CN112079899B - Method for separating antibacterial lipopeptide from bacillus amyloliquefaciens fermentation liquor - Google Patents
Method for separating antibacterial lipopeptide from bacillus amyloliquefaciens fermentation liquor Download PDFInfo
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Abstract
The invention discloses a method for separating antibacterial lipopeptide from bacillus amyloliquefaciens fermentation liquor, which comprises the steps of centrifuging the bacillus amyloliquefaciens fermentation liquor, foaming obtained fermentation clear liquor, collecting and cooling foam, and then carrying out acid precipitation, extraction, concentration and drying on foamless clear liquor generated by the foam to obtain a lipopeptide finished product. The method has the advantages of simple separation process, high yield and high purity of the lipopeptide.
Description
Technical Field
The invention relates to crude separation of antibacterial lipopeptide in microbial fermentation liquor, in particular to a simple, convenient, effective and low-cost treatment method of bacillus amyloliquefaciens fermentation liquor.
Background
Chemical preservatives used in the food industry, such as benzoic acid, sorbitol, etc., affect human health and also pollute the natural environment. Meanwhile, the wide and large-scale use of the antibiotics causes the obvious reduction of the inhibition effect of the antibiotics on pathogenic bacteria. With the increase of the drug resistance of pathogenic bacteria, development of new antibacterial substances is required. Lipopeptide compounds mainly exhibit properties including antibacterial, anti-hemagglutination, antiviral, anti-tumor, anti-mycoplasma, antiprotozoal, and stronger surface activity, and are resistant to pH, heat, lower in critical micelle concentration, capable of chelating metal ions, easily degradable, and not prone to generate drug resistance, etc.
It was found for the first time by Arima et al that bacillus subtilis (b.subtilis) can produce antibacterial lipopeptide Surfactin, and further studies found that bacillus amyloliquefaciens (b.amyloliquefaciens), bacillus coagulans (b.coagulars), bacillus pumilus (b.pumilus), bacillus cereus (b.cereus), bacillus thuringiensis (b.lhurrirsis), bacillus licheniformis (b.izchemforms), bacillus circulans (b.circulans), and bacillus paenibacillus (p.aesibacillus) can produce lipopeptide compounds. Although there are many strains producing antibacterial lipopeptide, the production of antibacterial lipopeptide reported at present is difficult to meet the requirement of commercial production, and the production cost of separation and purification in the extraction and purification process of antibacterial lipopeptide accounts for more than 60% of the total fermentation cost, so that the production and application of antibacterial lipopeptide in industrial scale are limited.
The extraction method of antibacterial lipopeptides varies depending on the fermentation mode, physicochemical properties and lipopeptide species. Meanwhile, the separation and purification play a key role in ensuring the quality and determining the economic benefit in the whole fermentation production process. Currently, the commonly used methods for isolating antimicrobial lipopeptides include precipitation and organic solvent extraction. Wherein, the precipitation method mainly comprises acid precipitation, salting out, organic solvent precipitation and the like, but the antibacterial lipopeptide product obtained by the precipitation method has lower purity and more impurities and does not meet the industrial application standard. The organic solvent extraction method uses chloroform and the like to extract the antibacterial lipopeptide, so that the extracted product inevitably contains residual toxic solvent, has certain potential safety hazard and simultaneously causes the reduction of the biological activity of the antibacterial lipopeptide.
In combination with the structural characteristics of the antibacterial lipopeptide and the advantages and disadvantages of different separation methods, two or more related separation and purification technologies are generally combined and applied to the extraction of the antibacterial lipopeptide at present, so that the aims of improving the yield and reducing the cost are fulfilled. For example, chinese patent CN107964557A discloses a fermentation method for increasing the yield of bacillus antibacterial lipopeptide, wherein the antibacterial lipopeptide separation method comprises: centrifuging, performing acid precipitation (hydrochloric acid, pH2.0, 4 deg.C overnight) on the fermented supernatant, centrifuging again, and collecting precipitate; freeze drying the precipitate, leaching with methanol, and concentrating the methanol extract under reduced pressure to obtain antibacterial lipopeptide crude extract; it also adds a small amount of fatty acids during fermentation to increase the yield of lipopeptides. However, the separation process of the antibacterial lipopeptide actually involves repeated leaching and concentration, which not only is complex and time-consuming to operate, but also does not lead to high purity and yield of the lipopeptide extract.
