Separation method and application of antibacterial peptide in lactobacillus fermentation product

Abstract

The invention provides a separation method of antibacterial peptide in a lactobacillus fermentation product and application of the antibacterial peptide. The method comprises the following steps: firstly, preparing a soybean meal substrate, then adding an activated lactobacillus paracasei FX-6 strain and anhydrous dextrose into the substrate, and fermenting to obtain fermentation liquor; then, extracting a crude peptide solution from the fermentation liquor, carrying out graded filtration on the crude peptide solution through ultrafiltration membranes with different molecular weights, and extracting fractionation liquor from the crude peptide solution; and purifying the fractionation liquid for multiple times to finally obtain the anti-helicobacter pylori antibacterial peptide. According to the invention, the anti-helicobacter pylori activity evaluation is carried out on different fractionation liquids, and the low-molecular-weight antibacterial peptide with significant helicobacter pylori growth inhibition capability is successfully separated, extracted and purified from the fractionation liquids. The novel antibacterial peptide prepared by the invention has a smaller molecular structure, is easy to absorb and metabolize by organisms, and can effectively inhibit the growth of helicobacter pylori.

Description

Technical Field

[0001] The invention relates to the technical field of biological component extraction and antimicrobial compounds, in particular to a method for separating antimicrobial peptides from lactobacillus fermentation products and application thereof. Background Art

[0002] Helicobacter pylori (HP) is a Gram-negative, spiral-shaped, microaerophilic bacterium that is widely present in the global population and is closely related to the occurrence of various gastrointestinal diseases. A large number of epidemiological and clinical studies have shown that HP infection is the main causative factor of peptic ulcers, chronic gastritis, and gastric mucosal damage. It is particularly closely related to the occurrence of gastric cancer and has been listed as a Class I carcinogen for gastric cancer by the World Health Organization. HP causes chronic gastritis through various mechanisms, destroys the gastric mucosal barrier, leads to epithelial cell damage and gastric gland atrophy. In particular, the pathogenic toxins secreted by HP, such as CagA and VacA, aggravate gastric mucosal damage and promote the transformation of chronic atrophic gastritis to gastric cancer. Therefore, eradication of HP infection is considered to be one of the effective means to reduce the risk of gastric cancer.

[0003] Currently, antibiotic therapy remains the mainstay of treatment for HP infection, particularly triple and quadruple therapy. However, HP resistance to commonly used antibiotics, such as clarithromycin and metronidazole, has been increasing year by year, and resistance has become a major obstacle to treatment effectiveness. Furthermore, the side effects of antibiotics, particularly gastrointestinal adverse reactions, pose new challenges to clinical treatment. Therefore, the development of novel anti-HP therapeutic strategies, particularly antimicrobial substances derived from natural sources, has become a research hotspot.

[0004] Antimicrobial peptides are a class of small molecule proteins composed of short amino acid chains, typically produced by the immune systems of plants, animals, microorganisms, or animals. They can exert their antimicrobial effects by interfering with bacterial cell membranes, inhibiting bacterial growth, or disrupting bacterial cell structures. Microbial fermentation, particularly the metabolites of lactic acid bacteria, has been shown to be an important pathway for the production of antimicrobial peptides. Antimicrobial peptides produced by lactic acid bacteria have significant inhibitory effects on a variety of pathogens. However, research on the application of antimicrobial peptides in the treatment of HP infections is still limited. Therefore, exploring the potential application of antimicrobial peptides in inhibiting HP growth is particularly important. Summary of the Invention

[0005] In order to overcome the problems existing in the related art, the purpose of the present invention is to provide a method for separating antimicrobial peptides from Lactobacillus fermentation products and its application, wherein the method separates antimicrobial peptides from a soybean meal base through fermentation, graded filtration and multiple purifications, and the antimicrobial peptides can inhibit the growth of Helicobacter pylori.

