CN115433258A - Antibacterial polypeptide and application thereof - Google Patents

Abstract

The invention belongs to the technical field of food preservation, and in particular relates to an antibacterial polypeptide screened from fermented walnut dregs and its application. The present invention uses compound probiotics to ferment walnut dregs to improve the antibacterial activity against dominant bacteria of Rosa roxburghii, explores the changes of polypeptides with antibacterial activity in fermented walnut dregs, and screens the polypeptides FGGDSTHP, ALGGGY, YVVPW, PLLRW with antibacterial activity. The interaction mechanism between antibacterial peptides and phenolic compounds was further analyzed, and the optimal antibacterial combination YVVPW‑SA was obtained to improve the synergistic antibacterial activity of the two against P.victoriae.

Description

A kind of antibacterial polypeptide and its application

Technical field:

The invention belongs to the technical field of food preservation, and in particular relates to an antibacterial polypeptide screened from fermented walnut dregs and an application thereof.

Background technique:

Studies have reported that the loss and waste of fruits and vegetables in the postharvest part of the supply chain is as high as 13% to 38%. Therefore, relevant research is urgently needed to extend the shelf life of fruits and vegetables, thereby reducing postharvest losses. The current research on postharvest preservation of fruits and vegetables focuses on adding some antibacterial agents such as organic acids (benzoic acid and sorbic acid, etc.), enzymes (lysozyme), bacteriocin (nisin) and polyphenols (curcumin and quercetin, etc.) , but the existing natural antibacterial agents have problems such as high cost and unclear targeting.

Based on previous research in the laboratory, the fermentation of walnut meal can produce metabolites such as organic acids, volatiles, polypeptides and free amino acids through microorganisms and enzymes. These metabolites can pass through the cell membrane of microorganisms and accumulate in the cytoplasm to exert antibacterial activity. In addition, the biological activity of plant extracts is usually not due to the existence of a single component, but the effect produced by the interaction of multiple active components.

Therefore, the present application will prepare a fermented product with antibacterial activity to the dominant bacteria of Rosa roxburghii, and use it to develop natural and low-cost antibacterial products, in order to prolong the storage period of food, especially fruits and vegetables, and to preserve the freshness of fruits and vegetables for fermented walnut meal. The application in provides a theoretical basis.

Invention content:

In this study, compound probiotics were used to ferment walnut meal to improve the antibacterial activity against the dominant bacteria of Rosa roxburghii, explore the changes of polypeptides with antibacterial activity in fermented walnut meal, and screen the peptides with antibacterial activity, and analyze the antibacterial peptides and phenolic compounds. The interaction mechanism between them, and obtain the optimal antibacterial combination.

Maestro软件分子对接探究抗菌肽与Rho1GTPase晶体结构(PDB ID:3A58)的结合方式。采用二倍稀释法复配抗菌肽与酚类化合物，测定复合物对P.victoriae的抗菌活性，并通过傅里叶红外光谱(FTIR)、荧光光谱、分子对接探究抗菌多肽与酚类化合物之间的互作机理。In order to solve the above-mentioned technical problems, the present invention takes the bacteriostasis effect on Penicillium victoriae (Penicillium.victoriae), the dominant bacterium of thorn pear spoilage, as an index, and adopts Bacillus subtilis (Bacillus Subtilis) and plant lactobacillus (Lactiplantibacillus plantarum) to compound ferment walnut dregs . The changes of phenolic compounds before and after fermentation were explored, and the phenolic compounds with the strongest antibacterial activity against P.victoriae in fermented walnut meal were screened. The antibacterial activity of fermented walnut meal with different molecular weights was compared, the peptide sequence of the fermented walnut meal with the strongest antibacterial activity was identified by LC-MS/MS, and the antibacterial activity was verified by virtual screening with PyRx software. pass

Molecular docking using Maestro software to explore the binding mode of antimicrobial peptides and the crystal structure of Rho1GTPase (PDB ID: 3A58). Antibacterial peptides and phenolic compounds were compounded by double dilution method, and the antibacterial activity of the compound against P.victoriae was determined, and the relationship between antibacterial peptides and phenolic compounds was explored by Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy, and molecular docking interaction mechanism.

