CN109182170B - Surfactin high-yield strain and application thereof - Google Patents
Surfactin high-yield strain and application thereof Download PDFInfo
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- CN109182170B CN109182170B CN201811026114.8A CN201811026114A CN109182170B CN 109182170 B CN109182170 B CN 109182170B CN 201811026114 A CN201811026114 A CN 201811026114A CN 109182170 B CN109182170 B CN 109182170B
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a high-yield Surfactin strain and application thereof. The strain is bacillus subtilis LM34S07 which is preserved in China general microbiological culture Collection center in 2018, 4 and 18 months, and the preservation number is CGMCC 15620. The Surfactin produced by the strain has the outstanding advantages of high synthesis rate and high yield, can be applied to the fields of microbial feed, feed additives, microbial fertilizers, plant disease control, food preservation and preservation, cosmetics, medicines, environmental protection and the like, and has huge application potential and economic value.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus subtilis strain for high yield of Surfactin and application thereof.
Background
Surfactin, the name of Chinese surfactant, is a secondary metabolite synthesized by bacillus bacteria through a non-ribosome pathway, and the basic structure is a cyclic lipopeptide formed by connecting 1 beta-hydroxy fatty acid and a polypeptide containing 7 amino acids through an inner ester bond, and one or a plurality of-CH (CH-CH) with different molecular weights can be generated due to different lengths of carbon chains in fatty acids2A homologue of (a).
The Surfactin has the outstanding characteristics of large surface activity and good emulsifying and foaming performances, and can remarkably reduce the surface tension of water and other liquid-liquid interfacial tension; in addition, Surfactin has been demonstrated to have antimicrobial activities such as antibacterial, antifungal, antiviral, anti-mycoplasma, antitumor, etc.; meanwhile, the Surfactin is a cyclic lipopeptide formed by connecting fatty acid and polypeptide chain, and has the excellent characteristics of easy biodegradation, no environmental pollution and the like.
Because of the outstanding characteristics of the cyclic lipopeptide Surfactin, the Surfactin and microbial strains for producing the Surfactin are developed and applied to various fields such as microbial feeds, feed additives, microbial fertilizers, plant disease control, food processing, medicines, cosmetics, environmental protection, oil exploitation and the like, and are favored by researchers, industrial developers and the like at home and abroad.
However, the Surfactin production cost is high due to the low yield of the Surfactin of the existing microbial strains, and the Surfactin price is high, so that the application range, large-scale production and commercial development and application of the Surfactin are greatly limited.
Disclosure of Invention
The invention provides a high-yield Surfactin strain, a production method and application thereof, aiming at solving the problems that the existing Surfactin has low yield and cannot meet the requirement of industrial production.
The invention provides a Surfactin high-producing strain, which is bacillus subtilis LM34S07 (Bacillus subtilis)Bacillus subtilis LM34S07), which has been deposited in China general microbiological culture Collection center at 18.4.2018 with the preservation number of CGMCC 15620 and the address of the institute of microbiology of China academy of sciences No. 3, West Lu No. 1 Hospital, North Cheng, the Chaoyang district, Beijing.
The Surfactin high-yield strain Bacillus subtilis LM34S07 provided by the invention is obtained by carrying out compound mutagenesis breeding on an original strain Bacillus subtilis through nitrosoguanidine and He-Ne laser irradiation.
The bacillus subtilis LM34S07 grows well on the nutrient agar plate, the bacterial colony is milky white and opaque, and the bacterial colony is round and has irregular edges; rod-shaped, gram-positive, spore-forming, oval cells.
The strain provided by the invention belongs to internationally recognized safe strains, is listed as one of commercially applicable microbial strains by the American FDA and the environmental protection agency, and is also one of primary strains which are specified by the Ministry of agriculture in China and do not need to be subjected to safety identification.
The invention provides a method for producing Surfactin by using bacillus subtilis LM34S07, which comprises the following steps: selecting a strain LM34S07 loop, inoculating the strain into a 250mL triangular flask filled with 30-100mL beef extract peptone culture medium, and performing shake culture at 30-35 ℃ and at 150-; inoculating the seed solution into a 500mL triangular flask containing 100-200mL fermentation medium according to the inoculation amount of 3-10% (v/v), and performing shake culture at 30-35 ℃ and 200rpm for 28-48h to obtain a fermentation solution; the fermentation medium comprises glucose 15-35g, glutamic acid 3-8g, yeast extract 1-4g, potassium dihydrogen phosphate 0.7-1.1g, potassium chloride 0.3-0.6g, magnesium sulfate heptahydrate 0.3-0.6g, copper sulfate 0.1-0.2mg, ferrous sulfate heptahydrate 0.1-0.2mg, manganese sulfate 3-10mg, water 1L, pH7.0, and sterilizing at 115 deg.C for 30 min.
The invention provides application of bacillus subtilis LM34S07 or a fermentation product thereof or a microbial inoculum containing bacillus subtilis LM34S07 in the fields of feed and agriculture.