Disclosure of Invention
The invention aims to provide a method for separating antibacterial lipopeptide from bacillus amyloliquefaciens fermentation liquor.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for separating antibacterial lipopeptide from thallus fermentation liquor comprises the following steps:
1) removing thalli in the fermentation liquor to obtain fermentation clear liquid;
2) allowing the fermented clear liquid to generate foam under the action of heat and/or machinery, separating the generated foam, and cooling the foam to be separated to obtain foamless clear liquid generated by the foam;
3) adjusting the pH value of the foamless clear liquid to be acidic and then standing; then separating the generated precipitate;
4) extracting the precipitate with alcohol solvent, and filtering (removing insoluble impurities) to obtain filtrate;
5) and concentrating the filtrate and drying to obtain a yellow-brown powdery antibacterial lipopeptide finished product.
Preferably, the step 1) specifically comprises the following steps: and centrifuging the thallus in the fermentation liquor, and collecting supernatant.
Preferably, the fermentation strain employed in the fermentation broth is a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain (e.g., strain wxy-b 3).
Preferably, the fermentation conditions of the strain are as follows: the shaking culture is carried out for more than or equal to 12 hours at the temperature of less than or equal to 48 ℃.
Preferably, the step 2) specifically comprises the following steps: foaming the fermentation clear liquid in a rotating container (for example, a laboratory can adopt a rotary evaporator) under the conditions that the temperature is 37-45 ℃, the pressure is 0.08-0.09 MPa and the rotating speed is 100-150 rpm, and collecting the foam; and standing the collected foam for 10-30 min at 4-10 ℃.
Preferably, in the step 3), 4-6 mo1/L HCL is used for adjusting the pH value of the foamless clear liquid to 1.5-2.5; the standing temperature is 4-10 ℃, and the standing time is 8-12 h.
Preferably, in the step 4), the precipitate is dissolved in methanol or ethanol with the volume of 5-10 per mill of the fermentation liquid, and then extracted for 2-3 hours, and an organic filter membrane with the diameter of 0.2-0.22 μm is adopted for filtration.
Preferably, the step 5) specifically comprises the following steps: evaporating the filtrate to dryness, dissolving the filtrate again by using water with the volume of 5-10 per mill of the fermentation liquor, prefreezing the filtrate at the temperature of-20 to-80 ℃ for 1-2 h, and then carrying out vacuum freeze drying.
The invention has the beneficial effects that:
the invention adopts a separation scheme of foaming, acid precipitation and extraction on the fermentation clear liquid to process the antibacterial lipopeptide fermentation liquid of thalli (such as bacillus amyloliquefaciens), simplifies the separation process of lipopeptide finished products, and ensures that the yield of the lipopeptide reaches more than 85 percent and the purity of the lipopeptide finished products reaches more than 67 percent.
Furthermore, the bacterial strain wxy-b3 adopted by the invention has high yield of the antibacterial lipopeptide, and the fermentation liquor is easy to process.
Furthermore, the invention utilizes the supernatant obtained by centrifuging the fermentation liquor to generate foam, so that most of lipopeptide generated by fermentation is attached to the foam, the lipopeptide is simply, conveniently and quickly enriched, and the concentration time is saved.
Drawings
FIG. 1 shows the results of liquid chromatography analysis of the lipopeptide preparation.
FIG. 2 is a schematic diagram of the structure of a portion of the lipopeptide component of the lipopeptide preparation; wherein, (a) Surfactin, (B) Fengycin B, (c) Fengycin A; n of Rn means the number of C atoms in the fatty acid chain.