[0006] A method for separating antimicrobial peptides from lactobacillus fermentation products, comprising:

[0007] S1: preparing a soybean meal base, adding the activated Lactobacillus paracasei FX-6 strain and anhydrous glucose to the soybean meal base for fermentation to obtain a fermentation liquid;

[0008] S2: extracting a crude peptide solution from the fermentation broth;

[0009] S3: using multiple ultrafiltration membranes to perform graded filtration on the crude peptide solution to extract and separate components to obtain a fractionated liquid;

[0010] S4: Purifying the fractionated liquid multiple times to obtain antimicrobial peptides.

[0011] In a preferred technical solution of the present invention, in step S3, a first ultrafiltration membrane with a molecular weight cutoff of 5 kDa and a second ultrafiltration membrane with a molecular weight cutoff of 10 kDa are used in sequence to perform graded filtration on the crude peptide solution, and components separated in the first molecular weight cutoff interval, the second molecular weight cutoff interval, and the third molecular weight cutoff interval are extracted to obtain a fractionated liquid.

[0012] In a preferred technical solution of the present invention, the first molecular weight cutoff interval is less than 5kDa, the second molecular weight cutoff interval is greater than or equal to 5kDa and less than or equal to 10kDa, and the third molecular weight cutoff interval is greater than 10kDa.

[0013] In a preferred technical solution of the present invention, step S4 includes using 0.1% trifluoroacetic acid aqueous solution and 100% acetonitrile as mobile phases, and eluting the fraction with acetonitrile at a concentration of 4%-8%; when the concentration of the sample solution to be purified is 100 g / mL and the injection volume is 10 mL, diluting it with ultrapure water and filtering it with a 0.22 μm filter to extract the first antibacterial activity fraction.

[0014] In a preferred technical solution of the present invention, after extracting the first antimicrobial activity fraction, the method further includes re-dissolving the first antimicrobial activity fraction in double-distilled water and loading it onto a C18 chromatographic column; using 0.1% trifluoroacetic acid aqueous solution and 100% methanol as the mobile phase, and using 3% methanol for isocratic elution at a flow rate of 100 mL / min for 120 minutes to extract the second antimicrobial activity fraction.

[0015] In a preferred technical solution of the present invention, the extraction of the second antimicrobial activity fraction further includes re-dissolving the second antimicrobial activity fraction in double-distilled water and loading it onto an ECOSIL C18 chromatographic column; using 100% pure water as the mobile phase, eluting with 100% pure water at a flow rate of 0.8 mL / min for 60 minutes to extract the third antimicrobial activity fraction; and concentrating, freezing and drying the third antimicrobial activity fraction to obtain the antimicrobial peptide.

[0016] In a preferred technical solution of the present invention, in step S2, the fermentation broth is centrifuged and the supernatant after centrifugation is collected; the centrifugal speed is 4000 rpm and the centrifugation time is 20 min; the supernatant after centrifugation is concentrated using a rotary evaporator to obtain a concentrated solution; the concentrated solution is sequentially frozen, dried and filtered to obtain a crude peptide solution.

[0017] In a preferred technical solution of the present invention, in step S1, defatted soybean meal and water are mixed at a material-liquid ratio of 1:12 to obtain a mixed liquid; the mixed liquid is boiled for 15 minutes, and the mixed liquid is filtered to remove residue in the mixed liquid to obtain a soybean meal base.

[0018] In a preferred technical solution of the present invention, in step S1, the inoculation amount of the Lactobacillus paracasei FX-6 strain is 1×10 8 CFU, the concentration of anhydrous glucose is 2%, the fermentation temperature is 37° C., and the fermentation time is 5 days.

[0019] The present invention also provides an application of antimicrobial peptides in lactobacillus fermentation products. The antimicrobial peptides are prepared by adopting the separation method of antimicrobial peptides in lactobacillus fermentation products, and the antimicrobial peptides are used to inhibit the growth of Helicobacter pylori.