One of the technical solutions provided by the present invention is an antimicrobial peptide extracted from fermented walnut dregs, the amino acid sequences of the antimicrobial peptide from the N terminal to the C terminal are respectively Phe-Gly-Gly-Asp-Ser-Thr-His-Pro (FGGDSTHP ), Ala-Leu-Gly-Gly-Gly-Tyr (ALGGGY), Tyr-Val-Val-Pro-Trp (YVVPW) and Pro-Leu-Leu-Arg-Trp (PLLRW);

Furthermore, the antimicrobial peptide YVVPW has the strongest antibacterial activity against P.victoriae, with an MIC of 16.0mg/mL; at the same time, it has antibacterial activity against S.aureus;

Further, the antibacterial peptide FGGDSTHP has antibacterial activity against P.victoriae;

Further, the antibacterial peptide ALGGGY has antibacterial activity against both P.victoriae and S.aureus;

Furthermore, the antimicrobial peptide PLLRW has antibacterial activity against S.aureus.

The second technical solution provided by the present invention is an antibacterial composition comprising antibacterial peptide YVVPW and salicylic acid (SA);

Further, the concentration of the antimicrobial peptide YVVPW in the composition is 1.0 mg/mL-4.0 mg/mL, and the concentration of SA is 0.27 mg/mL-1.10 mg/mL;

Preferably, the concentration of the antimicrobial peptide YVVPW in the composition is 1.0mg/mL, and the concentration of SA is 0.27mg/mL. At this time, the synergistic effect of the combination of the two is the best, and the graded inhibitory concentration index is 0.07, which is 0.07 for P. victoriae. The inhibition zone reached 7.35±0.20mm.

Preferably, the concentration of the antimicrobial peptide YVVPW in the composition is 2.0mg/mL, the concentration of SA is 0.55mg/mL, and the graded inhibitory concentration index FIC is 0.25. At this time, the combination of the two has the highest antibacterial activity against P. victoriae, and the antibacterial activity against The inhibition zone of P.victoriae reached 11.50±0.41mm.

The third technical solution provided by the present invention is the application of the antimicrobial peptide described in the first technical solution or the antibacterial composition described in the second technical solution, especially in food preservation, more particularly in the preservation of fruits and vegetables, and further, is Application in Rosa roxburghii preservation.

Beneficial effect:

The invention provides a group of fermented walnut dregs antibacterial peptides which have an inhibitory effect on dominant roxburghii spoilage bacteria. The antibacterial peptides have obvious antibacterial effects, and walnut dregs are used as raw materials, so the production cost is low and the economic benefit is high.

Based on the test results, among the phenolic compounds in fermented walnut meal, SA had the highest antibacterial activity against P. victoriae (MIC was 4.40 mg/mL). The antibacterial activity of fermented walnut meal by ultrafiltration was measured against P. victoriae. The results showed that the fermented walnut meal with a molecular weight of 0-3 kDa had the highest antibacterial activity (the MIC of the freeze-dried powder was 10 μg/mL), and peptides such as FGGDSTHP, ALGGGY and YVVPW all had the highest antibacterial activity against P. victoriae. victoriae has antibacterial activity, and PLLRW has antibacterial activity against S.aureus. The synthetic short peptide sequence YVVPW has been proved to have high antibacterial activity against P.victoriae (MIC is 16.0mg/mL). The concentrations of YVVPW and SA combined to exert synergistic antibacterial effect were 1.0-4.0 mg/mL YVVPW and 0.27-1.10 mg/mL SA, respectively, and the two had synergistic antibacterial activity against P.victoriae. The addition of SA quenched the fluorescence of the Trp and Tyr residues of YVVPW, and hydrogen bonds, hydrophobic interactions, and π-π stacking were the main interaction forces between them.

