CN112553276B - Production process for producing antibacterial peptide by using brevibacillus laterosporus - Google Patents

Production process for producing antibacterial peptide by using brevibacillus laterosporus Download PDF

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CN112553276B
CN112553276B CN202011468387.5A CN202011468387A CN112553276B CN 112553276 B CN112553276 B CN 112553276B CN 202011468387 A CN202011468387 A CN 202011468387A CN 112553276 B CN112553276 B CN 112553276B
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CN112553276A (en
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郄丽萍
张雪霞
胡卫国
任风芝
陈博
李丽红
安泰
杨鑫
马婕
段银箫
葛鹍鹏
朱京童
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North China Pharmaceutical New Drug R&d Co ltd
Cofco Nutrition and Health Research Institute Co Ltd
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Abstract

The invention discloses a fermentation process for producing antibacterial peptide by using brevibacillus laterosporus as a strain. The process improves the combination of fermentation organic nitrogen sources, and dynamically regulates and controls the fermentation process: fermenting for 10-20hr, adding organic nitrogen source, controlling dissolved oxygen, and regulating fermentation pH; controlling the foam at the later stage of fermentation; culturing for 12-25hr to obtain multi-component antibacterial peptide with antibacterial and antifungal activity, and fermenting to obtain fermentation yield of above 3500 ug/ml. Compared with the product before optimization, the yield of the antibacterial peptide is improved by 3 times, and the cost is greatly reduced.

Description

Production process for producing antibacterial peptide by using brevibacillus laterosporus
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation process for producing multi-component antibacterial peptide by using brevibacillus laterosporus.
Background
Antimicrobial peptides, also known as antimicrobial peptides and peptide antibiotics, are derived from a variety of organisms and are part of the natural defense system in the natural world. The antibacterial peptide is a micromolecule polypeptide with biological activity generated by induction in a living body, and most of the active polypeptides have the advantages of alkalinity, heat stability, wide PH tolerance range and broad-spectrum antibacterial property. Not only for gram-negative bacteria, but also on fungi, insects and certain viruses and tumors. Compared with antibiotics, the antibiotic has no harm to normal cells of human and animals, no side effect and no residue. Compared with the traditional antibiotics, the antibacterial agent has incomparable superiority, can not induce the generation of drug-resistant strains, is hopeful to become a new generation antibacterial agent after the national issue of the restriction order, and is the best choice for replacing antibiotics.
The Bacillus (Bacillus) has safe application history in food industry, especially the brevibacillus laterosporus, has the function of inhibiting various bacteria and fungi, has the characteristics of high safety, wide antimicrobial spectrum, good stability and no residue, and is a potential probiotic. Becomes a research hotspot in recent years, and has been widely used for food and food preservation and fresh-keeping; in the feed industry, the feed micro-ecological preparation has been approved by the Ministry of agriculture to be used for the cultivation of meat chicken, meat duck, pig and shrimp, and has extremely high safety; is used in the field of crop disease control to form green antibiotic-free agricultural production.
At present, the industrial process of the fermentation production of the antibacterial peptide has a plurality of problems, such as low strain yield, high cost, difficult separation of the antibacterial peptide and a culture medium, difficult control of the fermentation process due to bubbles generated in the fermentation process and the like. Therefore, each process of producing the antibacterial peptide by fermentation has a limitation on the yield and quality of the antibacterial peptide, for example, brevibacillus laterosporus is a bacterium, and the production period of the product is short, which has a limitation on the yield of the antibacterial peptide. CN 104004803 removes the feedback inhibition angle of the product by adding activated carbon to adsorb the antibacterial peptide product in the late stage of fermentation production, and increases the yield of the antibacterial peptide. CN 105255973 utilizes a magnetic field to promote the high yield of antibacterial peptide of bacillus amyloliquefaciens. Improving the yield and quality of the fermentation production of antimicrobial peptides requires a more industrially suitable process.
Disclosure of Invention
Aiming at the problems of low yield, difficult process control and the like in the conventional antibacterial peptide production fermentation process, the invention provides a fermentation process of Brevibacillus laterosporus antibacterial peptide, which can prolong the production period of bacterial fermentation and improve the fermentation yield by improving the fermentation formula and regulating the fermentation process.