[0020] The beneficial effects of the present invention are:

[0021] The present invention provides a method for isolating antimicrobial peptides from lactobacillus fermentation products, comprising preparing a soybean meal base, adding an activated Lactobacillus paracasei FX-6 strain and anhydrous glucose to the soybean meal base for fermentation to obtain a fermentation broth. A crude peptide solution is extracted from the fermentation broth, and the crude peptide solution is subjected to fractional filtration using multiple ultrafiltration membranes to extract and separate components to obtain a fractionated liquid. The fractionated liquid is purified multiple times to obtain the antimicrobial peptides. In the method, defatted soybean meal and water are mixed at a material-to-liquid ratio of 1:12, residue is removed, and the activated Lactobacillus paracasei FX-6 strain is inoculated into the soybean meal base. 2% anhydrous glucose is added for fermentation. After fermentation, the fermentation broth is centrifuged at 4000 rpm for 20 minutes, the supernatant is collected, and the supernatant is concentrated to obtain a crude peptide solution, which is a crude extract containing antimicrobial peptides. The crude peptide solution is subjected to fractional filtration using multiple ultrafiltration membranes to collect and separate the components, and the different components are evaluated for their anti-Helicobacter pylori activity. Antimicrobial peptides were extracted from the fraction using a preparative RP-HPLC system using a three-step purification process. Ultrafiltration separation yielded fractions with a molecular weight of less than 5 kDa, which exhibited the most significant antimicrobial activity against Helicobacter pylori. Low-molecular-weight peptides have smaller molecular structures and are more easily absorbed and metabolized by organisms, thus enhancing their biological effects. The antimicrobial peptides prepared by this invention possess an α-helical structure and can effectively inhibit the growth of Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of the method for separating antimicrobial peptides from lactobacillus fermentation products of the present invention;

[0023] Figure 2 This is an analytical liquid chromatogram of the first purification of the fractionated liquid according to the present invention;

[0024] Figure 3 This is an analytical liquid chromatogram of the second purification of the fractionated liquid according to the present invention;

[0025] Figure 4 This is an analytical liquid chromatogram of the third purification of the fractionated liquid according to the present invention;

[0026] Figure 5 This is an experimental diagram showing the growth inhibition of Helicobacter pylori by FxCy2 at different concentrations of the present invention. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although preferred embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to make the present invention more thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0028] Example 1

[0029] like Figure 1 As shown, this embodiment provides a method for separating antimicrobial peptides from Lactobacillus fermentation products, comprising:

[0030] S1: preparing a soybean meal base, adding the activated Lactobacillus paracasei FX-6 strain and anhydrous glucose to the soybean meal base for fermentation to obtain a fermentation liquid;

[0031] S2: extracting a crude peptide solution from the fermentation broth;

[0032] S3: using multiple ultrafiltration membranes to perform graded filtration on the crude peptide solution to extract and separate components to obtain a fractionated liquid;

[0033] S4: Purifying the fractionated liquid multiple times to obtain antimicrobial peptides.

[0034] In step S3, the crude peptide solution is subjected to graded filtration using a first ultrafiltration membrane with a molecular weight cutoff of 5 kDa and a second ultrafiltration membrane with a molecular weight cutoff of 10 kDa, respectively, to extract components separated in the first molecular weight cutoff interval, the second molecular weight cutoff interval, and the third molecular weight cutoff interval to obtain a fractionated liquid.

[0035] The first molecular weight cutoff interval is less than 5 kDa, the second molecular weight cutoff interval is greater than or equal to 5 kDa and less than or equal to 10 kDa, and the third molecular weight cutoff interval is greater than 10 kDa.

[0036] This study used Helicobacter pylori ATCC 43504 as an indicator bacterium to analyze the active ingredients in the fermentation broth and extract effective antibacterial substances. The crude peptide solution contained a crude extract, which exhibited significant antibacterial activity at concentrations of 32 mg / mL, 64 mg / mL, and 128 mg / mL. However, given the complex composition of the crude extract, further separation and purification were required to screen for effective antibacterial components.

