10ml test tube filled with 5ml of liquid activated culture medium and two solid activated culture mediums, sucking 0.5ml of liquid activated culture medium by a liquid transfer gun, pumping the liquid activated culture medium into a freeze-drying tube, fully mixing, pouring bacterial liquid back to the solid activated culture medium, uniformly mixing, sucking the bacterial liquid, uniformly coating the bacterial liquid on solid activated culture medium plates, coating 0.3ml of bacterial liquid on each plate, and finishing the whole process on a sterile operating platform. The liquid activation culture medium and the solid activation culture medium are placed in a constant temperature incubator at 37 ℃ and cultured for 36 hours; if the bacterial colony in the liquid activated culture medium is in a flat bacterial colony shape which is light pink, round, irregular in edge, capable of being folded and opaque and is changed from wetting to drying, and the bacterial colony detected by a solid activated culture medium through a mirror is obvious, the bacterial colony can be used as a primary engineering bacterial strain for synthesizing the biological foaming agent.
Secondly, taking a certain amount of culture solution in the liquid activation culture medium, and diluting the culture solution by a certain multiple by SCP culture medium in a grading way, wherein the dilution multiple is determined by an OD600 value, and the OD value is the absorbance measured by a spectrophotometer at 600 nm. After dilution, the mixture is evenly spread on a blood agar plate, and is cultured for 24 hours at 37 ℃, strains with large specific area of a hemolysis cycle are screened out, and are respectively inoculated with a liquid growth culture medium and are cultured for 24 hours at 37 ℃.
Then, 55mL of liquid fermentation medium is filled in a 250mL triangular flask, a liquid transfer device is used for precisely absorbing bacterial liquid in the liquid growth medium and injecting the bacterial liquid into the triangular flask, the inoculation amount is ensured to be 2.5%, the inoculated liquid fermentation medium is placed into a rotary water bath shaking table and is cultured for more than 72 hours under the conditions that the temperature is 37.6 ℃, the pH value is 8, the rotating speed of the shaking table is 220r/min and the oxygen introduction amount is 90 mL/min. After fermentation, the secondary metabolite can be obtained.
Thirdly, extracting active substances in the fermentation liquor by adopting an acidification grading precipitation method, and carrying out constant-temperature water bath on the fermentation liquor for 30min at low temperature (2 ℃); placing the fermentation liquor in a high-speed centrifuge, and centrifuging for 20min under the condition that the relative centrifugal force is 8000 g; adding 6mol/L concentrated hydrochloric acid into the supernatant to adjust the pH to 2.0, and standing overnight at the temperature of 2 ℃; centrifuging for 30min under the condition that the relative centrifugal force is 12000g, and removing supernatant; resuspending the precipitate with ultra-clean water, adjusting pH to 2.0, and acid-washing again; and centrifuging, and collecting precipitate to obtain the crude product of the surfactin. Adding 50mL of anhydrous methanol into the crude product of the surfactin, performing 50KHZ ultrasonic extraction for 25min, centrifuging for 20min under the condition that the relative centrifugal force is 18000g, and evaporating the supernatant to dryness under reduced pressure in a rotary evaporator at 60 ℃ and the vacuum degree of 0.6; adding ultra-clean water into the steamed dry matter, adjusting the pH to 12.0 by using 1mol/LNaOH, and filtering by using a filter membrane; adjusting the pH of the filtrate to 2.0 by using 6mol/L concentrated hydrochloric acid; the mixture was centrifuged at 12000g relative centrifugal force for 15min, and the precipitate was collected and dried. The precipitate is dried to obtain the pure active substance.
Finally, 0.005kg by weight of active substance was added to 99.995kg of water, heated and the temperature was controlled at 30-40 ℃; stirring with magnetic stirrer for 40-50min to mix active substances in water; stopping stirring, and standing the foaming agent solution to room temperature to form a white, transparent, uniform and stable biological foaming agent.
