CN113801186A - Method for extracting lipopeptide compounds from bacillus subtilis fermentation liquor - Google Patents
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a method for extracting an antibacterial lipopeptide compound from bacillus subtilis fermentation liquor, which comprises the following steps: centrifuging the bacillus subtilis fermentation liquor to obtain bacillus subtilis fermentation supernatant; preparing a double aqueous phase extraction system by adopting ethanol and ammonium sulfate; adding the bacillus subtilis fermentation supernatant into a double-aqueous-phase extraction system, adjusting the pH of the double-aqueous-phase extraction system to 2-8, stirring and mixing uniformly, standing for extraction and phase separation, and collecting an upper phase extraction liquid; adding ethanol with the volume 1-3 times of that of the upper phase extraction liquid into the upper phase extraction liquid, separating out ammonium sulfate precipitate, filtering the ammonium sulfate precipitate, and collecting filtrate; drying and concentrating the filtrate by using a rotary evaporator to obtain the bacillus subtilis antibacterial lipopeptide compound. Compared with the prior art, the method has the advantages of high extraction speed and high yield of the antibacterial lipopeptide compounds, and the extracted ethanol and ammonium sulfate can be recycled, so that the problem of environmental pollution is solved.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for extracting an antibacterial lipopeptide compound from a bacillus subtilis fermentation broth.
Background
The antibacterial lipopeptide compound is a natural protein antibacterial substance generated by the non-ribosome way in the growth process of bacillus, and has the characteristics of no toxicity, no residue, no drug resistance and the like, so that the antibacterial lipopeptide compound is widely concerned at home and abroad as a potential antibiotic substitute. So far, the antibacterial lipopeptide compounds which are well researched mainly comprise three major classes of camelina sativa element (Fengycin), surfactin (surfactin) and Iturin (Iturin), most of which are low molecular weight (300- & ltSUB & gt 3000Da) & lt SUB & gt lipopeptide compounds, and have unique physicochemical characteristics of low toxicity, degradability, no environmental pollution, acid and alkali resistance and the like.
The researches show that the camelina sativa extract is an antifungal inhibitor, can be used for treating dermatophytosis, and becomes an antifungal medicament with high value due to the characteristics of broad-spectrum antifungal property, low toxicity, low allergic reaction and the like. The surfactant is an amphiphilic molecule capable of reducing surface tension, has the characteristics of emulsification, washing, sterilization, moistening, dispersion, low irritation, high skin compatibility and the like, and is widely applied to the fields of daily necessities and cosmetics, such as facial cleanser, shampoo, toothpaste and the like. The iturin is an amphiphilic compound with a cyclic lipopeptide structure, consists of nonpolar fatty acid chains through combining polar amino acid fatty chains, and has the effects of surface activity, hemolysis, antifungal, antitumor and the like and unique multiple drug resistance. The iturin is also one of the antibiotics with antibacterial activity which are discovered at the earliest, and the homolog of the iturin, namely bacitracin D (Bacillus subtilis D), has a remarkable inhibiting effect on molds such as aspergillus flavus, and is the only active substance which is obtained from microorganisms at present and has a remarkable inhibiting effect on the aspergillus flavus. In addition, the antibacterial lipopeptide compound also has potential application value in the fields of biomedicine, aquaculture, food preservation, molecular probes and the like.
At present, the extraction processes of antibacterial lipopeptide compounds mostly adopt the extraction processes of acidification precipitation, organic solvent extraction, adsorption method, foam separation method and the like, and the extraction processes generally take a long time. Moreover, a large amount of acid is needed for acidification and precipitation, so that the environment is polluted, and the production cost is increased due to the need of subsequent treatment; the organic solvent extraction uses a large amount of toxic reagents, which are extremely harmful to human bodies and environment. Therefore, a method for rapidly separating the antibacterial lipopeptide, which is simple and feasible in process, suitable for industrial production, is urgently needed.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a method for extracting the lipopeptide compounds in the bacillus subtilis fermentation liquor, which has high extraction speed and high yield.