The technical problem to be solved by the invention is realized by the following technical scheme:
the invention provides a fermentation process for producing antibacterial peptide by fermentation, which comprises the following steps: the method comprises the following steps of taking brevibacillus laterosporus as a strain, inoculating the strain into seeds containing a carbon source, an inorganic and organic nitrogen source and inorganic salt and a fermentation culture medium, and fermenting to prepare the antibacterial peptide, wherein the method comprises the following steps:
1) Slant strain activation: inoculating the strain of the brevibacillus laterosporus cold-storage tube in an LB slant culture medium for culture;
2) Seed shake flask culture: inoculating the slant culture into a sterilized seed culture medium, and performing shake-flask culture to obtain a seed solution;
3) Seed tank culture: after sterilizing the seed culture medium in the first-stage seeding tank, inoculating the seed solution obtained in the step 2), and culturing to obtain a seed solution;
4) Fermentation culture: inoculating the seed liquid obtained in the step 2) or 3) into a fermentation culture medium, and controlling the dissolved oxygen to be more than 20%; during fermentation for 10-20hr, FP101 tryptone 0.2-2g per liter of fermentation liquid, pH 6.0-6.5, and foam control at foam high fermentation stage for 12-25hr to obtain antibacterial peptide.
The brevibacillus laterosporus is Bacillus laterosporus (Brevibacillus laterosporus) CGMCC No.16337.
Further preferred scheme:
slant strain activation: the Brevibacillus laterosporus cold storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at the temperature of 32-38 ℃.
Seed culture: inoculating slant culture or seed bottle into sterilized seed culture medium, shake-culturing at 32-38 deg.C and 200rpm for 8-16hr to obtain seed solution, shape Brevibacterium, and thallus concentration of 10 7 -10 8 cfu/ml。
Seed tank culture: sterilizing the first-stage seeding tank culture medium, inoculating the seed solution obtained in step 2), wherein the inoculation amount is 0.2-5%, and the temperature is 32%38 ℃ below zero, the stirring speed is 40-50HZ, the ventilation volume is 1.8-1.2, and the pressure in a tank is 0.04-0.05MPa; culturing for 8-16hr to obtain seed solution with thallus concentration of 10 7 -10 8 cfu/ml。
The seed culture medium is prepared as follows: 5-20g of yeast extract, 2-10g of corn steep liquor powder, 20-30g of glucose, 3-10g of sodium chloride, 0.1-1g of monopotassium phosphate, 2-5g of calcium carbonate, 1000ml of water, 6.0-7.0 of PH, 121 ℃, and sterilizing for 30min.
Fermentation culture:
inoculating the seed liquid obtained in the step 2) or 3) into a fermentation medium by the inoculation amount of 2% -15%. The fermentation tank culture process comprises the following steps: the culture conditions are 32-38 ℃, the stirring speed is 150-500rpm, the air flow is 1.2, and the tank pressure is 0.04-0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20hr, FP101 tryptone 0.2-1g per liter of fermentation liquid, pH 6.0-6.5, and composite defoaming agent T31 in the high foam stage to control foam, and total fermentation culture time is 12-25hr. T31 is a polyether (R- [ C) 2 H 4 ]m-[C 3 H 6 ]nH) and an organic silicon-synthesized defoaming agent for fermentation (manufactured by shijiazhuang tairun science and technology ltd).
Fermentation medium: 20-50g of glucose, 5-20g of glycerol, 2-5g of inorganic ammonium salt, 0.05-0.5g of dipotassium hydrogen phosphate, 3-10g of yeast powder (or corn steep liquor powder), 5-15g of industrial peptone, 5-10 g of sodium chloride, 1-3g of calcium chloride, 1-3g of Val, 1000ml of water and 6.0-7.0 of PH; wherein the inorganic ammonium salt can be ammonium sulfate, ammonium nitrate and ammonium chloride.
The invention has the following advantages:
the combination of inorganic and organic nitrogen sources is optimized, the fermentation process is finely regulated, the titer of the fermentation liquor reaches more than 3500ug/ml, compared with the fermentation liquor before optimization, the yield of the antibacterial peptide is improved by 3 times, the titer of the antibacterial peptide fermentation liquor is stable, and the cost is greatly reduced.
Drawings
FIG. 1: spectrogram of fermentation liquid
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention.
In the embodiment of the invention, the used strains are Brevibacillus laterosporus, the strains are from the group of Zhongliang, and the preservation number is CGMCC NO.16337.