FIG. 1 foaming Capacity of the invention and conventional chemical blowing agents
The foaming capacity FC is the ratio of the foam volume to the gas quantity, as shown in figure 1, when the mass concentration of the biological foaming agent is 0.03%, the foaming capacity is the same as that of a traditional chemical foaming agent with the mass concentration of 1 per mill, which indicates that the same foaming effect is achieved, and the biological foaming agent only needs one thirtieth of the traditional chemical foaming agent at most. And the concentration of the biological foaming agent is continuously increased, the foaming capacity of the biological foaming agent is not obviously improved, which indicates that 0.03 per mill is the optimal use concentration of the biological foaming agent.
FIG. 2 foam size of the present invention and conventional chemical blowing agents
Fig. 2 shows the distribution of foaming after 1min, 5min, 10min and 15min after the foaming is finished, and it can be seen from the figure that at the initial moment, the bubbles of the biological foaming agent are in a "small and dense" state, and the bubbles of the biological foaming agent are more uniform overall with the increase of time, and a more obvious Plateau boundary can be found, which shows that the bubbles are closely arranged, and the stability of the bubbles can be improved.
Claims (7)
1. The biological foaming agent is characterized by comprising a bioactive substance and water, and is prepared by the following steps:
a. obtaining an engineering strain: respectively adding an activation culture solution into standard bacillus subtilis strains and mixing to enable the strains to be dispersed in the activation culture solution, extracting a strain-activation solution mixture by using a liquid transfer device to be respectively inoculated into a liquid activation culture medium and a solid activation culture medium, uniformly coating bacterial liquid in the solid activation culture medium by using a triangular rod, culturing under a constant temperature condition, and when light pink grows out from the solid activation culture medium, if bacterial colonies in the activation culture medium are light pink, circular, irregular in edge, foldable, opaque and flat bacterial colonies which are turned from wetting to drying, and the bacterial colonies are obvious by microscopic examination of the solid activation culture medium, indicating that the strains can be used as primary engineering strains for synthesizing biological foaming agents.
b. Screening high-yield strains: the method comprises the steps of adding a small amount of engineering strain liquid in a liquid activated culture medium into a blood agar plate, uniformly coating the liquid by using a triangular rod, culturing under a constant temperature condition, obtaining the largest strain by calculating the specific area of a hemolysis ring, inoculating the strain into a solid growth culture medium and a liquid growth culture medium, and culturing under the constant temperature condition, wherein the solid culture medium is used for storage, and the liquid culture medium is used for enrichment culture.
c. Strain preservation and rejuvenation: the strain to be preserved is diluted fully with SCP culture medium, spread to solid growth culture medium, cultured at constant temperature and preserved in low temperature for short period. Fully diluting the strain to be preserved by using an SCP culture medium, inoculating the strain to a liquid growth culture medium, culturing at a constant temperature, fully mixing a bacterium solution and glycerol, placing the prepared solution in a freezing tube, quickly freezing, and refrigerating in a low-temperature refrigerator. Can be used for long-term storage. The deposited strain needs to be revived before use. The strain stored for a short period to a solid growth medium was cultured in a constant temperature incubator, and the colony morphology was observed. The strains, which were cryopreserved in glycerol tubes for a long period of time, were thawed before being retested. And uniformly mixing the bacterial liquid and the liquid growth culture medium according to a certain proportion. Then inoculating bacterial liquid and uniformly coating the bacterial liquid on a solid activation culture medium. The bacterial solution and the plate were incubated at constant temperature and observed for morphological changes. If the colony characteristics of the solid activation medium are obvious, and the solid activation medium is in a flat colony shape which is light pink, round, irregular in edge, capable of being folded and opaque and is changed from wetting to drying, the strain is proved to be successfully rejuvenated.
