A bacterial antimicrobial peptide fermentation optimization method and application

Abstract

The present invention discloses a method and application for optimizing fermentation of bacterial antimicrobial peptides, comprising the following steps: preparing seed liquid; inoculating the seed liquid into the initial culture medium to prepare sterile fermentation filtrate and adjusting the pH value, collecting the precipitate by standing and centrifugation, then extracting with ethanol, and obtaining the crude extract of lipopeptide substances by rotary evaporation; optimizing the initial culture medium and fermentation process, centrifuging the optimized culture medium at room temperature to remove the bacteria and other insoluble substances; filtering to obtain sterile fermentation supernatant; adjusting the pH value of the fermentation supernatant with concentrated hydrochloric acid; centrifuging again after standing and collecting the precipitate; extracting the precipitate with anhydrous ethanol, and concentrating the extract by rotary evaporation at 40°C to obtain a high-purity antimicrobial peptide compound. The antimicrobial peptide of the present invention is superior to the prior art in terms of efficient synthesis and extraction, diversity of application fields, etc., and has significant innovation and practicality, providing new technical support for the development and application of antimicrobial peptides.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of bacterial antimicrobial peptide compounds, and in particular to a bacterial antimicrobial peptide fermentation optimization method and application. Background Art

[0002] Antimicrobial peptides are a class of short-chain polypeptides synthesized by microorganisms, plants or animal cells, usually composed of 10 to 50 amino acids, with broad-spectrum antimicrobial activity. The mechanism of action of antimicrobial peptides mainly includes destroying the cell membrane structure by binding to the cell membrane, inducing target cell lysis, interfering with the synthesis of nucleic acids or proteins, etc. Due to the high inhibitory effect of antimicrobial peptides on bacteria, fungi and certain viruses, and the lack of drug resistance, they have shown great application potential in the fields of medicine, food safety, agricultural disease prevention and control, and are considered to be an important alternative to solve the problems of antibiotic abuse and drug resistance.

[0003] At present, the practical application of antimicrobial peptides is still subject to many limitations, mainly including high production costs, difficult separation and purification, poor activity stability and other problems. Although traditional chemical synthesis methods can achieve the production of a variety of antimicrobial peptide molecules, there are problems such as complex synthesis process, high cost, and environmental pollution. In addition, although antimicrobial peptides extracted from animals or plants are natural and safe, they have low yields and low efficiency and cannot meet the needs of large-scale industrialization. Antimicrobial peptide production technology based on microbial fermentation has become a hot topic in recent years. It has attracted widespread attention for its advantages such as low cost, high efficiency, and green environmental protection.

[0004] The fermentation production technology of functional bacteria provides an effective solution for the large-scale application of antimicrobial peptides. Functional bacteria can synthesize antimicrobial peptides through genetic coding and secrete them into the culture medium through the fermentation process to achieve product accumulation. This technology not only simplifies the production process of antimicrobial peptides, but also significantly increases the yield of antimicrobial peptides by optimizing the strains and fermentation conditions. However, there are still some key technical bottlenecks in current research and application, including the selection and optimization of antimicrobial peptide-producing bacteria, the improvement of fermentation conditions, the efficient separation and purification of antimicrobial peptides, and the improvement of the stability and function of antimicrobial peptides.

[0005] Existing technology Screening and utilization of natural antimicrobial peptide-producing bacteria: Natural antimicrobial peptide-producing bacteria are widely found in soil, ocean and other ecosystems, such as lactic acid bacteria, Bacillus and Streptomyces. These strains fight pathogenic microorganisms in the natural environment by secreting antimicrobial peptides, providing humans with natural antimicrobial resources; Technical features of this technology: The screening and fermentation methods of natural strains are simple, and the antimicrobial peptides produced are usually highly active and have good biocompatibility; This type of method relies on optimizing culture conditions, such as the ratio of carbon and nitrogen sources, temperature, pH value and culture medium formula, to promote the secretion of antimicrobial peptides; However, this type of method has some problems in industrial applications: First, the antimicrobial peptide yield of natural strains is usually low, which is difficult to meet commercial needs; second, some strains have strict requirements on culture conditions and are sensitive to environmental changes, making it difficult to achieve large-scale production; Finally, genetic modification of natural strains is difficult, limiting the possibility of further increasing the yield of antimicrobial peptides through metabolic engineering.