In order to achieve the purpose, the invention is implemented according to the following technical scheme:
a method for extracting lipopeptide compounds from bacillus subtilis fermentation liquor comprises the following steps:
s1, fermenting and culturing the bacillus subtilis, and centrifuging the bacillus subtilis fermentation liquor to obtain a bacillus subtilis fermentation supernatant;
s2, preparing a double-aqueous-phase extraction system by using ethanol and ammonium sulfate, wherein the volume fraction of the ethanol in the double-aqueous-phase extraction system is 20-40%, and the mass fraction of the ammonium sulfate is 10-30%;
s3, adding the bacillus subtilis fermentation supernatant into a double-aqueous-phase extraction system to obtain a mixed solution, adjusting the pH of the double-aqueous-phase extraction system to 2-8, wherein the volume fraction of the bacillus subtilis fermentation supernatant in the mixed solution is 50-70%, uniformly stirring and mixing, standing, extracting, splitting phases, and collecting an upper phase extraction liquid;
s4, adding ethanol with the volume 1-3 times of that of the upper phase extraction liquid, separating out ammonium sulfate precipitate, filtering the ammonium sulfate precipitate, and collecting filtrate;
s5, drying and concentrating the filtrate obtained in the step S4 by using a rotary evaporator to obtain the bacillus subtilis antibacterial lipopeptide compound.
Further, the fermentation medium used for fermentation culture of bacillus subtilis in the step S1 contains the following components per liter: 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of dipotassium hydrogen phosphate and the balance of water, wherein the pH value of the fermentation medium is 7.0.
Further, in the step S1, the centrifugation of the Bacillus subtilis fermentation broth is performed by solid-liquid separation in a centrifuge with a rotation number of 8000r/min for 10 min.
Further, in the step S3, the extraction temperature is 20-40 ℃, the stirring time is 10-20min, the rotation speed of the stirrer is 100-200rpm, and the standing phase separation time is 10-30 min.
Preferably, in the step S5, the rotary evaporator temperature is 60 ℃ and the pressure is 0.1 MPa.
Preferably, in step S2, the volume fraction of ethanol in the aqueous two-phase extraction system is 35%, the mass fraction of ammonium sulfate is 20%, and the pH of the aqueous two-phase extraction system is 6.
The principle of the invention is as follows: the method adopts a double-aqueous-phase extraction technology, selects an ethanol/ammonium sulfate system as a component of a component phase, the ethanol/ammonium sulfate system can provide positive and negative ions, and affects the distribution of the positive and negative ions in a solution between two phases, so as to change the distribution coefficient, thereby affecting the selective distribution of the antibacterial lipopeptide between the two phases, simultaneously uses ethanol as the component of the component phase, can effectively reduce the emulsification phenomenon, and obtains the antibacterial lipopeptide compound of bacillus subtilis after the filtrate after the phase formation is dried and concentrated by using a rotary evaporator.
Compared with the prior art, the invention has the following beneficial effects:
compared with the prior art for extracting the antibacterial lipopeptide compounds, the invention can keep the activity and conformation of bioactive substances and has wide application prospect in the aspect of extracting natural products.
The invention avoids the use of volatile organic solvent, has high extraction speed and high yield of the antibacterial lipopeptide compound, and the extracted ethanol and ammonium sulfate can be recycled, thereby avoiding the problem of environmental pollution.
The method does not need special equipment, is easy for continuous operation, has low production cost and is suitable for industrial production requirements of different scales.
Drawings
FIG. 1 shows the effect of different ethanol volume fractions on the extraction yield of the antibacterial lipopeptide compounds from Bacillus subtilis when ammonium sulfate (20% by mass) was used (note: error bars represent standard deviation (n ═ 3)).
FIG. 2 is a graph showing the effect of different ethanol volume fractions on the extraction rate of the antibacterial lipopeptide compounds from Bacillus subtilis when ammonium sulfate (25% by mass) (note: error bars represent standard deviation (n ═ 3)).
