CN111705098A - Efficient production, separation and extraction method of antibacterial lipopeptide - Google Patents
Efficient production, separation and extraction method of antibacterial lipopeptide Download PDFInfo
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Abstract
The invention discloses a high-efficiency production and separation method of antibacterial lipopeptide, which is produced by fermenting Bacillus subtilis GD (Bacillus subtilis) CGMCC No.8898 and comprises the following steps of preparing a fermentation culture medium; preparing a first-level seed solution; preparing a secondary seed solution; fermenting and culturing to obtain fermentation liquor; and (4) separating and extracting the fermentation liquor to obtain the antibacterial lipopeptide. Compared with the prior art of producing lipopeptide by using bacillus strains, the technical scheme disclosed by the invention can effectively shorten the production period, improve the production efficiency and reduce the production cost.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for efficiently producing, separating and extracting antibacterial lipopeptide.
Background
With the improvement of living standard of people, animal food becomes an indispensable important source in the food composition of consumers in China, and especially, the safety of biological food of meat food such as livestock, poultry, aquatic products and the like is important. The main problems causing the safety of the animal food in China are drug-resistant super bacteria caused by the abuse of antibiotics in the culture of livestock, poultry, aquatic products and the like, and drug residues of the livestock, poultry and aquatic products and the like.
With the release of No. 194 bulletin board of rural agricultural department in 7/10/2019, no growth-promoting drug feed additive is prohibited to be added into the feed from 7/1/2020, and the overall footfall of 'feed prohibited' is approaching day by day. From the aspect of European banning, acidifier, microecological preparation, plant essential oil, antibacterial peptide, antibacterial lipopeptide and bacteriocin are mainly used for replacing antibiotics. Therefore, the antibacterial lipopeptide substances attract high attention of scholars at home and abroad, and are cyclic lipopeptide compounds consisting of peptide bonds of 7-9 amino acids and lipophilic aliphatic hydrocarbon chains of 12-19 carbon chains. The antibacterial lipopeptide comprises iturin (Iturins), Fengycin (Fengycin) and Surfactin (Surfactin), in particular Surfactin lipopeptide, and has inhibiting and killing effects on various gram-positive bacteria, gram-negative bacteria and viruses. The structure of the antibiotic is composed of peptide bonds of amino acids and lipophilic fatty hydrocarbon chains, the antibiotic mechanism of the antibiotic is completely different from that of common antibiotics, and the antibiotic is characterized in that holes are formed on cell membranes of microorganisms to enable cell contents to flow out, so that bacteria die, and drug resistance is not easy to generate; but also has better effect on super bacteria with drug resistance. Lipopeptide compounds produced by the bacillus subtilis in the middle and later periods of natural growth and fermentation culture are the most important antibacterial substances. The lipopeptide compound has the characteristics of high antibacterial activity, wide antibacterial spectrum, stable structure, no harm to human, animals and livestock, easy decomposition in nature and the like, so the lipopeptide compound is a novel environment-friendly biological bacteriostatic agent with great development potential. But the antibacterial lipopeptide compounds are not widely applied in production at present.
"antimicrobial action and active ingredient analysis of lipopeptides produced by Bacillus subtilis GD [ J ]. animal and veterinary medicine, 2016, 48 (1): 16-20' study on the antibacterial activity of lipopeptide generated by Bacillus subtilis GD strain CGMCC No. 8898.
The invention patent of patent No. 201010256246.7 discloses a fermentation method and application of bacillus subtilis lipopeptide in feed, and the invention patent of patent No. 201110361652.4 discloses a preparation method and application of antibacterial lipopeptide in veterinary medicine, which has the advantages of long production period, low lipopeptide yield, low purity of lipopeptide products produced by extraction processes of acid precipitation and alcohol extraction, high production cost and serious influence on industrial production of antibacterial lipopeptide and application and popularization in livestock raising and aquatic products.
Disclosure of Invention
The invention provides a production method for producing antibacterial lipopeptide by utilizing Bacillus subtilis GD (Bacillus subtilis) strain through high-efficiency fermentation and a method for separating the antibacterial lipopeptide from fermentation liquor.