[0006] Existing technology: Construction and fermentation production of genetically engineered bacteria: The rapid development of genetic engineering technology has brought new strategies for the production of antimicrobial peptides; inserting antimicrobial peptide synthesis genes into efficient expression hosts such as Escherichia coli, Bacillus subtilis or yeast through gene editing can significantly increase the production of antimicrobial peptides; Technical features of this technology: Genetically engineered bacteria have the advantages of high expression efficiency, flexible operation, and strong adaptability. They can overcome some limitations of natural strains and achieve efficient synthesis of antimicrobial peptides by optimizing gene sequences and host metabolic pathways; However, the use of genetically engineered bacteria also faces certain challenges: First, some host bacteria may produce toxic reactions to antimicrobial peptides, resulting in decreased expression efficiency; Second, the metabolic burden of engineered bacteria is large, which may affect their growth and fermentation performance; In addition, in the food and agricultural fields, the use of genetically engineered bacteria is strictly regulated, which limits their actual application scope.

[0007] Existing technology metabolic engineering and synthetic biology strategies: Metabolic engineering technology improves the synthesis efficiency of antimicrobial peptides by optimizing the metabolic network of natural strains; for example, by knocking out competitive metabolic pathways or enhancing the activity of key enzymes, more metabolic flows can be directed to the synthesis of antimicrobial peptides; in addition, synthetic biology combined with metabolic engineering can achieve efficient synthesis of antimicrobial peptides by artificially designing and constructing new metabolic pathways; technical features of this technology: metabolic engineering and synthetic biology methods can significantly improve the yield and quality of antimicrobial peptides, and are more in line with industrial needs; such methods can also expand their application areas by designing specific functional modules to produce antimicrobial peptides with customized characteristics; however, despite great potential in theory, the implementation process of metabolic engineering is complicated and usually requires multiple rounds of experimental verification; in addition, the stability of the modified strains is poor and may gradually lose the target function as the generations pass on, affecting their long-term application.

[0008] Although the above schemes have achieved certain results in the production of antimicrobial peptides, they still have some common shortcomings, such as low yield, high production cost, difficulty in separation and purification, poor genetic stability, limited scope of application, low antimicrobial peptide yield, harsh culture conditions, and insufficient antimicrobial activity and stability.

[0009] Therefore, the present invention provides a method and application for optimizing the fermentation of bacterial antimicrobial peptides. The present invention overcomes the main bottlenecks in the prior art by screening or constructing a functional bacterium with high antimicrobial peptide production capacity and combining it with an optimized fermentation process. Specifically, the functional bacteria of the present invention can stably and efficiently produce antimicrobial peptides under a wide range of environmental conditions, while the fermentation process achieves a significant reduction in cost. Summary of the invention

[0010] The purpose of the present invention is to provide a method for optimizing the fermentation of bacterial antimicrobial peptides and its application, so as to fill the gap in the current technology.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] The BER1 bacteria of the present invention is prior art. The prior art CN114276957 discloses a biocontrol strain having the effect of preventing and controlling various plant pathogenic fungi and the BER1 bacteria in its application.