FIG. 3 is a graph showing the effect of different ethanol volume fractions on the extraction rate of the antibacterial lipopeptide compounds from Bacillus subtilis when ammonium sulfate (30% by mass) (note: error bars represent standard deviation (n ═ 3)).
FIG. 4 is a graph showing the effect of two aqueous phase extraction systems at different pH values on the extraction rate of the antibacterial lipopeptide compounds from Bacillus subtilis (note: error bars represent standard deviation (n-3)).
FIG. 5 is a high performance liquid chromatogram of a two aqueous phase extraction of a Bacillus subtilis antimicrobial lipopeptide compound.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
Example 1
(1) Fermenting and culturing bacillus subtilis by using a fermentation medium to obtain bacillus subtilis fermentation liquor, wherein the fermentation medium contains the following components per liter: 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of dipotassium hydrogen phosphate and the balance of water, wherein the pH value of the fermentation medium is 7.0; centrifuging the bacillus subtilis fermentation liquor at 8000rpm for 10min, removing the precipitated thallus, and collecting the supernatant for later use;
(2) and (2) adding a small amount of ammonium sulfate into the fermentation supernatant prepared in the step (1) for multiple times according to the proportion, magnetically stirring until the ammonium sulfate is completely dissolved, and slowly adding ethanol in a flowing manner to ensure that the total volume of the aqueous two-phase extraction system is 100 mL. Wherein the mass fraction of ammonium sulfate is 20%, the volume fractions of ethanol are 20%, 25%, 30%, 35% and 40%, respectively, a double-aqueous-phase extraction system is established, the pH of the double-aqueous-phase extraction system is adjusted to 7, magnetic stirring is carried out for 10min at room temperature, standing and phase splitting are carried out for 15min, and the antibacterial lipopeptide compound is dissolved in the upper phase; separating to remove the lower salt solution and retaining the upper phase extract.
(3) Adding 3 times of ethanol into the upper phase extraction liquid obtained in the step (2), and precipitating ammonium sulfate; precipitating ammonium sulfate, filtering, rotary evaporating the filtrate at 60 deg.C and 0.1Mpa under reduced pressure, and evaporating ethanol and water to obtain Bacillus subtilis antibacterial lipopeptide compound product.
Fig. 1 shows the effect of different ethanol volume fractions on the extraction rate of the antibacterial lipopeptide compound of bacillus subtilis when ammonium sulfate (mass fraction is 20%), and it can be seen from fig. 1 that the concentrations of ammonium sulfate and ethanol have certain effect on the yield of the antibacterial lipopeptide compound, wherein when the mass fraction of ammonium sulfate is 20% and the volume fraction of ethanol is 35%, the optimal extraction system is provided, and the extraction rate of the antibacterial lipopeptide compound reaches 91.78%.
Example 2
(1) Fermenting and culturing bacillus subtilis by using a fermentation medium to obtain bacillus subtilis fermentation liquor, wherein the fermentation medium contains the following components per liter: 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of dipotassium hydrogen phosphate and the balance of water, wherein the pH value of the fermentation medium is 7.0; centrifuging the bacillus subtilis fermentation liquor at 8000rpm for 10min, removing the precipitated thallus, and collecting the supernatant for later use;
(2) and (2) adding a small amount of ammonium sulfate into the fermentation supernatant prepared in the step (1) for multiple times according to the proportion, magnetically stirring until the ammonium sulfate is completely dissolved, and slowly adding ethanol in a flowing manner to ensure that the total volume of the aqueous two-phase extraction system is 100 mL. Wherein the mass fraction of ammonium sulfate is 25%, the volume fractions of ethanol are 20%, 25%, 30%, 35% and 40%, respectively, a double-aqueous-phase extraction system is established, the pH of the double-aqueous-phase extraction system is adjusted to 7, magnetic stirring is carried out for 10min at room temperature, standing and phase splitting are carried out for 15min, and the antibacterial lipopeptide compound is dissolved in the upper phase; separating to remove the lower salt solution and retaining the upper phase extract.