Description of drawings:

Fig. 1 unfermented walnut meal lyophilized powder negative ion (A) and positive ion (B) chromatogram;

Among them, 1. gallic acid; 2. salicylic acid; 3. protocatechin; 4. salicin; 5. chlorogenic acid; 6. procyanidins; 7. (-)-epicatechin; 8. iso Quercitrin; 9. Hyperin; 10. Caffeic acid; 11. Ellagic acid; 12. Trans-ferulic acid; 13. Phloridin; 14. Naringenin-7-O-glucoside; 15 . Naringenin.

Fig. 2 fermented walnut meal lyophilized powder negative ion (A) and positive ion (B) chromatogram;

Among them, 1. gallic acid; 2. salicylic acid; 3. protocatechin; 4. salicin; 5. chlorogenic acid; 6. procyanidins; 7. (-)-epicatechin; 8. iso Quercitrin; 9. Hyperin; 10. Caffeic acid; 11. Ellagic acid; 12. Trans-ferulic acid; 13. Phloridin; 14. Naringenin-7-O-glucoside; 15 . Naringenin.

Fig. 3 LC-MS analysis base peaks of 0-3kDa fermented walnut meal freeze-dried powder.

Fig. 4 MS/MS spectrum (A) and chemical structure (B) of peptide YVVPW.

Figure 5. The three-dimensional structure (A) of peptide YVVPW binding to Rho1 GTPase, the binding mode of the active site (B) and the schematic plan view of the best conformational interaction (C).

The fluorescence spectrum of polypeptide YVVPW in the presence of different concentrations of SA in Fig. 6;

Figure 7 Fourier transform infrared spectra of YVVPW, SA and YVVPW-SA.

Figure 8 The binding mode of YVVPW and SA, (A) the electrostatic surface of the complex; (B) the binding mode of the active site.

detailed description:

In order to make the purpose, technical solutions and advantages of this patent more clear, the following will further describe this patent in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain the patent, not to limit the present invention.

The polypeptide of the present invention can be obtained by those skilled in the art through artificial synthesis, and can also be obtained by fermenting and screening walnut dregs.

The plant Lactobacillus used for fermenting the walnut meal in the present invention is Lactobacillus plantarum (Lactiplantibacillus plantarum) CICC 23121, and the Bacillus subtilis used in the present invention is Bacillus subtilis (Bacillus Subtilis) CICC 10002.

The present invention uses B.Subtilis and L.plantarum to ferment walnut dregs for 26 hours at a fermentation temperature of 35° C., qualitatively and quantitatively analyze the structure of phenolic compounds in fermented walnut dregs, and detect their effects on Penicillium victoriae, Staphylococcus aureus ( Staphylococcus aureus) and Escherichia coli (Escherichia coli), the highest antifungal activity of SA was obtained; fermented walnut meal was separated by ultrafiltration to obtain different molecular weight ranges (0～3kDa, 3～5kDa, 5～10kDa, >10kDa) The components of fermented walnut meal were tested for their antibacterial activity against P.victoriae, S.aureus and E.coli, and the 0-3kDa fermented walnut meal with the highest antibacterial activity after ultrafiltration was obtained; the structure of the polypeptide of the lyophilized powder of fermented walnut meal was determined 143 peptides were selected by molecular docking technology to screen out the peptide sequences with low docking energy when docking with the Rho1 GTPase crystal structure (PDBID: 3A58), and then the corresponding peptides were artificially synthesized to detect their effect on P. victoriae, S. Antibacterial activity of aureus and E.coli, high antibacterial short peptide sequences FGGDSTHP, ALGGGY, YVVPW and PLLRW in fermented walnut meal. Molecular docking of the polypeptide YVVPW with the highest antibacterial activity and Rho1GTPase was carried out by molecular docking technology, and the results showed that the two were closely combined, and the interaction forces mainly included hydrogen bonds, hydrophobic interactions and π-π stacking.