The invention provides application of bacillus subtilis LM34S07 or a fermentation product thereof or a microbial inoculum containing bacillus subtilis LM34S07 in the fields of food, medicine and cosmetics.
The invention provides application of bacillus subtilis LM34S07 or a fermentation product thereof or a microbial inoculum containing bacillus subtilis LM34S07 in the field of environmental protection.
The invention has the beneficial effects that:
the invention provides a bacillus subtilis LM34S07 with high yield of Surfactin, the strain has high speed and high yield of Surfactin synthesis, and the Surfactin content of fermentation liquor fermented for 40h is up to 9.78 g/L; the strain LM34S07 is utilized to produce Surfactin, so that the production cost can be greatly reduced, the production efficiency is improved, and the method has a huge industrial application prospect.
Drawings
FIG. 1 Surfactin standard HPLC profile;
FIG. 2 HPLC chromatogram of a sample of methanol extract.
Detailed Description
In order that the invention may be more readily understood, the invention is further illustrated by the following examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1: compound mutation breeding of high-yield Surfactin strains
Selecting starting strains, inoculating the starting strains into a 50mL triangular flask containing 10mL beef extract peptone medium, shake-culturing at 33 ℃ and 175r/min for 16h, centrifuging at 4 ℃ and 8000r/min for 15min, collecting thallus, washing with phosphate buffer solution with pH of 7.0 for 3 times, adding a certain amount of phosphate buffer solution with pH of 7.0 to suspend the thallus, and obtaining bacterial suspension for compound mutagenesis (10)8cfu/mL); uniformly mixing 1mL of bacterial suspension and 1mL of nitrosoguanidine solution with the concentration of 180ug/mL in a small test tube, placing the mixture in a water bath at 33 ℃, irradiating the mixture by using a He-Ne laser (external cavity quasi-single mode He-Ne laser with the wavelength of 632.8nm and the irradiation power of 12mW) for 2min each time for 3 times, pouring the mixture into 48mL of sterile physiological saline after treating the mixture in the water bath for 12min, and stopping reaction; then, each dilution was applied to blood by a gradient dilution methodAnd (3) culturing on a plate at a constant temperature of 33 ℃ for 36h, selecting a colony with a large hemolytic cycle, transferring and storing, detecting the yield of the Surfactin of mutant strain fermentation liquid by using an HPLC (high performance liquid chromatography) method, and finally screening to obtain the Surfactin high-yield mutant strain LM34S 07.
Example 2: detection of Surfactin content
Centrifuging the fermentation broth at 4 deg.C and 12000rpm for 20min, collecting supernatant, adjusting pH of the supernatant to 2.0 with concentrated hydrochloric acid, standing overnight at 4 deg.C, centrifuging at 4 deg.C and 12000rpm for 20min, and collecting precipitate; adding 5mL of methanol into the precipitate, stirring for 30min on a magnetic stirrer, standing and extracting in a refrigerator at 4 ℃ for 12h, centrifuging at 4 ℃ and 12000rpm for 20min after extraction is finished, and collecting methanol extract; extracting repeatedly for 3 times, mixing methanol extractive solutions, filtering with 0.22um sterile microporous membrane, and storing at 4 deg.C.
Detecting the content of Surfactin by adopting HPLC; ZORBAX SB-C chromatographic column18(5 μm, 4.6X 250mm), column temperature 35 ℃, sample size 20 μ l, wavelength 205nm, flow rate 0.8mL/min, mobile phase A pure water (containing 0.1% TFA), mobile phase B acetonitrile (containing 0.1% TFA); the gradient elution conditions were: for 0-9 min, 40% of mobile phase A and 60% of mobile phase B; 9-20 min, 7% of mobile phase A and mobile phase B; HPLC (high performance liquid chromatography) shows that the HPLC peak time (figure 2) of the methanol extract sample is basically the same as the peak time (figure 1) of the Surfactin standard product, the regression equation of the standard curve of the Surfactin standard product is Y =0.08132X-41.76, and the correlation coefficient R is2=0.9937, substituting the HPLC peak area of the methanol extract sample into a regression equation to calculate the Surfactin concentration, and further converting the Surfactin content in the fermentation liquid.
Example 3: surfactin fermentation
The fermentation medium comprises 20g of glucose, 5g of glutamic acid, 4g of yeast extract, 1g of monopotassium phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate heptahydrate, 0.16mg of copper sulfate, 0.15mg of ferrous sulfate heptahydrate, 5mg of manganese sulfate, 1L of water, pH7.0 and sterilized at 115 ℃ for 30 min.
Selecting a strain LM34S07 loop, inoculating the strain into a 250mL triangular flask filled with 50mL beef extract peptone medium, and performing shake culture at 33 ℃ and 175rpm for 18h to obtain a seed solution; inoculating the seed solution into a 500mL triangular flask filled with 150mL fermentation medium according to the inoculation amount of 7% (v/v), and performing shake culture at 33 ℃ and 175rpm for 40h to obtain fermentation liquor; through detection, the content of Surfactin in the fermentation liquid is 9.78 g/L.