The technical solution of the invention is as follows: the Bacillus subtilis GD strain adopted in the technical scheme is CGMCC No.8898, belongs to Bacillus and Bacillus subtilis strains, has a strain code GD and a Latin literature name Bacillus subtilis, and is preserved in the China general microbiological culture Collection center (CGMCC) with a preservation address: west road No. 1, north west of chaoyang district, beijing, No. 3, strain preservation code: CGMCC No.8898, the preservation date of the strain is: 2014-03-07.
The production method for producing the antibacterial lipopeptide by high-efficiency fermentation comprises the following steps:
s1, preparing a fermentation medium, wherein the main components of the fermentation medium comprise a carbon source, a nitrogen source and metal ions, wherein the carbon source adopts one or more of glucose, glycerol, starch, sucrose, maltodextrin and maltose, the content of the carbon source is 0.4-8.0%, the nitrogen source adopts one or more of plant protein, animal protein and yeast extract powder, the content of the nitrogen source is 0.5-6.0%, the content of the metal ions is 0.01-0.1%, and the initial pH is adjusted to 5.5-8.5;
s2, preparing a first-stage seed solution by adopting an LB culture medium;
s3, preparing a secondary seed solution by adopting an LB culture medium;
s4, fermentation culture, wherein the secondary seed culture solution obtained in the S3 is inoculated into the fermentation culture medium obtained in the S1 in an inoculation amount of 0.01-10%, batch fermentation is adopted in the fermentation process, timed, quantitative or continuous feeding is adopted,wherein the fermentation medium initially contains a part of carbon source, the rest carbon source is added in a feeding way, the culture temperature is 32-39 ℃, and the ventilation volume is 50-150 m3The fermentation is carried out for 12-60 hours, namely fermentation broth is obtained after the fermentation is carried out for 8 hours at the rotating speed of 10-65 Hz and the rotating speed of 20-85 Hz and the tank pressure of 0.05-0.5 Mpa and the fermentation pH value of 5.0-8.5;
s5, separating and extracting the antibacterial lipopeptide from the fermentation liquor obtained in the S4.
Preferably, in step S1, the vegetable protein includes one or more of soy protein isolate, pea protein, corn protein, and rice milk protein.
Preferably, in step S1, the animal protein includes one or more of tryptone, casein peptone, beef peptone and fish peptone.
Preferably, in step S1, the metal ions include: na (Na)+、Mg2+、K+、 Mn2+、Fe3+、Fe2+、Zn2+、Ca2 +、Cu2+One or more of them.
Preferably, in the step S2, activating a Bacillus subtilis GD strain (cgmccno.8898) with an LB plate, inoculating the activated strain in an LB culture medium, performing shake culture at 30-39 ℃ and 100-240 r/min for 12-24 hours to obtain a first-order seed culture solution.
Preferably, in step S3, the primary seed culture solution obtained in step S2 is inoculated into LB medium at an inoculum size of 0.1-10%, and shake cultivation is performed at a cultivation temperature of 30-39 ℃ and a rotation speed of 100-240 r/min for 12-24 hours to obtain a secondary seed culture solution.
Preferably, in the step S1, the fermentation medium is further added with an antifoaming agent, the antifoaming agent is one or a mixture of a liquid antifoaming agent and a solid antifoaming agent, and the antifoaming agent is 0.001% -0.5%.
Separating and preparing a lipopeptide crude product from fermentation liquor:
and taking 1000ml of fermentation liquor containing the antibacterial lipopeptide of the GD strain of the bacillus subtilis at the optimal fermentation stage, subpackaging, centrifuging at 5000-15000 r/min for 15-60 minutes, discarding the strain, and collecting supernatant. Adding 1-8% of macroporous adsorption resin XAD, placing in a shaking table at room temperature, shaking at 80-240 r/min, adsorbing for 16-36 hours, taking supernatant, detecting the content of the antibacterial lipopeptide in the supernatant, and ensuring that the residual quantity of the antibacterial lipopeptide in the supernatant is the lowest. Standing, filtering, reserving resin XAD, washing with distilled water until no antibacterial lipopeptide fermentation liquor remains on the surface of the resin, then performing antibacterial elution with solvents such as ethanol or methanol, placing in a shaking table (40-240 r/min) at room temperature, performing oscillation elution for 8-24 hours, collecting eluent, performing vacuum drying at low pressure, recovering the eluent, and obtaining a solid which is an antibacterial lipopeptide extract crude product.