Detailed Description
The present invention will be described in further detail with reference to examples. The examples are for better understanding of the technical solutions of the present invention by those skilled in the art, and are not intended to limit the scope of the present invention.
Preparation of antibacterial lipopeptide fermentation liquor of bacillus amyloliquefaciens
1. Fermentation strains and media
Taking Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) wxy-b3 (strain wxy-b3 for short) as a fermentation strain; the strain wxy-b3 has been preserved in China general microbiological culture Collection center (CGMCC for short, address No. 3 Xilu No. 1 Beijing, Chaoyang district) in 2020 at 06.08, and the preservation number is CGMCC No. 20041.
LB liquid medium formula: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1L of distilled water and pH 7.0.
2. Preparation of seed solution of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) wxy-b3
The preserved bacillus amyloliquefaciens wxy-b3 was inoculated into 30mL of LB liquid medium and cultured at 37 ℃ and 200rpm for 12 hours to obtain an activated seed solution.
3. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) wxy-b3 fermentation
Inoculating the activated seed solution into a 1L triangular flask containing 500mL LB liquid culture medium according to the proportion of 3%, and culturing at 37 ℃ and 200rpm for 48h to obtain fermentation liquor.
Treatment of Bacillus amyloliquefaciens fermentation liquor
Centrifuging the fermentation broth at 4 deg.C and 8000rpm for 10min by using a refrigerated high speed centrifuge, removing thallus, and collecting supernatant.
In order to enrich lipopeptide, 500mL of supernatant is placed in a distillation flask of a rotary evaporator, high-speed (100rpm) rotary evaporation is carried out at 42 ℃ and 0.08MPa, the foam of fermentation liquor generated after foaming is sucked back into a liquid receiving flask (the foam can be sucked back automatically under high-speed rotation), and the rotary evaporation is finished until no foam is generated. The collected foam was then allowed to stand in a refrigerator at 4 ℃ for 10min to become a foamless clear solution.
To the clear foamless solution was added 6mo1/L HCl to adjust the pH to 2.0 and the mixture was allowed to stand overnight in a refrigerator at 4 ℃.
The precipitate formed during standing was collected by centrifugation at 8000rpm for 10min at 4 ℃ and 5mL of methanol was added to a 50mL centrifuge tube to dissolve and extract the precipitate for 2 hours, and the resulting extract was filtered through a 0.22 μm organic filter (nylon 66) to collect the filtrate (i.e., a clear filtrate).
And (2) concentrating the filtrate in vacuum by using a rotary evaporator under the conditions of 37-42 ℃ and 0.08-0.09 MPa, decompressing and evaporating to dryness, dissolving a solid film layer formed after evaporation to dryness by using equivalent distilled water (5mL), freezing the dissolved aqueous solution in a refrigerator at-80 ℃ for 2h in advance, and then performing vacuum freeze drying to obtain a brown yellow powdery lipopeptide finished product.
(III) detection of lipopeptide finished product
And performing liquid chromatography and mass spectrometry on the lipopeptide finished product, and comparing with the analysis result of the fermentation liquor. The analysis results show (see fig. 1): the invention combines the separation methods of foaming, acid precipitation and methanol extraction to process the antibacterial lipopeptide fermentation liquor of the bacillus amyloliquefaciens, so that the lipopeptide yield reaches more than 85 percent, and the purity of the lipopeptide finished product reaches more than 67 percent (because the lipopeptide is adsorbed by foam generated by heating and high-speed rotating and oscillating fermentation supernatant, the foam is collected for lipopeptide separation, the processing time and cost can be obviously reduced, the complicated processing steps are reduced, and the purity and the yield are improved).