Example 4: surfactin fermentation
The fermentation medium comprises 35g of glucose, 3g of glutamic acid, 1g of yeast extract, 1.1g of monopotassium phosphate, 0.3g of potassium chloride, 0.6g of magnesium sulfate heptahydrate, 0.2mg of copper sulfate, 0.2mg of ferrous sulfate heptahydrate, 3mg of manganese sulfate, 1L of water, pH7.0 and is sterilized at 115 ℃ for 30 min.
Selecting a strain LM34S07 loop, inoculating the strain into a 250mL triangular flask filled with 100mL beef extract peptone medium, and performing shake culture at 35 ℃ and 200rpm for 24h to obtain a seed solution; inoculating the seed solution into a 500mL triangular flask filled with 200mL fermentation medium according to the inoculation amount of 5% (v/v), and performing shake culture at 35 ℃ and 200rpm for 48h to obtain fermentation liquor; through detection, the content of Surfactin in the fermentation liquor is 9.71 g/L.
Example 5: surfactin fermentation
The fermentation medium comprises glucose 15g, glutamic acid 8g, yeast extract 4g, potassium dihydrogen phosphate 0.7g, potassium chloride 0.6g, magnesium sulfate heptahydrate 0.3g, copper sulfate 0.1mg, ferrous sulfate heptahydrate 0.1mg, manganese sulfate 10mg, water 1L, pH7.0, and sterilizing at 115 deg.C for 30 min.
Selecting a strain LM34S07 loop, inoculating the strain into a 250mL triangular flask filled with 30mL beef extract peptone culture medium, and performing shake culture at 30 ℃ and 150rpm for 16h to obtain a seed solution; inoculating the seed solution into a 500mL triangular flask filled with 100mL fermentation medium according to the inoculation amount of 10% (v/v), and performing shake culture at 30 ℃ and 150rpm for 28h to obtain fermentation liquor; through detection, the content of Surfactin in the fermentation liquor is 9.66 g/L.
The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications within the scope that can be easily conceived by those skilled in the art without departing from the intended scope are also included in the present invention.
Claims (4)
1. A Surfactin high-producing strain is Bacillus subtilis LM34S07 (R) ((R))Bacillus subtilisLM34S07), which has been deposited with the general microorganisms of China Committee for culture Collection of microorganismsThe preservation number of the strain is CGMCC 15620, and the strain has high production rate and high yield of Surfactin.
2. Use of the Bacillus subtilis LM34S07 of claim 1 for the production of Surfactin.
3. The use of the bacillus subtilis LM34S07 of claim 1 for microbial feed, feed additives, microbial fertilizer, plant disease control.
4. The bacillus subtilis LM34S07 of claim 1, wherein the bacillus subtilis LM34S07 is applied to environmental protection and oil exploitation.
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| CN110904092A (en) * | 2019-12-09 | 2020-03-24 | 济南大学 | Lactobacillus brevis compound mutation breeding method for high yield of GABA |
| CN112980747A (en) * | 2021-04-30 | 2021-06-18 | 南京理工大学 | Bacillus subtilis for producing lipopeptide biosurfactant |
| CN113278543A (en) * | 2021-05-06 | 2021-08-20 | 华南农业大学 | Bacillus subtilis capable of producing surfactin in high yield and obtained through compound mutagenesis and fermentation method thereof |
Citations (3)
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| CN103898038A (en) * | 2014-03-28 | 2014-07-02 | 清华大学 | Engineering bacterium for highly expressing lipopeptide biosurfactant and application thereof |
| CN104830713A (en) * | 2015-04-09 | 2015-08-12 | 河南工业大学 | Bacillus thuringiensis for combined production of antimicrobial lipopeptide and gamma-polyglutamic acid |
| CN105602863A (en) * | 2015-12-14 | 2016-05-25 | 南京工业大学 | Bacillus subtilis strain capable of high yield of lipopeptide antibiotic and poly-gamma-glutamic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103898038A (en) * | 2014-03-28 | 2014-07-02 | 清华大学 | Engineering bacterium for highly expressing lipopeptide biosurfactant and application thereof |
| CN104830713A (en) * | 2015-04-09 | 2015-08-12 | 河南工业大学 | Bacillus thuringiensis for combined production of antimicrobial lipopeptide and gamma-polyglutamic acid |
| CN105602863A (en) * | 2015-12-14 | 2016-05-25 | 南京工业大学 | Bacillus subtilis strain capable of high yield of lipopeptide antibiotic and poly-gamma-glutamic acid |
Non-Patent Citations (1)
| Title |
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| 响应面法优化Bcillus subtilis抗菌脂肽发酵工艺条件;陈亮等;《食品科技》;20151231;第40卷(第4期);17-21 * |
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