The production and separation of the antibacterial lipopeptide adopt batch fermentation and batch extraction treatment.
Compared with the prior art, the invention has the advantages that: the production method for producing the antibacterial lipopeptide by efficiently fermenting the Bacillus subtilis GD strain (Bacillus subtilis) CGMCC No.8898 can rapidly ferment and produce the lipopeptide, can effectively shorten the production period, improve the production efficiency and reduce the production cost compared with the prior art for producing the lipopeptide by using the Bacillus subtilis strain.
Drawings
FIG. 1 is a graph showing the separation of lipopeptides using different separation methods and the control of the drug sensitivity assay using the tube-dish method according to the present invention.
The reference numbers in the figures illustrate:
1. the use of a published prior art separation method and the use of a tube-dish method for susceptibility testing;
2. example 3 the disclosed separation method was used and the tube-dish method was used for susceptibility testing.
Detailed Description
The technical scheme in the embodiment of the invention will be clearly and completely described below with reference to the accompanying drawings in the embodiment of the invention; it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.
Example 1
The Bacillus subtilis GD strain (Bacillus subtilis) CGMCC No.8898 is streaked by an LB flat plate and cultured for 18 hours at 36 ℃, the activated GD strain is selected by an inoculating rod to be inoculated into a first-stage seed liquid LB sterile culture medium by a standard single colony, the loading of a 250ml triangular flask is 150ml of LB culture medium, the culture temperature is 36 ℃, the rotating speed is 150r/min, the shaking table culture is carried out for 20 hours, the gram stain and the microscopic examination are carried out, the mixed bacteria are avoided, and the mixed bacteria are stored for later use in a refrigerator at 4 ℃.
Inoculating the cultured first-stage seed culture solution into a second-stage seed solution LB sterile culture medium by using a sterile liquid transfer gun according to the inoculation amount of 10%, wherein the loading amount of a 5000ml triangular flask is 3000ml LB culture medium, the culture temperature is 36 ℃, the rotating speed is 150r/min, the culture is carried out for 20 hours, so as to obtain a second-stage seed solution, and the second-stage seed solution is subjected to gram staining, microscopic examination, free of infectious microbes and kept in a refrigerator at 4 ℃ for later use.
The bacillus subtilis GD strain fermentation medium mainly comprises the following components: carbon source (glucose, glycerol, starch, maltodextrin) 8.0%; nitrogen source: 6.0% of yeast extract powder, animal protein (tryptone, casein peptone, beef peptone, etc.); metal ions: na (Na)+、Mg2+、K+、Mn2+、Fe2+、Zn2+、Ca2+One or more of them, the content is 0.1%; the defoaming agent is prepared by mixing a liquid defoaming agent and a solid defoaming agent together, and the dosage is controlled to be about 0.5 percent.
The fermenter was a 50 liter pilot tank, initially adjusted to pH 8.2.
In the fermentation process of this embodiment, the carbon source in the fermentation medium is added in a feeding manner, before the secondary seed solution is inoculated into the culture medium of the sterilization fermentation tank in an inoculation amount of 10%, the carbon source and the metal ions are added in a certain proportion, the carbon source and the metal ions are separately sterilized before being added, the initial carbon source addition amount is 30% of the total amount of the required carbon source, after inoculation and fermentation, the remaining carbon source is added in a feeding manner, in this embodiment, 70% of the carbon source of the total amount of the remaining required carbon source is added ten times in an amount of 7% each time.