EXAMPLE 1 preparation of antimicrobial peptides
1) Slant strain activation: the brevibacillus laterosporus cold-storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at 35 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 35 deg.C and 200rpm for 8hr in shake flask to obtain seed solution, shape Brevibacterium, and thallus concentration of 7 × 10 7 cfu/ml；
The seed culture medium is prepared as follows: 10g of yeast extract, 15g of corn steep liquor powder, 15g of glucose, 30g of sodium chloride, 5g of potassium dihydrogen phosphate, 1g of calcium carbonate, 1000ml of tap water, 6.0 of PH, 121 ℃, and sterilizing for 30min;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by the inoculation amount of 2%.
The fermentation medium was prepared as follows:
30g of glucose, 15g of glycerol, 2g of ammonium chloride, 2g of yeast powder, 5g of industrial peptone, 15g of calcium chloride, 1g of sodium chloride, 10g of dipotassium hydrogen phosphate, 0.5g of Val, 3g of tap water, 1000ml of pH 6.0, 121 ℃ and sterilization for 30min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions 35 degrees, stirring speed 300rpm, air flow rate 1:1.2 the pot pressure is 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20hr, FP101 tryptone 0.2g per liter of fermentation liquid is supplemented, and water-soluble composite defoaming agent T31 is adopted to control foam in the foam high-foaming period, the addition is controlled below 0.5%, and the total fermentation culture time is 25hr.
The titer of fermentation liquor in tank is 3540ug/ml (the spectrum of fermentation liquor is shown in figure 1).
EXAMPLE 2 preparation of antimicrobial peptides
1) Activating strains: the strain of the Brevibacillus laterosporus cold storage tube is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C and 200rpm for 8hr to obtain seed solution, shaped Brevibacterium, and thallus concentration of 8 × 107cfu/ml;
the seed culture medium is prepared as follows: 5g of yeast extract, 20g of corn steep liquor powder, 20g of glucose, 10g of sodium chloride, 0.5g of monopotassium phosphate, 3g of calcium carbonate, 1000ml of tap water, 6.5 of PH and 121 ℃, and sterilizing for 30min;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by the inoculation amount of 5%.
The fermentation medium was prepared as follows:
40g of glucose, 10g of glycerol, 3g of ammonium sulfate, 7g of yeast powder, 10g of industrial peptone, 3g of calcium chloride, 7g of sodium chloride, 0.2g of dipotassium hydrogen phosphate, 0.2g of Val, 1000ml of tap water, 6.5 PH, 121 ℃, and sterilizing for 30min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions of 37 degrees, stirring speed of 400rpm, air flow rate of 1:1.2 tank pressure 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20hr, FP101 tryptone 0.3g per liter of fermentation liquid is supplemented, and water-soluble composite defoaming agent T31 is adopted to control foam in the foam high-foaming period, the addition is controlled below 0.5%, and the total fermentation culture time is 25hr.
The titer of the fermentation liquor in the tank is 3960ug/ml by HPLC.
EXAMPLE 3 preparation of antimicrobial peptides
1) Activating strains: the Brevibacillus laterosporus cold storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at 32 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 32 deg.C and 200rpm for 16hr to obtain seed solution, shaped Brevibacterium, and thallus concentration of 5 × 107cfu/ml;
the seed culture medium is prepared as follows: 20g of yeast extract, 5g of corn steep liquor powder, 20g of glucose, 3g of sodium chloride, 0.1g of monopotassium phosphate, 2g of calcium carbonate, 1000ml of tap water, 7 of PH (potential of Hydrogen), 121 ℃, and sterilizing for 30min;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by 10 percent of inoculation amount.
The fermentation medium was prepared as follows:
20g of glucose, 20g of glycerol, 4g of ammonium sulfate, 10g of yeast powder, 8g of industrial peptone, 2g of calcium chloride, 7g of sodium chloride, 0.05g of dipotassium hydrogen phosphate, 0.05g of Val 2g of tap water, 1000ml of pH 7.0 and 121 ℃, and sterilizing for 30min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions 32 degrees, stirring speed 400rpm, air flow 1:1.2 tank pressure 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-12 hr, FP101 tryptone 1g per liter of fermentation liquid is supplemented, and water-soluble composite defoamer T31 is adopted to control foam in the foam high-foaming period, the addition is controlled below 0.5%, and the total fermentation culture time is 12hr.