d. And (3) microbial fermentation: inoculating a small amount of liquid growth culture medium to a certain amount of liquid fermentation culture medium, placing the liquid culture medium in a water bath shaking table, and culturing for more than 24h under the conditions of a certain temperature, pH value and shaking table rotation speed by adopting different shaking modes. After fermentation, the secondary metabolite can be obtained.
e. Active substance extraction: extracting active substances in the fermentation liquor by using an acidification fractional precipitation method, placing a conical flask filled with the fermentation liquor in a low-temperature water bath kettle to ensure that microorganisms do not continue to ferment, pouring the fermentation liquor into a high-speed centrifuge for centrifugation, taking supernate, adding hydrochloric acid to adjust the pH value to acidity, and standing overnight under a low-temperature condition; repeatedly centrifuging and acidifying for several times, and collecting precipitate to obtain crude product of surface active peptide. Adding anhydrous methanol into the crude product of the surface active peptide, fully dissolving the crude product by using ultrasonic waves, centrifuging the solution by using a high-speed centrifuge, taking supernatant, evaporating the supernatant to dryness by using a rotary evaporator under reduced pressure, adding ultrapure water into the evaporated material, adjusting the pH value to be alkaline by using NaOH, and filtering by using a filter membrane; adjusting the pH of the filtrate to acidity with concentrated hydrochloric acid, centrifuging in a high-speed centrifuge, collecting precipitate, and drying. The precipitate is dried to obtain the pure product of the surface active peptide.
f. The biological foaming agent consists of the following components: active substance, water.
2. The biotype foaming agent according to claim 1, wherein the engineering strain is obtained by the following steps:
preparing a 10ml test tube filled with 5ml of liquid activated medium and two solid activated media, sucking 0.5ml of liquid activated medium by a pipette gun, pumping into a freeze-drying tube, fully mixing, pouring bacterial liquid back into the solid activated medium, uniformly mixing, sucking the bacterial liquid, uniformly coating the bacterial liquid on solid activated medium plates, coating 0.1-0.3ml of bacterial liquid on each plate, and finishing the whole process on a sterile operating platform. Placing the liquid activation culture medium and the solid activation culture medium in a constant temperature incubator at 30-40 ℃ for culturing for 12-36 h; if the bacterial colony in the liquid activated culture medium is in a flat bacterial colony shape which is light pink, round, irregular in edge, capable of being folded and opaque and is changed from wetting to drying, and the bacterial colony detected by a solid activated culture medium through a mirror is obvious, the bacterial colony can be used as a primary engineering bacterial strain for synthesizing the biological foaming agent.
3. The biotype foaming agent according to claim 1, wherein the screening of high producing strains comprises the following steps:
the surface active peptide of the secondary metabolite of the bacillus subtilis has hemolytic property, and the higher the concentration of the substance, the stronger the hemolytic ability. According to the characteristics, bacteria with stronger surface active peptide synthesis capacity can be preliminarily screened. Taking a certain amount of culture solution in the liquid activation culture medium, and diluting with SCP culture medium by a certain multiple in a grading way, wherein the dilution multiple is determined by an OD600 value, and the OD value is the absorbance measured by a spectrophotometer at 600 nm. Uniformly coating the diluted strain on a blood agar plate, culturing at 30-40 ℃ for 6-18h, screening out a strain with a large specific area of a hemolysis cycle, respectively inoculating the strain to a solid growth culture medium and a liquid growth culture medium, culturing at 30-40 ℃ for 6-18h, storing and marking the solid culture medium in a refrigerator at 2-5 ℃, and using the liquid culture medium for enrichment culture of the screened strain.