Controlling fermentation parameters in the fermentation process: the culture temperature is 36-38 ℃, and the ventilation volume is 120m3The rotation speed is 25Hz, after 8 hours, the rotation speed is 45Hz, the tank pressure is 0.5Mpa, the pH is adjusted to 7.5-8.5, the fermentation culture is carried out for 26-50 hours, and the batch fermentation is carried out.
Sampling is carried out for 26 hours of fermentation, sporulation is observed, the content of the antibacterial lipopeptide is detected, and the time period with the optimal content is determined.
Preparing a fermentation liquid lipopeptide crude product:
and (3) taking 200ml of fermentation liquor of the Bacillus subtilis GD strain containing the antibacterial lipopeptide in the optimal fermentation stage, centrifuging at 10000r/min for 45 minutes, discarding the strain, and collecting supernatant. Adding a certain amount of macroporous adsorption resin XAD, shaking in a shaking table at room temperature, 150r/min, adsorbing for 18 hours, taking the supernatant to detect the content of the antibacterial lipopeptide, and ensuring the minimum residual quantity of the antibacterial lipopeptide in the supernatant. Standing, filtering, retaining resin XAD, washing with distilled water until no antibacterial lipopeptide fermentation liquor remains on the surface of the resin, then performing antibacterial elution with solvents such as ethanol or methanol, placing in a shaking table (140r/min) at room temperature, performing shaking elution for 20 hours, collecting the eluent, performing vacuum drying at low pressure, recovering the eluent, and obtaining a solid which is an antibacterial lipopeptide extract crude product.
And (3) detecting the purity and the content of the sample:
centrifuging the fermentation liquid for 10 min at 5000r/min, removing thallus, freeze drying the fermentation liquid, extracting with methanol or ethanol, and detecting with High Performance Liquid Chromatography (HPLC).
Detection conditions are as follows: RP-C18 column (4.6 mm. times.250 mm, 5 μm);
mobile phase A: water (0.1% Trifluoroacetic acid (TFA));
mobile phase B: acetonitrile (ACN);
0～50min，A：40％～95％，B：60％～5％
flow rate: 1 ml/min; column temperature: 30 ℃;
sample introduction volume: 20 mu l of the mixture;
detection wavelength: 210 nm.
The standard is purchased from sigma company and has a purity of more than 98 percent.
The detection result is as follows: 476.3342 mg/L.
Example 2
The Bacillus subtilis GD strain (Bacillus subtilis) CGMCC No.8898 is streaked by an LB flat plate and cultured for 18 hours at 36 ℃, the activated GD strain is selected by an inoculating rod to be inoculated into a first-stage seed liquid LB sterile culture medium by a standard single colony, the loading of a 250ml triangular flask is 150ml of LB culture medium, the culture temperature is 36 ℃, the rotating speed is 150r/min, the shaking table culture is carried out for 20 hours, the gram stain and the microscopic examination are carried out, the mixed bacteria are avoided, and the mixed bacteria are stored for later use in a refrigerator at 4 ℃.
Inoculating the cultured first-stage seed culture solution into a second-stage seed solution LB sterile culture medium by using a sterile liquid transfer gun according to the inoculation amount of 10%, wherein the loading amount of a 5000ml triangular flask is 3000ml LB culture medium, the culture temperature is 36 ℃, the rotating speed is 150r/min, the culture is carried out for 20 hours, so as to obtain a second-stage seed solution, and the second-stage seed solution is subjected to gram staining, microscopic examination, free of infectious microbes and kept in a refrigerator at 4 ℃ for later use.
The bacillus subtilis GD strain fermentation medium mainly comprises the following components: carbon source (glucose, glycerol, starch, maltodextrin) 4.0%; nitrogen source: 3.0% of yeast extract powder, vegetable proteins (soybean protein isolate, rice milk protein, etc.), animal proteins (tryptone, casein peptone, beef peptone); metal ions: na (Na)+、Mg2+、K+、Mn2+、Fe2+、Zn2+、Ca2+One or more of the components, the content is 0.08%; the defoaming agent is prepared by mixing a liquid defoaming agent and a solid defoaming agent together, and the dosage is controlled to be about 0.02 percent.