(3) Adding 3 times of ethanol into the upper phase extraction liquid obtained in the step (2), and precipitating ammonium sulfate; precipitating ammonium sulfate, filtering, rotary evaporating the filtrate at 60 deg.C and 0.1Mpa under reduced pressure, and evaporating ethanol and water to obtain Bacillus subtilis antibacterial lipopeptide compound product.
FIG. 2 shows the effect of different ethanol volume fractions on the extraction rate of the antibacterial lipopeptide compound from Bacillus subtilis (note: error bars represent standard deviations (n ═ 3)) when ammonium sulfate (mass fraction: 25%) is present, and it can be seen from FIG. 2 that the concentrations of ammonium sulfate and ethanol have a certain effect on the yield of the antibacterial lipopeptide compound, wherein the extraction rate of the antibacterial lipopeptide compound is 87.12% when the mass fraction of ammonium sulfate is 25% and the mass fraction of ethanol is 30% in the optimum extraction system.
Example 3
(1) Fermenting and culturing bacillus subtilis by using a fermentation medium to obtain bacillus subtilis fermentation liquor, wherein the fermentation medium contains the following components per liter: 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of dipotassium hydrogen phosphate and the balance of water, wherein the pH value of the fermentation medium is 7.0; centrifuging the bacillus subtilis fermentation liquor at 8000rpm for 10min, removing the precipitated thallus, and collecting the supernatant for later use;
(2) and (2) adding a small amount of ammonium sulfate into the fermentation supernatant prepared in the step (1) for multiple times according to the proportion, magnetically stirring until the ammonium sulfate is completely dissolved, and slowly adding ethanol in a flowing manner to ensure that the total volume of the aqueous two-phase extraction system is 100 mL. Wherein the mass fraction of ammonium sulfate is 30%, the volume fractions of ethanol are 20%, 25%, 30%, 35% and 40%, respectively, a double-aqueous-phase extraction system is established, the pH of the double-aqueous-phase extraction system is adjusted to 7, magnetic stirring is carried out for 10min at room temperature, standing and phase splitting are carried out for 15min, and the antibacterial lipopeptide compound is dissolved in the upper phase; separating to remove the lower salt solution and retaining the upper extraction liquid;
(3) adding 3 times of ethanol into the upper phase extraction liquid obtained in the step (2), and precipitating ammonium sulfate; precipitating ammonium sulfate, filtering, rotary evaporating the filtrate at 60 deg.C and 0.1Mpa under reduced pressure, and evaporating ethanol and water to obtain Bacillus subtilis antibacterial lipopeptide compound product.
Fig. 3 shows the effect of different ethanol volume fractions on the extraction rate of the bacilus subtilis antimicrobial lipopeptide compound when ammonium sulfate (30% by mass) (note: error bars represent standard deviation (n ═ 3)), and fig. 3 shows that the concentrations of ammonium sulfate and ethanol have certain effect on the yield of the antimicrobial lipopeptide compound, wherein the extraction rate of the antimicrobial lipopeptide compound is 83.86 when the mass fraction of ammonium sulfate is 30% and the volume fraction of ethanol is 25% in the optimal extraction system.
From the extraction rates of the antimicrobial lipopeptide compounds obtained in examples 1, 2 and 3, it is found that the extraction rate of the antimicrobial lipopeptide compound is the highest when the mass fraction of ammonium sulfate is 20% and the volume fraction of ethanol is 35% in the optimum extraction system in the same manner as in the other bars.