The part test method that the present invention relates to is as follows:

(1) Inhibition zone test:

Penicillium victoriae was the dominant rot bacterium of Rosa roxburghii, and the antibacterial effect on P. victoriae, Escherichia coli and Staphylococcus aureus was used as the screening index to determine the antibacterial effect of different groups. bacteriostatic activity. Pipette 100 μL of P.victoriae spore suspension with a concentration of 10 ⁵ CFU/mL on PDA medium, and 100 μL of S.aureus and E.coli spore suspension with a concentration of 10 ⁵ CFU/mL on LB medium. Use a sterile Oxford cup to punch holes and add 100 μL of fermented walnut meal (you can replace it when measuring the inhibition zone of other components), use sterile water as a negative control and natamycin as a positive control. The diameter of the inhibition zone was measured after the PDA and LB plates were cultured in a 28°C incubator for 5 days and 24 hours, respectively.

(2) Determination of polyphenol content of fermented walnut meal

Referring to the method of Folin and Ciocalteu (1927), accurately weigh 10 mg of gallic acid, dissolve it in ultrapure water and dilute it into a 100 mL volumetric flask, shake it up for later use, and obtain the gallic acid standard solution. Accurately draw standard solutions 0, 0.5, 1.0, 1.5, 2.0 and 2.5mL into a 10mL colorimetric tube, add 1.0mL (50%) of Folin’s phenol reagent and 2mL (15%) of NaCO ₃ solution in turn, and set the volume to 10mL, After mixing well, let it stand at room temperature for 1 hour, measure the absorbance value at a wavelength of 760nm, and establish a standard curve. Draw 1.0 mL of fermented walnut meal to determine the polyphenol content according to the standard curve determination method, and use the fermented walnut meal without reaction solution as a blank sample.

(3) Determination of polypeptide content of fermented walnut meal

With reference to Lu Wei et al. (2005), the yield of the polypeptide was determined by the biuret method. Mix 1 mL of Gly-Gly-Tyr-Arg tetrapeptide with different concentrations and 4 mL of biuret reagent, let it stand for 60 min, measure the absorbance value at a wavelength of 540 nm, and establish a standard curve. Take 1 mL of fermented walnut meal, add 1 mL of 10% (w/v) trichloroacetic acid solution, shake well, let stand at room temperature for 10 min, and centrifuge at 6000 r/min for 10 min to obtain supernatant. Take 1 mL of supernatant and mix with 4 mL of biuret reagent, let it stand for 60 min, and measure the absorbance value at 540 nm wavelength. The polypeptide concentration of fermented walnut meal was obtained from the standard curve equation and the polypeptide content was calculated.

(4) Composition and changes of free amino acids before and after fermentation

The composition and change of free amino acids before and after walnut meal fermentation were determined according to "Determination of Amino Acids in Food" (GB5009.124-2016), and the instrument used was an amino acid analyzer (ninhydrin post-column derivatization ion exchange chromatography).

(5) Composition and changes of phenolic compounds before and after fermentation

Metabolite extraction:

Take 100 mg of fermented walnut dregs freeze-dried powder, add 900 μL of deionized water, sonicate for 15 minutes in an ice bath, centrifuge at 12000 r/min for 10 minutes, take the supernatant and filter it with a 0.45 μm membrane before injecting samples for detection. Take 0.1 g of unfermented walnut meal freeze-dried powder, add 1 mL of deionized water, sonicate for 15 min in an ice bath, centrifuge at 12000 r/min for 10 min, take the supernatant and filter it with a 0.45 μm membrane before injecting samples for detection.