[0037] The present invention uses a first ultrafiltration membrane and a second ultrafiltration membrane to perform ultrafiltration separation on a crude peptide solution to separate three components with different molecular weights, wherein the size of the first component is less than 5 kDa, the size of the second component is greater than or equal to 5 kDa and less than or equal to 10 kDa, and the size of the third component is greater than 10 kDa.

[0038] After processing through two ultrafiltration membranes, the three size fractions were evaluated for their antimicrobial activity against Helicobacter pylori. Fractions exhibiting significant antimicrobial activity were frozen, dried, and stored at -20°C. Fractions smaller than 5 kDa exhibited the most significant antimicrobial activity against Helicobacter pylori. There is a close correlation between the molecular weight of protein hydrolysates and their antimicrobial activity. Low-molecular-weight peptides have smaller molecular structures and are more easily absorbed and metabolized by organisms, thereby enhancing their biological effects.

[0039] The multiple purification steps in step S4 include gradually adding anhydrous ethanol to the fermentation supernatant until it reaches 70% saturation in an ice bath, and then allowing the mixture to stand overnight at 4°C. The mixture is then centrifuged to separate the precipitate from the supernatant. The supernatant is then extracted, frozen, dried, and stored at -20°C.

[0040] The present invention monitors all eluted fractions during the purification process and uses a preparative RP-HPLC system to separate peptides through a three-step purification procedure. The first purification uses 0.1% trifluoroacetic acid aqueous solution, i.e., solvent A, and 100% acetonitrile, i.e., solvent B, as mobile phases, and acetonitrile with a concentration of 4%-8% is used to elute the fraction, and the elution process lasts for 80 minutes. The concentration of the sample solution to be purified is 100 mg / mL, the injection volume is 10 mL, and it is appropriately diluted with ultrapure water and filtered using a 0.22 μm filter membrane. The sample is eluted at a flow rate of 100 mL / min, and the absorbance value at a wavelength of 214 nm is measured in real time. The three fractions are collected and concentrated, frozen, and dried, and then tested for antibacterial activity.

[0041] The dried fraction with the highest antimicrobial activity from the first purification step was redissolved in double-distilled water and reloaded onto a C18 column for a second purification step. The conditions were changed to solvent A: 0.1% trifluoroacetic acid in water and solvent B: 100% methanol. Isocratic elution was performed with 3% methanol at a flow rate of 100 mL / min for 120 minutes. The five fractions eluted from the second purification step were collected, concentrated, frozen, and dried before being assayed for antimicrobial activity.

[0042] The dried fraction with the highest antimicrobial activity from the second purification step was redissolved in double-distilled water and reloaded onto an ECOSIL C18 column for a third purification step. The purification process was performed for 60 minutes using 100% pure water as the mobile phase at a flow rate of 0.8 mL / min. The four fractions eluted from the third purification step were collected, concentrated, frozen, and dried to yield the antimicrobial peptide.

[0043] This embodiment provides a method for isolating antimicrobial peptides from a lactobacillus fermentation product, comprising preparing a soybean meal base, adding an activated Lactobacillus paracasei FX-6 strain and anhydrous glucose to the soybean meal base for fermentation to obtain a fermentation broth. A crude peptide solution is extracted from the fermentation broth, and the crude peptide solution is subjected to fractional filtration using multiple ultrafiltration membranes to extract and separate components to obtain a fractionated liquid. The fractionated liquid is then purified multiple times to obtain the antimicrobial peptides. In the present invention, defatted soybean meal and water are mixed at a material-to-liquid ratio of 1:12, residue is removed, and the activated Lactobacillus paracasei FX-6 strain is inoculated into the soybean meal base. Fermentation is then added with 2% anhydrous glucose. After fermentation, the fermentation broth is centrifuged at 4000 rpm for 20 minutes, the supernatant is collected, and the supernatant is concentrated to obtain a crude peptide solution, which serves as a crude extract containing antimicrobial peptides. The crude peptide solution is subjected to fractional filtration using multiple ultrafiltration membranes to collect and separate the components, and the different components are evaluated for their anti-Helicobacter pylori activity. Antimicrobial peptides were extracted from the fraction using a preparative RP-HPLC system using a three-step purification process. Ultrafiltration separation yielded fractions with a molecular weight of less than 5 kDa, which exhibited the most significant antimicrobial activity against Helicobacter pylori. Low-molecular-weight peptides have smaller molecular structures and are more easily absorbed and metabolized by organisms, thus enhancing their biological effects. The antimicrobial peptides prepared by this invention possess an α-helical structure and can effectively inhibit the growth of Helicobacter pylori.