[0013] A method for preparing an antimicrobial peptide compound comprises the following steps:

[0014] (1) Preparation of seed solution: BER1 strain was streaked on NA medium for activation; a single colony was picked and inoculated into NB liquid medium, and cultured in a shaking incubator at 30°C and 180 rpm for 12 to 18 hours to prepare seed solution;

[0015] (2) Preparation of sterile fermentation filtrate: inoculate the seed liquid into the initial culture medium, place it in a shaker for culture until the OD600 reaches 0.8 to 1.0, and obtain the culture liquid; centrifuge the culture liquid, remove the bacterial cells, and filter the supernatant to obtain the sterile fermentation filtrate;

[0016] (3) Extraction method: The pH value of the sterile fermentation filtrate was adjusted with HCl, allowed to stand, and then centrifuged to collect the precipitate, which was then extracted with ethanol and rotary evaporated to obtain a crude extract of lipopeptides;

[0017] (4) Optimization of fermentation method:

[0018] The initial culture medium and fermentation process were optimized by single factor experiments with different carbon sources, different nitrogen sources, different inorganic salts, different inoculation amounts, different fermentation temperatures, different fermentation times, pH and rotation speeds.

[0019] (5) Isolation and purification of antimicrobial peptides:

[0020] a. Preparation of sterile fermentation supernatant: inoculate the seed liquid into the optimized initial culture medium to obtain the culture solution, centrifuge at room temperature to remove the bacteria and other insoluble substances; filter to obtain the sterile fermentation supernatant;

[0021] b. Antimicrobial peptide precipitation: adjust the pH value of the fermentation supernatant with concentrated hydrochloric acid; centrifuge again after standing to collect the precipitate;

[0022] c. Extraction and concentration: The precipitate was extracted with anhydrous ethanol, and the extract was concentrated by rotary evaporation at 40°C to obtain a high-purity antimicrobial peptide compound.

[0023] Preferably, in step (1), a single bacterial colony is picked and inoculated into NB liquid culture medium, and cultured in a shaking incubator at 30° C. and 180 rpm for 12 to 18 hours to prepare a seed solution.

[0024] Preferably, in step (2), the seed liquid is inoculated into the initial culture medium at an inoculation rate of 1-2%, and cultured in a shaking incubator at 30°C and 180-200 r / min for 48 hours.

[0025] Preferably, in step (2), the culture solution is centrifuged at 4°C and 10,000-12,000 r / min for 30 minutes; and the supernatant is filtered through a 0.22-micron microporous filter membrane to obtain a sterile fermentation filtrate.

[0026] Preferably, in step (3), the sterile fermentation filtrate is adjusted to pH 2.0 with 6 mol / L HCl, allowed to stand at 4°C for 18 hours, and then centrifuged at 10,000-12,000 r / min for 30 minutes.

[0027] Preferably, in step (5), the mixture in step a is centrifuged at 10,000 to 12,000 r / min for 30 minutes at room temperature to remove the bacteria and other insoluble substances; and filtered through a 0.22 micron filter membrane to obtain a sterile fermentation supernatant.

[0028] Preferably, in step (5), the pH value of the fermentation supernatant is adjusted to 2.0 with concentrated hydrochloric acid in step b; after standing at 4°C for 18 hours, the supernatant is centrifuged again at 10,000-12,000 r / min for 30 minutes to collect the precipitate.

[0029] The present application also claims to protect an antimicrobial peptide compound, which is an antimicrobial peptide compound prepared by the above-mentioned preparation method of the antimicrobial peptide compound.

[0030] The present application also claims to protect an application of the above antimicrobial peptide compound, and the present invention further expands the application of antimicrobial peptides in the fields of medicine, food safety and agriculture.