The titer of fermentation liquor in a tank is 3576ug/ml by HPLC measurement.
EXAMPLE 4 preparation of antimicrobial peptides
1) Activating strains: the Brevibacillus laterosporus cold storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C and 200rpm for 10hr to obtain seed solution, shaped Brevibacterium, and thallus concentration of 8.5 × 107cfu/ml;
the seed culture medium is prepared as follows: 5g of yeast extract, 20g of corn steep liquor powder, 20g of glucose, 20g of sodium chloride, 5g of potassium dihydrogen phosphate, 0.5g of calcium carbonate, 3g of 1000ml of tap water, 6.5 of PH, 121 ℃, and sterilizing for 30min;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by 10 percent of inoculation amount.
The fermentation medium was prepared as follows:
40g of glucose, 10g of glycerol, 3g of ammonium nitrate, 3g of yeast powder, 3g of industrial peptone, 15g of calcium chloride, 5g of sodium chloride, 0.2g of dipotassium hydrogen phosphate, 0.1g of Val, 1000ml of tap water, 6.5 pH and 121 ℃, and sterilizing for 30min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions of 37 degrees, stirring speed of 400rpm, air flow rate of 1:1 pot pressure of 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20hr, FP101 tryptone 0.2g per liter of fermentation liquid is supplemented, and water-soluble composite defoamer T31 is adopted to control foam in the high foam period, the addition amount is controlled to be below 0.5%, and the total fermentation culture time is 25hr.
The titer of the fermentation liquor in the tank is 3845ug/ml by HPLC measurement.
EXAMPLE 5 preparation of antimicrobial peptides
1) Activating strains: the Brevibacillus laterosporus cold storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C and 200rpm for 16hr to obtain seed solution, shaped Brevibacterium, and thallus concentration of 8.3 × 107cfu/ml;
the seed culture medium is prepared as follows: 10g of yeast extract, 15g of corn steep liquor powder, 30g of glucose, 10g of sodium chloride, 10g of potassium dihydrogen phosphate, 1g of calcium carbonate, 5g of 1000ml of tap water, pH 6.0, 121 ℃, and sterilizing for 30min;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by 15 percent of inoculation amount.
The fermentation medium was prepared as follows:
50g of glucose, 5g of glycerol, 5g of ammonium sulfate, 3g of corn steep liquor powder, 3g of industrial peptone, 5g of calcium chloride, 1g of sodium chloride, 7g of dipotassium hydrogen phosphate, 0.05g of Val, 3g of tap water, 1000ml of pH 6.0, 121 ℃ and sterilization for 30min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions of 37 degrees, stirring speed of 400rpm, air flow rate of 1:0.8 tank pressure 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20hr, FP101 tryptone 0.5g per liter of fermentation liquid is supplemented, and water-soluble composite antifoaming agent T31 is adopted to control foam in foam high-foaming period, the addition amount is controlled below 0.5%, and the total fermentation culture time is 25hr.
The titer of the fermentation liquor in the tank is 3680ug/ml by HPLC measurement.
EXAMPLE 6 preparation of antimicrobial peptides
1) Activating strains: the Brevibacillus laterosporus cold storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C and 200rpm in 16 bottles for 10hr to obtain seed solution, shape Brevibacterium, and thallus concentration of 1.1 × 10 8 cfu/ml；
The seed culture medium is prepared as follows: 20g of yeast extract, 5g of corn steep liquor powder, 20g of glucose, 5g of sodium chloride, 0.1g of monopotassium phosphate, 2g of calcium carbonate, 1000ml of tap water, 6.5 of PH, 121 ℃, and sterilizing for 30min;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by an inoculation amount of 5%.
The fermentation medium was prepared as follows:
30g of glucose, 20g of glycerol, 2g of ammonium nitrate, 10g of corn steep liquor powder, 5g of industrial peptone, 1g of calcium chloride, 10g of sodium chloride, 0.4g of dipotassium hydrogen phosphate, 0.4g of Val 4g of tap water, 1000ml of pH 6.5, 121 ℃, and sterilizing for 30min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions 38 degrees, stirring speed 400rpm, air flow 1:1.2 the pot pressure is 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-14 hr, FP101 tryptone 2g per liter of fermentation liquid is supplemented, and water-soluble composite defoamer T31 is adopted to control foam in the high foam period, the addition is controlled below 0.5%, and the total fermentation culture time is 16hr.