Liquid chromatography-mass spectrometry analysis conditions:

The instrument used in this experiment is a Shimadzu ultra-high performance liquid chromatography (LC-30) connected to a SCIEX 5600+ mass spectrometer. The specific parameters are as follows: chromatographic column SHIMADZU InerSustain C 18 (100×2.1 mm, 2 μm), column temperature 35° C., flow rate 0.300 mL/min. Mobile phase A was acetonitrile, and mobile phase B was 0.1% acetic acid solution. The elution gradient is: 0~2min, 5%A; 2~4min, 5~20%A; 4~12min, 20~25%A; 12~14min, 25~46%A; 14~26min, 46~100%A %A; 26-28min, 100%A; 28-30min, 5%A. Mass spectrometry was performed using electrospray ionization (ESI) in positive and negative ion modes. Electrospray ion source (ESI) conditions: ion source temperature 500°C (positive ion) and 450°C (negative ion), ion spray voltage (ISVF) 5500V (positive ion) and 4400V (negative ion), TOF MS scan range 100～1200Da, The product ion scanning range is 50-1000Da, the TOF MS scanning accumulation time is 0.2s, and the product ion scanning accumulation time is 0.01s. The declustering voltage of MS/MS is ±60V, and the collision energy is 35±15eV.

The present invention will be further explained by specific examples below.

Embodiment 1 fermented walnut meal

(1) Strain activation: Lactobacillus plantarum CICC 23121 was picked and transferred to 100 mL of MRS liquid medium, and cultured in a 37°C incubator for 24 hours. Bacillus subtilis CICC 10002 was picked and transferred to 100mL LB liquid medium, and cultured in a 28°C incubator for 24h.

(2) Fermentation substrate of walnut dregs: Take walnut dregs powder that has passed through a 60-mesh sieve and sterilized by ultraviolet light, mix with sterile water (8.0%, w/v), after repeated freezing and thawing, mix with glucose solution sterilized at 115°C for 15 minutes Mix aseptically so that the final concentration of glucose is 2.0% (w/v).

(3) Fermented walnut meal:

① The activated strains Lactobacillus plantarum and Bacillus subtilis were cultured in the seed medium for 18 hours;

②According to the inoculation amount of 6.5% (v/v) respectively, take the above two kinds of bacterial solutions, centrifuge at 4000rpm/min for 10min, discard the supernatant, and resuspend the precipitated bacteria in an equal volume of 0.9% (w/v) in saline;

③The above bacterial suspensions were mixed and inoculated in the walnut meal fermentation substrate, and placed in an incubator at 35°C for fermentation by shaking (180r/min) for 26 hours.

④ Take the shaken walnut meal fermentation broth and perform ultrasonic crushing in an ice bath. Set the ultrasonic crushing conditions as operating frequency 20kHz, power 375W, time 30min, and time interval (working time: interval time) 5:5 (s/s). After the ultrasonication is completed, centrifuge at 8000r/min for 20min (4°C), the obtained supernatant is the fermented walnut meal, and the freeze-dried powder of the supernatant is the fermented walnut meal freeze-dried powder. That is, in the present invention, the supernatant after the ultrasonic crushing of the walnut meal fermentation liquid is the fermented walnut meal. The fermented walnut meal is vacuum freeze-dried to obtain a freeze-dried powder of the fermented walnut meal, which is stored at -80°C.

(4) Determination of antibacterial performance

The supernatant of the walnut meal fermented liquid was ultrasonically crushed for the zone of inhibition test. The zone of inhibition for P.victoria was 13.70±0.10mm, and the zone of inhibition for S.aureus was 25.25±0.50mm.

(5) Determination of polyphenols and polypeptides in fermented walnut meal

The contents of polyphenols and polypeptides in the ultrasonic crushing supernatant of walnut meal fermentation 0h were 50.53±0.22mg/100mL and 7.64±0.15mg/mL, respectively, and the polyphenols and polypeptides in the ultrasonic crushing supernatant after fermentation were 74.25 ±0.09mg/100mL and 15.27±0.15mg/mL.