The fermenter was a 50 liter pilot tank, initially adjusted to pH 6.5.
In the fermentation process of this embodiment, the carbon source in the fermentation medium is added in a feeding manner, before the secondary seed solution is inoculated into the culture medium of the sterilization fermentation tank in an inoculation amount of 2%, the carbon source and the metal ions are added in a certain proportion, the carbon source and the metal ions are separately sterilized before being added, the initial carbon source addition amount is 30% of the total amount of the required carbon source, after inoculation and fermentation, the remaining carbon source is added in a feeding manner, in this embodiment, 70% of the carbon source of the total amount of the remaining required carbon source is added ten times in an amount of 7% each time.
Controlling fermentation parameters in the fermentation process: the culture temperature is 33-36 ℃, and the ventilation volume is 100m3The rotation speed is 15Hz, after 8 hours, the rotation speed is 25Hz, the tank pressure is 0.5Mpa, the pH is adjusted to 6.0-7.0, the fermentation culture is carried out for 26-50 hours, and the batch fermentation is carried out.
Sampling is carried out after 30 hours of fermentation, sporulation is observed, the content of the antibacterial lipopeptide is detected, and the time period with the optimal content is determined.
Preparing a fermentation liquid lipopeptide crude product:
200ml of fermentation liquor containing the antibacterial lipopeptide in the optimal fermentation stage of the Bacillus subtilis GD strain is taken, centrifuged for 45 minutes at 8000r/min, the strain is discarded, and the supernatant is collected. Adding a certain amount of macroporous adsorption resin XAD, shaking in a shaking table at room temperature, adsorbing at 150r/min for 30 hours, taking the supernatant to detect the content of the antibacterial lipopeptide, and ensuring the minimum residual quantity of the antibacterial lipopeptide in the supernatant. Standing, filtering, retaining resin XAD, washing with distilled water until no antibacterial lipopeptide fermentation liquor remains on the surface of the resin, then performing antibacterial elution with solvents such as ethanol or methanol, placing in a shaking table (140r/min) at room temperature, performing shaking elution for 20 hours, collecting the eluent, performing vacuum drying at low pressure, recovering the eluent, and obtaining a solid which is an antibacterial lipopeptide extract crude product.
And (3) detecting the purity and the content of the sample:
centrifuging the fermentation liquid for 10 min at 5000r/min, removing thallus, freeze drying the fermentation liquid, extracting with methanol or ethanol, and detecting with High Performance Liquid Chromatography (HPLC).
Detection conditions are as follows: RP-C18 column (4.6 mm. times.250 mm, 5 μm);
mobile phase A: water (0.1% Trifluoroacetic acid (TFA));
mobile phase B: acetonitrile (ACN);
0～50min，A：40％～95％，B：60％～5％
flow rate: 1 ml/min; column temperature: 30 ℃;
sample introduction volume: 20 mu l of the mixture;
detection wavelength: 210 nm.
The standard is purchased from sigma company and has a purity of more than 98 percent.
The detection result is as follows: 620.7784 mg/L.
Example 3
The Bacillus subtilis GD strain (CGMCC No.8898) is streaked by an LB flat plate and cultured for 18 hours at 36 ℃, the activated GD strain is selected by an inoculating rod to be inoculated into a primary seed liquid LB sterile culture medium by a standard single colony, the content of a 250ml triangular flask is 150ml of LB culture medium, the culture temperature is 36 ℃, the rotating speed is 150r/min, the GD strain is cultured for 20 hours by a shaking table, the strain is subjected to gram staining and microscopic examination, and the strain is free of infectious microbes and is stored for later use in a refrigerator at 4 ℃.
Inoculating the cultured first-stage seed culture solution into a second-stage seed solution LB sterile culture medium by using a sterile liquid transfer gun according to the inoculation amount of 10%, wherein the loading amount of a 5000ml triangular flask is 3000ml LB culture medium, the culture temperature is 36 ℃, the rotating speed is 150r/min, the culture is carried out for 20 hours, so as to obtain a second-stage seed solution, and the second-stage seed solution is subjected to gram staining, microscopic examination, free of infectious microbes and kept in a refrigerator at 4 ℃ for later use.