[0044] Example 2

[0045] This example is based on Example 1 and describes the differences from Example 1. This example provides a method for separating antimicrobial peptides from Lactobacillus fermentation products, comprising:

[0046] S1: preparing a soybean meal base, adding the activated Lactobacillus paracasei FX-6 strain and anhydrous glucose to the soybean meal base for fermentation to obtain a fermentation liquid;

[0047] S2: extracting a crude peptide solution from the fermentation broth;

[0048] S3: using multiple ultrafiltration membranes to perform graded filtration on the crude peptide solution to extract and separate components to obtain a fractionated liquid;

[0049] S4: Purifying the fractionated liquid multiple times to obtain antimicrobial peptides.

[0050] In step S1, defatted soybean meal and water were mixed at a material-liquid ratio of 1:12 (w / v), boiled for 15 min, and then filtered to remove the residue. The activated Lactobacillus paracasei FX-6 strain was inoculated into the soybean meal substrate at an inoculum size of 1×10 8CFU, 2% anhydrous glucose was added, and the mixture was fermented at 37°C for 5 days. After the fermentation, the fermentation broth was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected and concentrated using a rotary evaporator. The concentrated supernatant was then frozen and dried to obtain a crude peptide solution.

[0051] like Figure 2 As shown, in this example, the small molecule antimicrobial peptide component obtained by ultrafiltration was purified by preparative RP-HPLC. 0.1% trifluoroacetic acid aqueous solution was used as solvent A and 100% acetonitrile was used as solvent B. The crude peptide solution containing the crude peptide extract was separated to obtain three main peaks, among which the F1 peak showed a clear antimicrobial zone, indicating that the small molecule antimicrobial peptide component had antimicrobial activity.

[0052] like Figure 3 To further purify the active components of peak F1, the fractions corresponding to peak F1 were purified a second time on a C18 column, yielding five peaks: F1-1, F1-2, F1-3, F1-4, and F1-5. Antimicrobial activity testing was performed on the five freeze-dried fractions, and the results showed that fraction F1-2 exhibited significant antimicrobial activity, exhibiting a clear antimicrobial zone.

[0053] like Figure 4 As shown, F1-2 was further purified by analytical high-performance liquid chromatography (HPLC), i.e., the third purification, yielding four significant peaks: F1-2-1, F1-2-2, F1-2-3, and F1-2-4. In the antibacterial activity evaluation experiment, F1-2-2 exhibited the strongest antibacterial activity and was designated FxCy2. Figure 2 This is the analytical liquid chromatogram of the first purification of the fraction. Figure 3 This is the analytical liquid chromatogram of the second purification of the fraction. Figure 4 This is the analytical liquid chromatogram of the third purification of the fraction. Figure 2-Figure 3 The horizontal axis is the reaction time, the unit is min, and the vertical axis is the absorbance intensity, the unit is mAU, that is, milliabsorptivity. Figure 4 The horizontal axis is the reaction time, the unit is min, and the vertical axis is the voltage, the unit is millivolt.

[0054] Mobile phase A was 2% acetonitrile and 0.1% formic acid in water, and mobile phase B was 80% acetonitrile and 0.1% formic acid in water. A linear gradient was used to identify the substance of F1-2-2 over an 8-minute gradient at a flow rate of 300 nL / min. The linear gradient was as follows: 0-1 minute, 4% to 12.5%; 1-3.6 minutes, 12.5% ​​to 22.5%; 3.6-5.8 minutes, 22.5% to 45%; and 5.8-8 minutes, 45% to 99%.