The titer of fermentation liquor in a tank is 3515ug/ml by HPLC measurement.
EXAMPLE 7 preparation of antimicrobial peptides
1) Activating strains: the strain of the Brevibacillus laterosporus cold storage tube is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C and 200rpm for 8hr to obtain seed solution, shaped Brevibacterium, and thallus concentration of 8.4 × 107cfu/ml;
the seed culture medium is prepared as follows: 5g of yeast extract, 20g of corn steep liquor powder, 20g of glucose, 10g of sodium chloride, 0.5g of potassium dihydrogen phosphate, 3g of calcium carbonate, 1000ml of tap water, 6.5 of PH, 121 ℃, and sterilizing for 30min;
2) Seed tank culture: the volume of the tank is 300L, and the loading capacity is 150L; culture conditions 37 degrees, rotation speed 40hz, air flow 1.2, and tank pressure 0.05MPa. The culture medium is the same as the seed culture medium of the seed bottle. Sterilizing the first-stage seeding tank culture medium, inoculating the seed solution obtained in step 2), culturing at 0.3% and 37 deg.C for 16hr to obtain seed solution;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 3) into a fermentation medium by 10 percent of inoculation amount.
The fermentation medium was prepared as follows:
40g of glucose, 10g of glycerol, 3g of ammonium sulfate, 7g of yeast powder, 10g of industrial peptone, 3g of calcium chloride, 7g of sodium chloride, 0.2g of dipotassium hydrogen phosphate, 0.2g of Val, 1000ml of tap water, 6.5 PH, 121 ℃, and sterilizing for 30min.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 2T, and the filling amount is 1.2T; culture conditions at 37 degrees, stirring speed at 50hz, air flow rate at 1:1.2 tank pressure 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20hr, FP101 tryptone 0.3g per liter of fermentation liquid is supplemented, and water-soluble composite defoaming agent T31 is adopted to control foam in the foam high-foaming period, the addition is controlled below 0.5%, and the total fermentation culture time is 25hr.
The titer of the fermentation liquor in the tank is 3880ug/ml by HPLC measurement.
EXAMPLE 8 preparation of antimicrobial peptides
1) Activating strains: the strain of the Brevibacillus laterosporus cold storage tube is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C and 200rpm for 10hr to obtain seed solution, shaped Brevibacterium with thallus concentration of 9.0 × 10 7 cfu/ml；
The seed culture medium is prepared as follows: 5g of yeast extract, 20g of corn steep liquor powder, 20g of glucose, 20g of sodium chloride, 5g of potassium dihydrogen phosphate, 0.5g of calcium carbonate, 3g of 1000ml of tap water, 6.5 of PH, 121 ℃, and sterilizing for 30min;
3) Seed tank culture: the volume of the tank is 300L, and the filling amount is 150L; culture conditions 37 degrees, rotation speed 40hz, air flow 1.2, and tank pressure 0.05MPa. The culture medium is the same as the seed culture medium of the seed bottle. Sterilizing the first-stage seeding tank culture medium, inoculating the seed solution obtained in step 2), culturing at 0.3% and 37 deg.C for 16hr to obtain seed solution;
4) Fermentation culture:
inoculating the seed solution obtained in the step 3) into a fermentation medium by 10 percent of inoculation amount.
The fermentation medium was prepared as follows:
40g of glucose, 10g of glycerol, 3g of ammonium nitrate, 3g of yeast powder, 3g of industrial peptone, 15g of calcium chloride, 5g of sodium chloride, 0.2g of dipotassium hydrogen phosphate, 0.1g of Val, 1000ml of tap water, 6.5 pH and 121 ℃, and sterilizing for 30min.
5) The fermentation tank culture process comprises the following steps: the volume of the tank is 2T, and the filling amount is 1.2T; culture conditions at 37 degrees, stirring speed at 50hz, air flow rate at 1:1.2 the pot pressure is 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. During fermentation for 10-20hr, FP101 tryptone 0.2g per liter of fermentation liquid is supplemented, and water-soluble composite defoamer T31 is adopted to control foam in the high foam period, the addition amount is controlled to be below 0.5%, and the total fermentation culture time is 25hr.
The titer of fermentation liquor discharged from the tank is 3780ug/ml by HPLC measurement.