Example 2 polyphenol identification

(1) Determination of phenols

LC-MS/MS was used to detect phenolic compounds in unfermented walnut meal powder and fermented walnut meal freeze-dried powder, and a total of 15 phenolic compounds were identified (Table 1, Figures 1 and 2), including 7 phenolic acids compounds, 7 flavonoids and 1 phenolic glycoside. Among them, phenolic compounds include gallic acid, salicylic acid (SA), chlorogenic acid, protocatechuic acid, caffeic acid, ellagic acid, and trans-ferulic acid; Theophylline, isoquercitrin, hyperoside, phloridin, naringenin-7-O-glucoside and naringenin, phenolic glycoside compound salicin, and there is a certain structure between these compounds connect.

Among them, gallic acid, caffeic acid, SA and chlorogenic acid increased the most after walnut meal was fermented with probiotics, which were 125 times, 119 times, 117 times and 112 times that of before fermentation, but the content of isoquercitrin decreased by about 50 %, the content of hyperoside was reduced by 90%, and naringenin-7-O-glucoside disappeared.

Table 1 LC-MS/MS analysis of phenolic compounds in unfermented and fermented walnut meal freeze-dried powder

(2) Antibacterial levels of different phenolic compounds

Literature review on the antibacterial activity of 15 phenolic compounds in fermented walnut meal found that gallic acid, SA, naringenin, caffeic acid, ferulic acid, protocatechuic acid and chlorogenic acid may have antibacterial effects. The above seven phenolic compounds were treated with E.coli, S.aureus and P.victoriae (Table 2), and it was found that gallic acid had an antibacterial effect on S.aureus at a concentration of 5.0 mg/mL, and the inhibition zone reached 15.0 ±0.50mm, but no antibacterial effect on E.coli and P.victoriae. When the concentration of SA was 5.0mg/mL, SA had antibacterial effect on S.aureus, the inhibition zone reached 13.0±0.58mm, and it also had antibacterial effect on P.victoriae, the inhibition zone reached 10.0±0.29mm, but it had no effect on E.coli Inhibitory effect. The remaining 5 phenolic compounds including naringenin, caffeic acid, ferulic acid, protocatechuic acid and chlorogenic acid had no antibacterial effect on S.aureus, E.coli and P. victoriae at a concentration of 5.0 mg/mL Effect. The minimum inhibitory concentration (MIC) of SA was determined, and it was found that the MIC of SA to P. victoriae was 4.40 mg/mL.

Table 2 Antibacterial levels of phenolic compounds in fermented walnut meal freeze-dried powder

Note: Different lowercase letters (a~c) and capital letters (A~B) respectively indicate the significant difference (p<0.05) in the antibacterial activity of different phenolic compounds at 5.0 mg/mL against S.aureus and P.victoriae. Sterile water and natamycin were used as negative and positive controls, respectively.

Embodiment 3 polypeptide identification

In this example, ultrafiltration and LC-MS/MS were used to purify and identify the peptide sequence, PyRx virtually screened the characteristic peptides for antibacterial activity, artificially synthesized to verify its antibacterial activity, and molecular docking technology was used to analyze its interaction with receptor proteins.

(1) Antibacterial activity of fermented walnut meal freeze-dried powder with different molecular weight

Unfermented walnut meal and fermented walnut meal freeze-dried powder were sterilized by ultraviolet light, transferred to a centrifuge tube, dissolved in sterile water, and set aside.

Use ultrafiltration membranes with molecular weight cut-offs of 3kDa, 5kDa, and 10kDa to carry out ultrafiltration treatment on fermented walnut meal to purify and enrich to prepare fermented walnut meal with molecular weights of 0-3kDa, 3-5kDa, 5-10kDa and greater than 10kDa . After the end, the fermented walnut meal of each molecular weight was freeze-dried, sterilized by ultraviolet light, transferred to a centrifuge tube and dissolved in sterile water, and set aside.

The antibacterial activity of the above-mentioned different components and the freeze-dried powder of fermented walnut meal with different molecular weights was determined.