[0055] In this example, defatted soybean meal was mixed with water at a solid-liquid ratio of 1:12 (w / v), boiled for 15 minutes, and then filtered to remove the residue. The activated Lactobacillus paracasei FX-6 strain was inoculated into the soybean meal substrate at an inoculum size of 1×10 8 CFU, 2% anhydrous glucose was added and fermented at 37°C for 5 days. After fermentation, the crude peptide solution was obtained by centrifugation, concentration, freezing and drying.

[0056] Example 3

[0057] This example discloses an application of an antimicrobial peptide from a Lactobacillus fermentation product. The antimicrobial peptide is prepared using the method for isolating the antimicrobial peptide from a Lactobacillus fermentation product in Example 1 or Example 2. The antimicrobial peptide is used to inhibit the growth of Helicobacter pylori. The amino acid sequence of the antimicrobial peptide prepared in this example is FGANALLGVSLAVKCG.

[0058] The culture conditions for Helicobacter pylori in this example are as follows: Helicobacter pylori was provided by Ningbo Mingzhou Biotechnology Co., Ltd., strain number ATCC 43504, and was stored at -80°C in BHI liquid medium containing 20% ​​glycerol. To obtain an inoculum, the cells were cultured on Columbia blood agar medium containing 7% sterile defibrinated sheep blood and incubated under microaerophilic conditions (5% O2, 10% CO2, 85% N2) at 37°C for 3 days.

[0059] The inhibitory effect of different concentrations of FxCy2 on Helicobacter pylori: Figure 5As shown, treatment with different concentrations of FxCy2 revealed significant changes during the rapid growth phase (36-72 hours) of H. pylori. As the concentration of FxCy2 increased, H. pylori growth was gradually inhibited, and the inhibitory effect increased with increasing concentration. At a high concentration of 2 MIC, FxCy2 exhibited the most significant inhibitory effect on bacterial growth; at a lower concentration of 1 / 2 MIC, FxCy2 only exhibited a more modest inhibitory effect on bacterial growth. Furthermore, at concentrations of 1× and 2× MIC, all bacterial growth was completely inhibited within 60 hours. These results demonstrate that FxCy2 exhibits concentration-dependent antibacterial activity against H. pylori, suggesting its promising research and development as a potential antibacterial agent. The minimum inhibitory concentration (MIC) is a key indicator in microbiology and antibacterial testing. It measures the efficacy of an antibacterial agent against a specific microorganism; lower MIC values ​​indicate stronger antibacterial activity.

[0060] This example uses a method for isolating antimicrobial peptides from lactobacillus fermentation products to prepare antimicrobial peptides, which are then used to inhibit the growth of Helicobacter pylori. The comparative experiment in this example uses reaction time (in hours) as the independent variable and OD (optical density) at a wavelength of 600 nm as the dependent variable, as measured by a microplate reader. The experimental results show that the greater the MIC value, i.e., the higher the FxCy2 concentration, the more significant the growth inhibition of Helicobacter pylori. At concentrations of 1×MIC and 2×MIC, all bacteria were completely inhibited within 60 hours, demonstrating that FxCy2 has concentration-dependent antimicrobial activity against Helicobacter pylori.

[0061] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, apparatus, article, or method comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, apparatus, article, or method. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, apparatus, article, or method comprising the element.

[0062] The above description is only a preferred embodiment of the present application and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for separating antimicrobial peptides from lactobacillus fermentation products, characterized in that: include: S1: preparing a soybean meal base, adding the activated Lactobacillus paracasei FX-6 strain and anhydrous glucose to the soybean meal base for fermentation to obtain a fermentation liquid; S2: extracting a crude peptide solution from the fermentation broth; S3: using different ultrafiltration membranes to perform graded filtration on the crude peptide solution, extracting and separating components to obtain fractionated liquids; S4: Purifying the fractionated liquid multiple times to obtain antimicrobial peptides.