Example 4
(1) Fermenting and culturing bacillus subtilis by using a fermentation medium to obtain bacillus subtilis fermentation liquor, wherein the fermentation medium contains the following components per liter: 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of dipotassium hydrogen phosphate and the balance of water, wherein the pH value of the fermentation medium is 7.0; centrifuging the bacillus subtilis fermentation liquor at 8000rpm for 10min, removing the precipitated thallus, and collecting the supernatant for later use;
(2) and (2) adding a small amount of ammonium sulfate into the fermentation supernatant prepared in the step (1) for multiple times according to the proportion, magnetically stirring until the ammonium sulfate is completely dissolved, and slowly adding ethanol in a flowing manner to ensure that the total volume of the whole aqueous two-phase extraction system is 100 mL. Wherein the mass fraction of the ammonium sulfate is 20 percent, the volume fraction of the ethanol is 35 percent respectively, and a double aqueous phase extraction system is established;
(3) adjusting the pH value of the ethanol/ammonium sulfate double-aqueous phase extraction system prepared in the step (2) to 2-8, magnetically stirring for 10min, standing for phase separation for 15min, dissolving the antibacterial lipopeptide compounds in the upper phase, separating liquid to remove the lower-phase salt solution, and retaining the upper-phase extraction liquid;
(4) adding 3 times of ethanol into the upper phase extraction liquid obtained in the step (3) to precipitate ammonium sulfate; precipitating ammonium sulfate, filtering, rotary evaporating the filtrate at 60 deg.C and 0.1Mpa under reduced pressure, and evaporating ethanol and water to obtain Bacillus subtilis antibacterial lipopeptide compound product.
Fig. 4 shows the effect of different pH two-aqueous phase extraction systems on the extraction rate of the antibacterial lipopeptide compounds of bacillus subtilis (note: error line represents standard deviation (n ═ 3)), and fig. 4 shows that the pH of the two-aqueous phase extraction system has a certain effect on the yield of the antibacterial lipopeptide compounds, wherein the optimal extraction system is the one when the pH of the whole system is 6, and the extraction rate of the antibacterial lipopeptide compounds is 94.34%.
Therefore, by combining examples 1, 2, 3 and 4, it is understood that: under the same conditions of other strips, when the mass fraction of ammonium sulfate is 20%, the volume fraction of ethanol is 35%, and the pH value is 6.0, the optimal aqueous two-phase extraction system is provided, and the extraction rate of the antibacterial lipopeptide compound is the highest.
Example 5
(1) Fermenting and culturing bacillus subtilis by using a fermentation medium to obtain bacillus subtilis fermentation liquor, wherein the fermentation medium contains the following components per liter: 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of dipotassium hydrogen phosphate and the balance of water, wherein the pH value of the fermentation medium is 7.0; centrifuging the bacillus subtilis fermentation liquor at 8000rpm for 10min, removing the precipitated thallus, and collecting the supernatant for later use;
(2) and (2) adding a small amount of ammonium sulfate into the fermentation supernatant prepared in the step (1) for multiple times according to the proportion, magnetically stirring until the ammonium sulfate is completely dissolved, and slowly adding ethanol in a flowing manner to ensure that the total volume of the whole aqueous two-phase extraction system is 100 mL. Wherein the mass fraction of the ammonium sulfate is 20 percent, the volume fraction of the ethanol is 35 percent respectively, and a double aqueous phase extraction system is established;
(3) adjusting the pH value of the ethanol/ammonium sulfate aqueous two-phase extraction system prepared in the step (2) to 6, magnetically stirring for 10min, standing for phase separation for 15min, dissolving the antibacterial lipopeptide compound in the upper phase, separating liquid to remove the lower-phase salt solution, and retaining the upper-phase extraction liquid;
(4) adding 3 times of ethanol into the upper phase extraction liquid obtained in the step (3) to precipitate ammonium sulfate; precipitating ammonium sulfate, filtering, rotary evaporating the filtrate at 60 deg.C and 0.1Mpa under reduced pressure, and evaporating ethanol and water to obtain Bacillus subtilis antibacterial lipopeptide compound product, wherein the extraction rate of antibacterial lipopeptide compound in this example is 94.34%.