The bacillus subtilis GD strain fermentation medium mainly comprises the following components: carbon source (glucose, glycerol, starch, maltodextrin) 5.0%; nitrogen source: 6.0% of yeast extract powder, animal protein (tryptone, casein peptone, beef peptone, etc.); metal ions: na (Na)+、Mg2+、K+、Mn2+、Fe2+、Zn2+、Ca2+One or more of them, the content is 0.1%; the defoaming agent is prepared by mixing a liquid defoaming agent and a solid defoaming agent together, and the dosage is controlled to be about 0.01 percent.
The fermenter was a 50 liter pilot tank, initially adjusted to pH 6.8.
In the fermentation process of this embodiment, the carbon source in the fermentation medium is added in a feeding manner, before the secondary seed solution is inoculated into the culture medium of the sterilization fermentation tank in an inoculation amount of 5%, the carbon source and the metal ions are added in a certain proportion, the carbon source and the metal ions are separately sterilized before being added, the initial carbon source addition amount is 30% of the total amount of the required carbon source, after inoculation and fermentation, the remaining carbon source is added in a feeding manner, in this embodiment, 70% of the carbon source of the total amount of the remaining required carbon source is added ten times in an amount of 7% each time.
Controlling fermentation parameters in the fermentation process: the culture temperature is 37-38 ℃, and the ventilation volume is 120m3And h, rotating speed is 20Hz, after 8 hours, rotating speed is 35Hz, tank pressure is 0.5Mpa, pH is adjusted to 6.8-7.5, fermentation culture is carried out for 26-40 hours, and batch fermentation is carried out.
Sampling is carried out after 35 hours of fermentation, sporulation is observed, the content of the antibacterial lipopeptide is detected, and the time period with the optimal content is determined.
Preparing a fermentation liquid lipopeptide crude product:
200ml of fermentation liquor containing the antibacterial lipopeptide in the optimal fermentation stage of the Bacillus subtilis GD strain is taken, centrifuged at 5000r/min for 45 minutes, the strain is discarded, and the supernatant is collected. Adding a certain amount of macroporous adsorption resin XAD, shaking in a shaking table at room temperature, adsorbing at 120r/min for 20 hours, taking the supernatant to detect the content of the antibacterial lipopeptide in the supernatant, and ensuring that the residual quantity of the antibacterial lipopeptide in the supernatant is the minimum. Standing, filtering, retaining resin XAD, washing with distilled water until no antibacterial lipopeptide fermentation liquor remains on the surface of the resin, then performing antibacterial elution with solvents such as ethanol or methanol, placing in a shaking table (120r/min) at room temperature, performing shaking elution for 16 hours, collecting the eluent, performing vacuum drying at low pressure, recovering the eluent, and obtaining a solid which is an antibacterial lipopeptide extract crude product.
And (3) detecting the purity and the content of the sample:
centrifuging the fermentation liquid for 10 min at 5000r/min, removing thallus, freeze drying the fermentation liquid, extracting with methanol or ethanol, and detecting with High Performance Liquid Chromatography (HPLC).
Detection conditions are as follows: RP-C18 column (4.6 mm. times.250 mm, 5 μm);
mobile phase A: water (0.1% Trifluoroacetic acid (TFA));
mobile phase B: acetonitrile (ACN);
0～50min，A：40％～95％，B：60％～5％
flow rate: 1 ml/min; column temperature: 30 ℃;
sample introduction volume: 20 mu l of the mixture;
detection wavelength: 210 nm.
The standard is purchased from sigma company and has a purity of more than 98 percent.
The detection result is as follows: 683.4909 mg/L.
Control group 1:
the content of the antimicrobial lipopeptide in the fermentation process of patent No. 201010576515.8 and patent No. 3 is compared, and is shown in Table 1.
The fermentation liquid is obtained by adopting a fermentation process method disclosed in the patent with the patent number of 201010576515.8, namely 'a production method of bacillus subtilis lipopeptide and application of the bacillus subtilis lipopeptide in piglet feed'.