[0031] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0032] 1. The present invention has established a set of efficient fermentation processes by optimizing multiple factors such as carbon source, nitrogen source, inorganic salt, fermentation conditions, etc., so that the yield of antimicrobial peptides reaches more than 500 mg / L, which is 20% to 30% higher than the existing technology. In particular, soluble corn starch and soybean meal are used as carbon source and nitrogen source, which significantly improves the fermentation efficiency and reduces the production cost;

[0033] 2. The lipopeptide antimicrobial peptides produced by the functional bacterium BER1 in the present invention exhibit broad-spectrum and potent antimicrobial activity against a variety of plant pathogens. Compared with some antimicrobial peptides in the prior art that are only effective against specific species, the antimicrobial peptides of the present invention have a wider range of applications and higher practical value;

[0034] 3. The ethanol extraction technology used in the present invention can significantly retain the biological activity of antimicrobial peptides, providing a simpler and more efficient solution for industrial production;

[0035] 4. The antimicrobial peptide products of the present invention have a wider application potential in the fields of agriculture and medicine. For example, they can effectively replace traditional chemical pesticides in the prevention and control of plant diseases;

[0036] 5. The present invention optimizes the culture medium formula and fermentation conditions, and uses a large amount of cheap raw materials (such as soluble corn starch and soybean meal powder), which greatly reduces the production cost; at the same time, the fermentation cycle is short and the output is high, which has significant advantages in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be made based on these drawings without paying any creative work.

[0038] Figure 1 This is a regression model diagram of Example 1 of the present invention. DETAILED DESCRIPTION

[0039] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, a specific implementation scheme is now described in detail.

[0040] The present invention is further described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to the different requirements of specific use, and the implementation conditions not indicated are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0041] Example 1

[0042] See attached Figure 1 This embodiment provides a method and application for optimizing the fermentation of bacterial antimicrobial peptides; the method for preparing the antimicrobial peptide compound comprises the following steps:

[0043] (1) Preparation of seed solution: BER1 strain was streaked on NA medium for activation; a single colony was picked and inoculated into NB liquid medium, and cultured in a shaking incubator at 30°C and 180 rpm for 12 to 18 hours to prepare seed solution;

[0044] (2) Preparation of sterile fermentation filtrate: inoculate the seed liquid into the initial culture medium at an inoculum rate of 2%, and culture at 30° C. and 180 r / min on a shaking incubator for 48 hours until the OD600 reaches 0.8 to 1.0; centrifuge the culture medium at 4° C. and 12,000 r / min for 30 minutes to remove the bacterial cells, and filter the supernatant through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate;

[0045] (3) Extraction method: The pH of the sterile fermentation filtrate was adjusted to 2.0 with 6 mol / L HCl, and allowed to stand at 4°C for 18 h. The precipitate was then collected by centrifugation at 12,000 r / min for 30 min, and then extracted with ethanol. After rotary evaporation at 40°C, a crude extract of lipopeptides was obtained.

[0046] (4) Optimization of fermentation method:

[0047] The initial culture medium and fermentation process were optimized by single factor experiments with different carbon sources, different nitrogen sources, different inorganic salts, different inoculation amounts, different fermentation temperatures, different fermentation times, pH and rotation speeds.

[0048] (5) Isolation and purification of antimicrobial peptides:

[0049] a. Preparation of sterile fermentation supernatant: inoculate the seed liquid into the optimized initial culture medium to obtain the culture solution, centrifuge at 12000r / min for 30 minutes at room temperature to remove the bacteria and other insoluble substances; filter with a 0.22 micron filter membrane to obtain a sterile fermentation supernatant;

[0050] b. Antimicrobial peptide precipitation: The pH value of the fermentation supernatant was adjusted to 2.0 with concentrated hydrochloric acid; after standing at 4°C for 18 hours, the supernatant was centrifuged again at 12000 r / min for 30 minutes to collect the precipitate;

[0051] c. Extraction and concentration: The precipitate is extracted with anhydrous ethanol, and the extract is concentrated by rotary evaporation at 40°C to obtain a high-purity antimicrobial peptide compound;

[0052] Specifically, the optimization of the fermentation method:

[0053] Through single-factor experiments, different carbon sources, different nitrogen sources, different inorganic salts, different inoculation amounts, different fermentation temperatures, different fermentation times, pH and rotation speeds were explored to select the optimal fermentation process. The single-factor experimental design is shown in Table 1, and the single-factor experimental results are shown in Table 2.