Comparative example 1 preparation of antibacterial peptide
1) Activating strains: the strain of the Brevibacillus laterosporus cold storage tube is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C and 200rpm in 16 bottles for 16hr to obtain seed solution, shaped Brevibacterium, and thallus concentration of 6 × 10 8 cfu/ml；
The seed culture medium is prepared as follows: 1000ml of LB culture medium tap water, pH 6.5, 121 ℃, sterilizing for 30min;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by an inoculation amount of 5%.
The fermentation medium was prepared as follows:
fermentation medium: sterilizing LB culture medium with tap water 1000ml, pH 6.5 and 121 deg.C for 30min; the culture media are all in mass-to-volume ratio.
4) The fermentation tank culture process comprises the following steps: the volume of the tank is 50L, and the filling amount is 30L; culture conditions of 37 degrees, stirring speed of 400rpm, air flow rate of 1:1.2 the pot pressure is 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. The total fermentation culture time is 25hr.
The titer of the fermentation liquor in the tank is 630ug/ml by HPLC.
Comparative example 2 preparation of antimicrobial peptide
1) Activating strains: the Brevibacillus laterosporus cold storage tube strain is inoculated in an LB slant culture medium and cultured for 3 days at 37 ℃.
2) Seed shake flask culture: inoculating the slant culture into sterilized seed culture medium, culturing at 37 deg.C at 200rpm in 16 bottles for 16hr to obtain seed solution, shaped Brevibacterium, and thallus concentration of 6.5 × 108cfu/ml;
the seed culture medium is prepared as follows: 1000ml of LB culture medium tap water, pH 6.5, 121 ℃, sterilizing for 30min;
3) Fermentation culture:
inoculating the seed liquid obtained in the step 2) into a fermentation medium by the inoculation amount of 5%.
The fermentation medium was prepared as follows:
glucose 20g, yeast powder 5g, industrial peptone 10g, sodium chloride 10g, tap water 1000ml, pH 6.5, 121 ℃, and sterilization for 30min.
1000ml of LB culture medium tap water, pH 6.5, 121 ℃, and sterilizing for 30min 4) fermentation tank culture process: the volume of the tank is 50L, and the filling amount is 30L; culture conditions of 37 degrees, stirring speed of 400rpm, air flow rate of 1:1.2 the pot pressure is 0.05MPa; the dissolved oxygen is controlled to be more than 20 percent. The total fermentation time is 25hr.
The titer of the fermentation liquor in the tank is 1210ug/ml by HPLC measurement.
Example 9 antimicrobial Activity assay
The activity of the antibacterial peptide is measured by a Minimum (MIC) method, which comprises the following steps:
1) Sample group: respectively adding samples to be tested into a 96-well plate, and respectively adding 10 corresponding holes 5 Bacterial suspension of 200 ul/well in total volume.
2) Detection of bacteria control group: adding solvents with the same volume as the sample to be detected in the sample group into a 96-well plate respectively, and adding 10 corresponding holes respectively 5 Bacterial suspension of 200 l/well in total volume.
3) Medium blank control group: 200. Mu.l/well of assay medium.
4) Bacteria were cultured at 37 degrees for 24h before reading OD values per well using a 1420Victor2 multi-standard counter at 600nm wavelength.
5) The fungus was incubated at 26 ℃ for 48h and OD readings were taken per well at 600nm using a 1420Victor2 multiscale counter.
6) Each antibiotic substance was repeated three times, and the MIC90 was determined from the OD value.
By using the method, staphylococcus aureus, sarcina lutea, bacillus cereus, shigella dysenteriae, escherichia coli, salmonella enteritidis, salmonella paratyphi b, fusarium and other bacteria are taken as detection bacteria for detection, and the results are shown in table 1.