Further, in order to compare the advantages of the method for extracting antibacterial lipopeptide compounds of the present invention with the existing extraction method, example 5 is taken as an example, and comparative example 1 is designed for comparison.
Comparative example 1
The existing method for extracting the antibacterial lipopeptide compound by acidification and precipitation comprises the following steps: taking 1L of fermentation supernatant, adjusting pH of the fermentation supernatant to 2.0 with 6moL/L diluted hydrochloric acid, stirring well, standing at 4 deg.C for 12h, centrifuging at 8000rpm for 10min, and collecting precipitate; and leaching with methanol, filtering to obtain filtrate, performing reduced pressure rotary evaporation at 40 ℃, drying, and weighing to obtain the antibacterial lipopeptide compound product.
The results of comparing the quality of the extracted antibacterial lipopeptide compounds of example 5 with those of comparative example 1 are shown in Table 1.
TABLE 1
As can be seen from Table 1, the yield of the antibacterial lipopeptide compound extracted by the acidification precipitation method is 0.61g/L, and the yield of the antibacterial lipopeptide compound extracted by the ethanol/ammonium sulfate two-aqueous-phase system is 0.93g/L, so that the yield is improved by 52.46%.
And (3) carrying out high performance liquid chromatography analysis on the bacillus subtilis antibacterial lipopeptide compound extracted in the example 5:
(1) 10mg of the antimicrobial lipopeptide compound product was taken, 1mL of chromatographic grade methanol was added to dissolve it completely, 700. mu.L of ultrapure water was added, and the mixture was passed through a 0.22 μm organic filter.
(2) The detection conditions of the antibacterial lipopeptide compound by high performance liquid chromatography are as follows: a C18 (4.6X 250mm Unit C18 column) column was used, the mobile phase was acetonitrile and water, the detection wavelength was 200nm, and the flow rate was 1 mL/min. A gradient elution mode was used in which acetonitrile was gradually increased from 5% to 95%.
The high performance liquid chromatogram of the two aqueous phase extraction of the antibacterial lipopeptide compound of bacillus subtilis is shown in figure 5, and as can be seen from figure 5, the absorption peak of the high performance liquid chromatogram within 0-4 min is a solvent peak; two strong absorption peaks appear within 16-20 min, and the main component of the two strong absorption peaks is identified to be a C14-C15 homologue of bacillomycin D in iturin through LC-MS analysis; more absorption peaks appear within 23-28 min, and are mainly identified as lysobactin, camelina and iturin. The peak area normalization method is used for calculating to obtain that the peak group of the lipopeptide compound accounts for more than 90% of the total area, and the peak group of the bacillomycin D accounts for more than half of the total peak group area of the lipopeptide compound, so that further separation and purification of the bacillomycin D are facilitated.
In order to verify the inhibition effect of the extracted antibacterial lipopeptide compound of the bacillus subtilis on different indicator bacteria, the antibacterial spectrum is measured as follows:
10mg of the antimicrobial lipopeptide compound prepared in example 5 is weighed and dissolved in sterile water to prepare 10mg/mL antimicrobial lipopeptide solution for later use. Common pathogenic bacteria in the processes of food and aquaculture are selected as indicator bacteria, and the antibacterial activity of the antibacterial lipopeptide compound is measured by adopting an oxford cup method. The indicator bacteria include gram-negative bacteria, gram-positive bacteria and molds. The inhibitory effect of the antimicrobial lipopeptide compounds on different indicator bacteria is shown in table 2.