[0054] Table 1

[0055] Single Factor Different levels of a single factor Carbon source type Sucrose, fructose, galactose, mannose, soluble corn starch Type of nitrogen source <![CDATA[Soybean meal powder, corn flour, (NH4)2SO4, NH4Cl, NaNO3]]> Inorganic salt types <![CDATA[K2CO3、KH2PO4、NaH2PO4、MnSO4、MgCl2]]> Inoculation volume 0.5％、1％、2％、3％、4％、5％ Fermentation temperature (℃) 30、32、34、36、38、40 Fermentation time (h) 20、24、28、32、36、40 pH 6.4、6.8、7.2、7.6、8 Speed 150、180、200、220、250

[0056] Table 2

[0057]

[0058]

[0059] The single factor results showed that carbon source: soluble corn starch, nitrogen source: soybean meal, inorganic salt: MnSO4, inoculation amount 1%, fermentation temperature 32℃, fermentation time 32h, pH 7.2, and rotation speed 220rpm were the best single factors, with the highest Fengycin production and concentration of 430-480mg / ml.

[0060] Response surface experimental design, based on the results of single factor experiment, took A (inoculation amount), B (fermentation temperature), C (fermentation time) three factors that have important influence on Fengycin yield as independent variables, and Y (Fengycin yield) as response value, and designed a three-factor three-level response surface experiment using DesignExpert8.0.6 software. The Box-Behnken experimental design and results are shown in Table 3.

[0061] Table 3

[0062]

[0063] Model equation:

[0064] Y=450+5.1A+4.3B+3.2C-0.8A2-1.1B2-0.9C2+1.5AB+2.2AC+1.8BC; The regression model is calculated using the model equation as shown in the figure Figure 1 shown.

[0065] From the regression model, the predicted optimal fermentation conditions are: inoculation amount 1.324%, fermentation temperature 32.734℃, fermentation time 33.578h; the best carbon source is soluble corn starch, the nitrogen source is soybean meal, the inorganic salt is MnSO4, pH 7.2, and the rotation speed is 220rpm;

[0066] The fermentation conditions were set as follows: 5 g / L soluble corn starch, 3 g / L soybean meal, 2 g / L MnSO4, 3 g / L calcium carbonate, 2 g / L sodium chloride, 1.3% inoculation amount, 32.7°C fermentation temperature, and 33.5 h fermentation time for a verification test. The Fengycin yield obtained in the verification experiment was (507.11±0.18) mg / L.

[0067] In summary, the present invention has established a set of efficient fermentation processes by optimizing multiple factors such as carbon source, nitrogen source, inorganic salt, fermentation conditions, etc., so that the antimicrobial peptide yield reaches more than 500 mg / L, which is 20% to 30% higher than the prior art. In particular, soluble corn starch and soybean meal powder are used as carbon source and nitrogen source, which significantly improves the fermentation efficiency and reduces the production cost. The lipopeptide antimicrobial peptides produced by the functional bacteria BER1 in the present invention show broad-spectrum and strong antibacterial activity against a variety of plant pathogens. Compared with some antimicrobial peptides in the prior art that are only effective against specific species, the antimicrobial peptides of the present invention are The peptide has a wider range of applications and higher practical value; the ethanol extraction technology used in the present invention can significantly retain the biological activity of the antimicrobial peptide, providing a simpler and more efficient solution for industrial production; the antimicrobial peptide product of the present invention shows a wider application potential in the fields of agriculture and medicine, for example, in the prevention and control of plant diseases, it can effectively replace traditional chemical pesticides; the present invention optimizes the culture medium formula and fermentation conditions, and uses a large amount of cheap raw materials (such as soluble corn starch and soybean meal powder), which greatly reduces the production cost; at the same time, the fermentation cycle is short and the yield is high, which has significant advantages in industrial production.