Meanwhile, according to the analysis result, the lipopeptide compound contained in the lipopeptide finished product has the following composition and functions: the finished lipopeptide product contains lipopeptide compounds of two families, namely Surfactin and Fengycin (figure 2). The Surfactin family lipopeptide has a good antibacterial effect on bacteria, and the Fengycin family lipopeptide has an inhibitory effect on filamentous fungi. The fermentation liquor is applied to carry out bacteriostatic tests on various food-borne pathogenic bacteria and agricultural pathogenic fungi, and the bacteriostatic activity of the fermentation liquor on escherichia coli, bacillus cereus, wheat scab pathogenic bacteria Fusarium graminearum, potato dry rot pathogenic Fusarium sambucinum and apple rot mould Cytospora mandshurica is found to be better.
In a word, the invention obviously reduces the treatment cost of the bacillus amyloliquefaciens fermentation liquid, has quick, simple and convenient treatment process, is suitable for continuous production, and the lipopeptide finished product obtained by treatment has high purity, has broad-spectrum antibacterial and fungal pathogen resisting functions, has application potential in the fields of biological control of crops and food, and has huge industrial production value.
Claims (6)
1. A method for separating antibacterial lipopeptide from thallus fermentation liquor is characterized in that: the method comprises the following steps:
1) removing thalli in the fermentation liquor to obtain fermentation clear liquid; the fermentation strain adopted by the fermentation liquid is a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain;
2) allowing the fermented clear liquid to foam under the action of heat and machinery, separating the generated foam, and cooling the separated foam until a foam-free clear liquid is generated from the foam;
the step 2) specifically comprises the following steps: foaming the fermentation clear liquid in the rotary container under the conditions that the temperature is 37-45 ℃, the pressure is 0.08-0.09 MPa and the rotating speed is 100-150 rpm, and collecting the foam; standing the collected foam for 10-30 min at 4-10 ℃;
3) adjusting the pH value of the foamless clear liquid to be acidic and then standing; then separating the generated precipitate;
4) extracting the precipitate with alcohol solvent, and filtering to obtain filtrate;
5) and concentrating the filtrate and drying to obtain a lipopeptide finished product, wherein the lipopeptide finished product contains lipopeptide compounds of two families, namely Surfactin and Fengycin.
2. The method of claim 1, wherein the method comprises the steps of: the step 1) specifically comprises the following steps: and centrifuging the thallus in the fermentation liquor, and collecting supernatant.
3. The method of claim 1, wherein the method comprises the steps of: the fermentation conditions of the strain are as follows: the shaking culture is carried out for more than or equal to 12 hours at the temperature of less than or equal to 48 ℃.
4. The method of claim 1, wherein the method comprises the steps of: in the step 3), adjusting the pH value of the foamless clear liquid to 1.5-2.5 by using 4-6 mo1/L HCL; the standing temperature is 4-10 ℃, and the standing time is 8-12 h.
5. The method of claim 1, wherein the method comprises the steps of: in the step 4), the precipitate is dissolved in methanol or ethanol with the volume of 5-10 per mill of the fermentation liquor, then extracted for 2-3 h, and an organic filter membrane with the diameter of 0.2-0.22 mu m is adopted for filtration.
6. The method of claim 1, wherein the method comprises the steps of: the step 5) specifically comprises the following steps: evaporating the filtrate to dryness, dissolving the filtrate again by using water with the volume of 5-10 per mill of the fermentation liquor, prefreezing the filtrate at the temperature of-20 to-80 ℃ for 1-2 h, and then carrying out vacuum freeze drying.
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| CN104321422A (en) * | 2012-01-27 | 2015-01-28 | Gfs澳大利亚股份有限公司 | Method of producing biosurfactants |
| US20180020676A1 (en) * | 2014-12-29 | 2018-01-25 | Fmc Corporation | Bacillus velezensis rti301 compositions and methods of use for benefiting plant growth and treating plant disease |
| CN108841903A (en) * | 2018-06-29 | 2018-11-20 | 董骅 | A kind of method of strain fermentation production biosurfactant |
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