2. The method for separating antimicrobial peptides from lactobacillus fermentation products according to claim 1, wherein In step S3, the crude peptide solution is subjected to graded filtration using a first ultrafiltration membrane with a molecular weight cutoff of 5 kDa and a second ultrafiltration membrane with a molecular weight cutoff of 10 kDa, respectively, to extract components separated in the first molecular weight cutoff interval, the second molecular weight cutoff interval, and the third molecular weight cutoff interval to obtain a fractionated liquid.

3. The method for separating antimicrobial peptides from lactobacillus fermentation products according to claim 2, wherein: The first molecular weight cutoff interval is less than 5 kDa, the second molecular weight cutoff interval is greater than or equal to 5 kDa and less than or equal to 10 kDa, and the third molecular weight cutoff interval is greater than 10 kDa.

4. The method for separating antimicrobial peptides from lactobacillus fermentation products according to claim 1, wherein In step S4, 0.1% trifluoroacetic acid aqueous solution and 100% acetonitrile are used as mobile phases, and the fraction is eluted with 4%-8% acetonitrile; when the concentration of the sample solution to be purified is 100 mg / mL and the injection volume is 10 mL, it is diluted with ultrapure water and filtered with a 0.22 μm filter to extract the first antibacterial activity fraction.

5. The method for separating antimicrobial peptides from lactobacillus fermentation products according to claim 4, wherein: After extracting the first antibacterial activity fraction, the method further includes re-dissolving the first antibacterial activity fraction in double-distilled water and loading it onto a C18 chromatographic column; using 0.1% trifluoroacetic acid aqueous solution and 100% methanol as mobile phases, and using 3% methanol for isocratic elution at a flow rate of 100 mL / min for 120 minutes to extract the second antibacterial activity fraction.

6. The method for separating antimicrobial peptides from lactobacillus fermentation products according to claim 5, characterized in that: The extracting of the second antimicrobial activity fraction further includes re-dissolving the second antimicrobial activity fraction in double-distilled water and loading the column onto an ECOSIL C18 chromatographic column; using 100% pure water as a mobile phase, eluting with 100% pure water at a flow rate of 0.8 mL / min for 60 minutes to extract the third antimicrobial activity fraction; The third antimicrobial activity fraction is concentrated, frozen and dried to obtain antimicrobial peptides.

7. The method for separating antimicrobial peptides from lactobacillus fermentation products according to claim 1, characterized in that: In step S2, the fermentation broth is centrifuged and the supernatant after centrifugation is collected; the centrifugation speed is 4000 rpm and the centrifugation time is 20 min; the supernatant after centrifugation is concentrated using a rotary evaporator to obtain a concentrate; the concentrate is sequentially frozen, dried and filtered to obtain a crude peptide solution.

8. The method for separating antimicrobial peptides from lactobacillus fermentation products according to claim 1, characterized in that: In step S1, defatted soybean meal and water are mixed at a material-liquid ratio of 1:12 to obtain a mixed solution; the mixed solution is boiled for 15 minutes, and the mixed solution is filtered to remove residue in the mixed solution to obtain a soybean meal base.

9. The method for separating antimicrobial peptides from lactobacillus fermentation products according to claim 1, characterized in that: In step S1, the inoculation amount of the Lactobacillus paracasei FX-6 strain is 1×10 8 CFU, the concentration of anhydrous glucose is 2%, the fermentation temperature is 37° C., and the fermentation time is 5 days.

10. An application of antimicrobial peptides in lactobacillus fermentation products, characterized in that: The antimicrobial peptide is prepared by the method for separating the antimicrobial peptide from the lactobacillus fermentation product according to any one of claims 1 to 9, and the antimicrobial peptide is used to inhibit the growth of Helicobacter pylori.

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