4. The biotype foaming agent according to claim 1, wherein the specific steps of strain preservation and rejuvenation are as follows:
fully diluting the strain to be preserved with SCP culture medium, coating onto solid growth culture medium slant, culturing at 30-40 deg.C for 6-18h, and storing in 2-5 deg.C refrigerator for a short period of no more than 20 days; fully diluting a strain to be preserved by using an SCP culture medium, inoculating the strain to a liquid growth culture medium, culturing for 6-18h at 30-40 ℃, fully mixing a bacterium solution with 40% glycerol to prepare 1ml of solution in a freezing tube, freezing quickly, and refrigerating in a low-temperature refrigerator, wherein the solution can be used for long-term preservation, but the preservation time is not longer than 400 days; the deposited strain needs to be revived before use. The strain preserved for a short period to a solid growth medium was cultured in a constant temperature incubator at 30-40 ℃ for 6-18 hours, and the colony morphology was observed. The strains, which were cryopreserved in glycerol tubes for a long period of time, were thawed before being retested. Then 0.5ml of bacterial liquid is taken to be uniformly mixed with the liquid growth culture medium according to the proportion of 1:1 (v/v). Then inoculating 50-200 mul of bacterial liquid and uniformly coating the bacterial liquid on a solid activation culture medium. Placing the bacterial liquid and the flat plate in a constant temperature incubator at 30-40 ℃, culturing for 12-24h, and observing morphological change. If the colony characteristics of the solid activation medium plate are obvious, and the plate colony is light pink, round, irregular in edge, capable of being folded and opaque, and is in a flat colony form which is changed from wetting to drying, the strain is successfully rejuvenated.
5. The biological foaming agent as claimed in claim 1, wherein the microorganism fermentation comprises the following steps:
50-70mL of liquid fermentation medium is filled in a 250mL triangular flask, a liquid transfer device is used for accurately sucking bacterial liquid in the liquid growth medium and injecting the bacterial liquid into the triangular flask, the inoculation amount is ensured to be 1.5-3%, the inoculated liquid fermentation medium is placed into a rotary water bath shaking table and is cultured for more than 48h under the conditions that the temperature is 30-40 ℃, the pH value is 7-8, the rotation speed of the shaking table is 200 and 240r/min, and the oxygen introduction amount is 60-100 mL/min. After fermentation, the secondary metabolite can be obtained.
6. The biological foaming agent as claimed in claim 1, wherein the active substance extraction comprises the following steps:
extracting active substances from the fermentation liquor by an acidification, grading and precipitation method, and carrying out thermostatic water bath on the fermentation liquor for 25-35min at low temperature (4 ℃); placing the fermentation liquor in a high-speed centrifuge, and centrifuging for 15-25min under the condition that the relative centrifugal force is 6000-8000 g; adding 6mol/L concentrated hydrochloric acid into the supernatant to adjust the pH to 2.0, and standing overnight at the temperature of 2-5 ℃; centrifuging for 15-25min under the condition that the relative centrifugal force is 10000-; resuspending the precipitate with ultra-clean water, adjusting pH to 2.0, and acid-washing again; and centrifuging, and collecting precipitate to obtain the crude product of the surfactin. Adding 20-40mL of anhydrous methanol into the crude product of the surface active peptide, performing 20-60KHZ ultrasonic extraction for 25-35min to fully dissolve the crude product in a methanol reagent, filtering by using a filter membrane to obtain a residual active macromolecular structure, adding ultrapure water into the filtered residual substance, centrifuging for 15-25min under the condition that the relative centrifugal force is 16000-; adding ultra-clean water into the steamed dry matter, adjusting the pH to 12.0 by using 1mol/LNaOH, and filtering by using a filter membrane; adjusting the pH of the filtrate to 2.0 by using 6mol/L concentrated hydrochloric acid; centrifuging for 15-30min under the condition that the relative centrifugal force is 8000-11000g, collecting the precipitate and drying. The precipitate is dried to obtain the pure active substance.
7. The biological foaming agent as claimed in claim 1, wherein the biological foaming agent is prepared by the following steps:
adding 0.01-0.05 per mill of active substance into water, heating and controlling the temperature at about 30-40 deg.C; stirring with magnetic stirrer for 40-50min to mix active substances in water; stopping stirring, and standing the foaming agent solution to room temperature to form a white, transparent, uniform and stable biological foaming agent.
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