TABLE 1 bacteriostatic Activity of antimicrobial peptides in fermentation broths
Indicator strain MIC 90 (ug/ml)
Staphylococcus aureus (Staphylococcus aureus) 0.78
Bacillus subtilis 1.56
Sarcina lutea (berk.) Kuntze 3.12
Bacillus cereus 3.12
Shigella dysenteriae ﹤12.5
Escherichia coli 12.5
Salmonella enteritidis 50
Salmonella paratyphi B 50
Fusarium 6.25
Example 10 stability study
1) Stability of the antibacterial peptide fermentation broth (example 1) produced by fermentation of Brevibacillus laterosporus is examined at 100 ℃, the fermentation broth is kept stand at 100 ℃ for different times, and the titer of the fermentation broth is measured by HPLC after cooling. And (4) conclusion: the antibacterial peptide fermentation liquor is placed at 100 ℃ for 60min, and the titer is stable. See Table 2
TABLE 2 investigation of temp. stability of antimicrobial peptides in fermentation broths
Standing time (min) 0 20 40 60
Potency (ug/ml) 3540 3551 3512 3530
2) Stability examination of the antibacterial peptide fermentation broth (example 1) produced by fermentation of Brevibacillus laterosporus at different pH values showed that the potency was stable when the fermentation broth was left at pH2-11 for 48 hours. See Table 3
TABLE 3 stability of antimicrobial peptides in fermentation broths at different pH
Condition of pH value pH2 pH3 pH4 pH5 pH6 pH7 pH8 pH9 pH10 pH11
Potency (ug/ml) 3558 3566 3550 3571 3540 3545 3535 3566 3542 3554
EXAMPLE 11 liquid phase chromatogram of fermentation broth
The antibacterial peptide produced by the fermentation process is a group of peaks with similar ultraviolet absorption, as shown in figure 1.

Claims (6)

1. A method for producing antibacterial peptide by fermenting Brevibacillus laterosporus (Brevibacilli laterosporus) serving as a strain is characterized by comprising the following steps:
1) Slant strain activation: inoculating the strain of the brevibacillus laterosporus cold-storage tube in an LB slant culture medium for culture;
2) Seed shake flask culture: inoculating the slant culture into a sterilized seed culture medium, and performing shake culture to obtain a seed solution;
3) Seed tank culture: after sterilizing the seed culture medium in the first-stage seeding tank, inoculating the seed solution obtained in the step 2), and culturing to obtain a seed solution;
4) Fermentation culture: inoculating the seed solution obtained in the step 2) or 3) into a fermentation culture medium, wherein the dissolved oxygen is controlled to be more than 20%; during fermentation for 10-20hr, adding FP101 tryptone 0.2-2g per liter of fermentation liquid, pH 6.0-6.5, controlling foam in foam high fermentation period, and culturing for 12-25hr to obtain antibacterial peptide;
the fermentation medium formula is as follows: 20-50g of glucose, 5-20g of glycerol, 2-5g of inorganic ammonium salt, 0.05-0.5g of dipotassium phosphate, 3-10g of yeast powder or corn steep liquor powder, 5-15g of industrial peptone, 5-10 g of sodium chloride, 1-3g of calcium chloride, 1-4g of Val, 1000ml of water and 6.0-7.0 of PH;
defoaming with a composite defoaming agent T31 in a foam high-rise period;
the brevibacillus laterosporus is Brevibacillus laterosporus (Brevibacillus laterosporus) CGMCC No.16337.
2. The method of claim 1, wherein: the seed culture medium formula comprises: 5-20g of yeast extract, 5-20g of corn steep liquor powder, 20-30g of glucose, 3-10g of sodium chloride, 0.1-1g of monopotassium phosphate, 2-5g of calcium carbonate, 1000ml of water and 6.0-7.0 of PH.
3. The method of claim 1, wherein: culturing at 32-38 deg.C at 200rpm for 8-16hr to obtain seed liquid, brevibacterium, and thallus concentration of 10 7 -10 8 cfu/ml。
4. The method of claim 1, wherein: the seeding tank culture conditions comprise inoculum size of 0.2-5%, temperature of 32-38 deg.C, stirring rotation speed of 40-50HZ, ventilation amount of 1.8-1.2, tank pressure of 0.04-0.05MPa, and culture time of 8-16hr to obtain seed solution with thallus concentration of 10% 7 -10 8 cfu/ml 。
5. The method of claim 1, wherein: the inorganic ammonium salt is ammonium sulfate, ammonium nitrate or ammonium chloride.
6. The method of claim 1, wherein: the fermentation process comprises the following steps: inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 2-15%, wherein the culture condition is 32-38 ℃, the stirring speed is 150-400rpm, and the air flow is 1:0.8-1.2, and the tank pressure is 0.05-0.06MPa; during the fermentation for 10-20 hours, 0.2-2g of FP101 tryptone is added to each liter of fermentation liquor.
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