[0068] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for preparing an antimicrobial peptide compound, characterized in that: The steps include: (1) Preparation of seed solution: BER1 strain was streaked on NA medium for activation; a single colony was picked and inoculated into NB liquid medium, and cultured in a shaking incubator at 30°C and 180 rpm for 12 to 18 hours to prepare seed solution; (2) Preparation of sterile fermentation filtrate: inoculate the seed liquid into the initial culture medium, place it in a shaker for culture until the OD600 reaches 0.8 to 1.0, and obtain the culture liquid; centrifuge the culture liquid, remove the bacterial cells, and filter the supernatant to obtain the sterile fermentation filtrate; (3) Extraction method: The pH value of the sterile fermentation filtrate was adjusted with HCl, allowed to stand, and then centrifuged to collect the precipitate, which was then extracted with ethanol and rotary evaporated to obtain a crude extract of lipopeptides; (4) Optimization of fermentation method: The initial culture medium and fermentation process were optimized by single factor experiments with different carbon sources, different nitrogen sources, different inorganic salts, different inoculation amounts, different fermentation temperatures, different fermentation times, pH and rotation speeds. (5) Separation and purification of antimicrobial peptides: a. Preparation of sterile fermentation supernatant: inoculate the seed liquid into the optimized initial culture medium to obtain the culture solution, centrifuge at room temperature to remove the bacteria and other insoluble substances; Filter to obtain sterile fermentation supernatant; b. Antimicrobial peptide precipitation: adjust the pH value of the fermentation supernatant with concentrated hydrochloric acid; After standing, centrifuge again to collect the precipitate; c. Extraction and concentration: The precipitate was extracted with anhydrous ethanol, and the extract was concentrated by rotary evaporation at 40°C to obtain a high-purity antimicrobial peptide compound.

2. The method for preparing an antimicrobial peptide compound according to claim 1, characterized in that: In step (1), a single colony is picked and inoculated into NB liquid culture medium, and cultured in a shaking incubator at 30° C. and 180 rpm for 12 to 18 hours to prepare a seed solution.

3. The method for preparing an antimicrobial peptide compound according to claim 1, characterized in that: In step (2), the seed solution is inoculated into the initial culture medium at an inoculation rate of 1-2%, and cultured in a shaking incubator at 30°C and 180-200 r / min for 48 hours.

4. The method for preparing an antimicrobial peptide compound according to claim 1, characterized in that: In step (2), the culture solution is centrifuged at 4° C. and 10,000 to 12,000 r / min for 30 minutes; the supernatant is filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

5. The method for preparing an antimicrobial peptide compound according to claim 1, characterized in that: In step (3), the pH of the sterile fermentation filtrate is adjusted to 2.0 with 6 mol / L HCl, allowed to stand at 4°C for 18 hours, and then centrifuged at 10,000-12,000 r / min for 30 minutes.

6. The method for preparing an antimicrobial peptide compound according to claim 1, characterized in that: In step (5), the mixture in step a is centrifuged at 10,000 to 12,000 r / min for 30 minutes at room temperature to remove the bacteria and other insoluble substances; and filtered through a 0.22 micron filter membrane to obtain a sterile fermentation supernatant.

7. The method for preparing an antimicrobial peptide compound according to claim 1, characterized in that: In step (5), the pH value of the fermentation supernatant in step b is adjusted to 2.0 with concentrated hydrochloric acid; after standing at 4° C. for 18 hours, the supernatant is centrifuged again at 10,000 to 12,000 r / min for 30 minutes to collect the precipitate.

8. An antimicrobial peptide compound, characterized in that: An antimicrobial peptide compound prepared by the method for preparing an antimicrobial peptide compound according to any one of claims 1 to 6.

9. An application of an antimicrobial peptide compound, characterized in that: Use of an antimicrobial peptide compound prepared by the method for preparing an antimicrobial peptide compound according to any one of claims 1 to 6 or an antimicrobial peptide compound according to claim 8.

Images