Centrifuging the fermentation liquor for 10 minutes at 5000r/min, removing thallus, freeze drying the fermentation liquor,
extracting with methanol or ethanol, and detecting with High Performance Liquid Chromatography (HPLC).
Detection conditions are as follows: RP-C18 column (4.6 mm. times.250 mm, 5 μm);
mobile phase A: water (0.1% Trifluoroacetic acid (TFA));
mobile phase B: acetonitrile (ACN);
0～50min，A：40％～95％，B：60％～5％
flow rate: 1 ml/min; column temperature: 30 ℃;
sample introduction volume: 20 mu l of the mixture;
detection wavelength: 210 nm.
The standard is purchased from sigma company and has a purity of more than 98 percent.
The detection result is as follows: 155.0261 mg/L.
TABLE 1 control of production of antimicrobial lipopeptides by different fermentation processes
Control group 2:
the comparison between the extraction process of patent No. 201110361652.4 and the extraction process of this patent application 3 shows the bacteriostatic effect, which is shown in Table 2 and FIG. 1.
The extraction method of the crude lipopeptide adopts a preparation method disclosed in patent No. 201110361652.4 ' a preparation method of antibacterial lipopeptide and application thereof in veterinary medicine ', namely ' the zymocyte liquid is centrifuged at 7000rpm for 10 minutes under the condition of pH 2.0-5.0, bacteria are removed, supernatant is taken and is respectively adjusted to the required pH by 6mol/L HCl, then is centrifuged at 7000rpm for 10 minutes, precipitate is collected, and the precipitate is dried in an oven at 70 ℃ for 20 hours, thus obtaining the lipopeptide.
The tube-disc method is adopted for drug sensitivity experiment comparison: the invention provides a method for preparing crude antibacterial lipopeptide products in example 3 and patent No. 201110361652.4.
Namely, 0.05ml of newly cultured Escherichia coli was aspirated at a concentration of 10%5Indicator bacteria of cfu/ml, coated on LB agar plate; 1g of the crude lipopeptide is dissolved by 5ml of buffer solution to prepare a sample, the bacteriostatic effect is compared on an LB agar plate coated with escherichia coli by adopting a cup-dish method, 300 mu l of the sample of the antibacterial lipopeptide is added, the culture temperature is 37 ℃, and the result is observed for 18 hours.
TABLE 2 comparison of antibacterial effect of different extraction processes for antibacterial lipopeptides in TABLE 2
In summary, it can be seen that the method for efficiently producing antibacterial lipopeptide disclosed by the invention utilizes Bacillus subtilis GD (Bacillus subtilis) to efficiently ferment and produce antibacterial lipopeptide, so that compared with the prior art, the production period is effectively shortened, the production efficiency is improved, and the production cost is reduced.
The foregoing is only a preferred embodiment of the present invention; the scope of the invention is not limited thereto. Any person skilled in the art should be able to cover the technical solutions and modifications of the present invention within the technical scope of the present disclosure, and all equivalent substitutions and modifications of the technical solutions and modifications of the present invention should be covered by the protection scope of the present invention.
Claims (9)
1. An efficient production method of antibacterial lipopeptide is characterized in that: the method uses Bacillus subtilis GD strain (CGMCC No.8898) for fermentation production and comprises the following steps:
s1, preparing a fermentation medium, wherein the main components of the fermentation medium comprise a carbon source, a nitrogen source and metal ions, wherein the carbon source adopts one or more of glucose, glycerol, starch, sucrose, maltodextrin and maltose, the content of the carbon source is 0.4-8.0%, the nitrogen source adopts one or more of plant protein, animal protein and yeast extract powder, the content of the nitrogen source is 0.5-6.0%, the content of the metal ions is 0.01-0.1%, and the initial pH is adjusted to 5.5-8.5;
s2, preparing a first-stage seed solution by adopting an LB culture medium;
s3, preparing a secondary seed solution by adopting an LB culture medium;
s4, fermentation culture, wherein the secondary seed culture solution obtained in the step S3 is inoculated into the fermentation culture medium obtained in the step S1 in an inoculation amount of 0.01-10%, batch fermentation is adopted in the fermentation process, and feeding is performed regularly, quantitatively or continuously, wherein the fermentation culture medium initially contains a part of carbon source, the rest carbon source is added in a feeding mode, the culture temperature is 32-39 ℃, and the ventilation volume is 50-150 m3The fermentation is carried out for 12-60 hours, namely fermentation broth is obtained after the fermentation is carried out for 8 hours at the rotating speed of 10-65 Hz and the rotating speed of 20-85 Hz and the tank pressure of 0.05-0.5 Mpa and the fermentation pH value of 5.0-8.5;
s5, separating and extracting the antibacterial lipopeptide from the fermentation liquor obtained in the S4.