TABLE 2
| Indicator bacterium | Species of | Bacteriostatic effect |
| Listeria monocytogenes (Listeria monocytogenes) | Gram-negative bacteria | + |
| Escherichia coli (Escherichia coli) | Gram-negative bacteria | + |
| Shewanella putrefaction (Shewanella putrefacesens) | Gram-negative bacteria | +++ |
| Pseudomonas aeruginosa (Pseudomonas aeruginosa) | Gram-negative bacteria | +++ |
| Vibrio parahaemolyticus (Vibro parahaemolyticus) | Gram-negative bacteria | +++ |
| Brilliant vibrio (Vibro spleendidus) | Gram-negative bacteria | ++ |
| Micrococcus lysodeikticus (Micrococcus lysodeikticus) | Gram-positive bacteria | +++ |
| Staphylococcus aureus (Staphylococcus aureus) | Gram-positive bacteria | + |
| Bacillus cereus (Bacillus cereus ATCC 14579) | Gram-positive bacteria | - |
| Aspergillus flavus (Aspergillus flavus) | Mould fungus | +++ |
In table 2: -, no bacteriostatic effect; +, the diameter of the inhibition zone is less than 10 mm; + +, which represents that the diameter of the inhibition zone is 10-15 mm; and +++, the diameter of the inhibition zone is larger than 15 mm.
As can be seen from Table 2, the antibacterial lipopeptide compound has good inhibitory effect on Shewanella putrefaciens, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Micrococcus muralis and Aspergillus flavus, and shows that the antibacterial lipopeptide compound has application prospects in the fields of food, feed, aquaculture and the like.
The technical solution of the present invention is not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solution of the present invention fall within the protection scope of the present invention.
Claims (6)
1. A method for extracting an antibacterial lipopeptide compound in a bacillus subtilis fermentation broth is characterized by comprising the following steps:
s1, fermenting and culturing the bacillus subtilis, and centrifuging the bacillus subtilis fermentation liquor to obtain a bacillus subtilis fermentation supernatant;
s2, preparing a double-aqueous-phase extraction system by using ethanol and ammonium sulfate, wherein the volume fraction of the ethanol in the double-aqueous-phase extraction system is 20-40%, and the mass fraction of the ammonium sulfate is 10-30%;
s3, adding the bacillus subtilis fermentation supernatant into a double-aqueous-phase extraction system to obtain a mixed solution, adjusting the pH of the double-aqueous-phase extraction system to 2-8, wherein the volume fraction of the bacillus subtilis fermentation supernatant in the mixed solution is 50-70%, uniformly stirring and mixing, standing, extracting, splitting phases, and collecting an upper phase extraction liquid;
s4, adding ethanol with the volume 1-3 times of that of the upper phase extraction liquid, separating out ammonium sulfate precipitate, filtering the ammonium sulfate precipitate, and collecting filtrate;
s5, drying and concentrating the filtrate obtained in the step S4 by using a rotary evaporator to obtain the bacillus subtilis antibacterial lipopeptide compound.
2. The method for extracting lipopeptide compounds from a bacillus subtilis fermentation broth according to claim 1, wherein the fermentation medium used in the fermentation culture of the bacillus subtilis in step S1 contains the following components per liter: 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of dipotassium hydrogen phosphate and the balance of water, wherein the pH value of the fermentation medium is 7.0.
3. The method for extracting lipopeptide compounds from bacillus subtilis fermentation broth according to claim 1, wherein in the step S1, the centrifugation of the bacillus subtilis fermentation broth is performed by solid-liquid separation in a centrifuge with 8000r/min of rotation for 10 min.
4. The method for extracting lipopeptide compounds from Bacillus subtilis fermentation broth as claimed in claim 1, wherein in step S3, the extraction temperature is 20-40 ℃, the stirring time is 10-20min, the stirrer rotation speed is 100-200rpm, and the standing phase separation time is 10-30 min.
5. The method for extracting lipopeptide compounds from a fermentation broth of Bacillus subtilis according to claim 1, wherein the rotary evaporator temperature is 60 ℃ and the pressure is 0.1MPa in step S5.
6. The method for extracting lipopeptide compounds from bacillus subtilis fermentation broth according to claim 1, wherein in step S2, the volume fraction of ethanol in the aqueous two-phase extraction system is 35%, the mass fraction of ammonium sulfate is 20%, and the pH of the aqueous two-phase extraction system is 6.
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