2. The method of claim 1, wherein the method comprises the steps of: in step S1, the vegetable protein includes one or more of soy protein isolate, pea protein, corn protein, and rice milk protein.
3. The method of claim 1, wherein the method comprises the steps of: in step S1, the animal protein includes one or more of tryptone, casein peptone, beef peptone, and fish peptone.
4. The method of claim 1, wherein the method comprises the steps of: in step S1, the metal ions include: na (Na)+、Mg2+、K+、Mn2+、Fe3+、Fe2+、Zn2+、Ca2+、Cu2+One or more of them.
5. The method of claim 1, wherein the method comprises the steps of: in the step S2, activating a Bacillus subtilis GD strain (Bacillus subtilis) CGMCC No.8898 by an LB plate, inoculating the activated Bacillus subtilis GD strain in an LB culture medium, culturing at the temperature of 30-39 ℃ and the rotating speed of 100-240 r/min, and performing shake culture for 12-24 hours to obtain a primary seed culture solution.
6. The method of claim 1, wherein the method comprises the steps of: in the step S3, the primary seed culture solution obtained in the step S2 is inoculated into an LB culture medium in an inoculum size of 0.1-10%, the culture temperature is 30-39 ℃, the rotating speed is 100-240 r/min, and shake culture is carried out for 12-24 hours to obtain a secondary seed culture solution.
7. The method of claim 1, wherein the method comprises the steps of: in the step S1, a defoaming agent is further added to the fermentation medium, the defoaming agent is one or a mixture of a liquid defoaming agent and a solid defoaming agent, and the content of the defoaming agent is 0.001-0.5%.
8. A method for separating and extracting antibacterial lipopeptide is characterized by comprising the following steps: centrifuging fermentation liquor of Bacillus subtilis GD strain (Bacillus subtilis) CGMCC No. 8898; adsorbing for 16-36 hours by using adsorption resin after removing thalli; standing and filtering, and keeping the resin; eluting for 8-24 hours by using ethanol or methanol, and drying the elution liquid in vacuum at low pressure to obtain a solid, namely the antibacterial lipopeptide crude product.
9. The method for separating and extracting antibacterial lipopeptide according to claim 8, wherein the method comprises the following steps: the adsorption resin adopts macroporous adsorption resin XAD, and the dosage is 1-8%.
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| CN113293110A (en) * | 2021-06-01 | 2021-08-24 | 大连海象生物工程有限公司 | Preparation method of antibacterial lipopeptide compound |
| CN114931166A (en) * | 2022-05-18 | 2022-08-23 | 焦作市佰役安生物工程有限公司 | Application of bacillus subtilis antibacterial lipopeptide extract in corn storage |
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| CN113293110A (en) * | 2021-06-01 | 2021-08-24 | 大连海象生物工程有限公司 | Preparation method of antibacterial lipopeptide compound |
| CN113293110B (en) * | 2021-06-01 | 2023-12-22 | 大连海象生物工程有限公司 | Preparation method of antibacterial lipopeptid compound |
| CN114931166A (en) * | 2022-05-18 | 2022-08-23 | 焦作市佰役安生物工程有限公司 | Application of bacillus subtilis antibacterial lipopeptide extract in corn storage |
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