The results are as follows: compare the antibacterial effect of fermented walnut meal freeze-dried powder with different components and molecular weights on P.victoriae, as shown in Table 3. The results showed that unfermented walnut meal had no antibacterial activity against P.victoriae, and the MIC of fermented walnut meal freeze-dried powder against P.victoriae was 40μg/mL, and the inhibition zone reached 12.23±0.25mm.

After the fermented walnut meal is subjected to ultrafiltration and freeze-drying with 10kDa, 5kDa and 3kDa membranes, the freeze-dried powder of the fermented walnut meal larger than 10kDa, 5-10kDa, 3-5kDa and 0-3kD is obtained. Comparing the antibacterial activity of 4 groups of lyophilized fermented walnut meal with different molecular weights against P.victoriae, it was found that 5-10kDa and 3-5kDa fermented walnut meal lyophilized powder had no antibacterial activity against P.victoriae. The MIC of freeze-dried powder of fermented walnut meal greater than 10kDa to P.victoriae was 20μg/mL, and the inhibition zone reached 10.33±0.26mm. The MIC of 0～3kDa fermented walnut meal freeze-dried powder to P.victoriae is 10μg/mL, and the inhibition zone reaches 13.18±0.28mm, which is lower than the MIC of fermented walnut meal freeze-dried powder to P.victoriae, which shows that the fermented walnut meal frozen The antibacterial activity of dry powder against P.victoriae mainly comes from the components of 0～3kDa. Subsequently, the identification and analysis of the peptide will be carried out on the 0-3kDa lyophilized powder fraction.

Table 3 The antibacterial level of different fermented walnut meal components freeze-dried powder to P. victoriae

Note: Different capital letters (A～B) in the same column indicate significant differences in the antibacterial activity of different fermented walnut meal components freeze-dried powders against P.victoriae at the same concentration (p<0.05). Sterile water and natamycin were used as negative and positive controls, respectively.

(2) LC-MS/MS peptide composition identification

The freeze-dried powder of fermented walnut dregs of 0~3kDa was subjected to reductive alkylation pretreatment, passed through the pre-column (Acclaim PepMap RPLC C 18, 300μm×5mm, 5μm), and then injected into the analytical column (Acclaim PepMap RPLC C18, 150μm×150mm, 1.9μm). Mobile phase A was 0.1% formic acid and mobile phase B was 0.1% formic acid and 80% acetonitrile. The elution gradient is: 0~2min, 4~8%B; 2~45min, 8~28%B; 45~55min, 28~40%B; 55~56min, 40~95%B; 56~66min, 95%B ~95%B. Primary mass spectrometry parameters: primary mass spectrometry resolution is 70000, AGC target is 3e6, MaximumIT is 40ms, and scanning range is 100-1500m/z. Secondary mass spectrometry parameters: Secondary mass spectrometry resolution is 17500, AGC target is 1e5, Maximum IT is 60ms, TopN is 20, NCE/stepped NCE is 27. Use the software PEAKS Studio8.5 Denovo to analyze the peptide sequence.

Results: LC-MS/MS (Fig. 3) was performed on the freeze-dried powder of fermented walnut meal of 0-3 kDa (Fig. 3) and compared with the database. A total of 143 polypeptide sequences were obtained, including 122 polypeptide sequences with the number of amino acids ≤ 10.

(3) Virtual screening of peptides

Use Chem3D Ultra 14.0 software to draw the 3D structure of the peptide, format conversion and energy minimization. The crystal structure of Rho1 GTPase (PDB ID: 3A58) was obtained from the RCSB database (https://www.rcsb.org). The protein structure was pretreated by hydrogenation with AutodockTools 1.5.6 software, and saved in pdbqt format. Use the PyRx software to set all the peptides as ligands, perform docking scoring and virtual screening with the enzyme in sequence, and judge the strength of the interaction by the docking energy to screen the peptides. Download the walnut protein sequence from the Uniprot protein database (https://www.uniprot.org/proteomes), and use the Geneious software to compare whether the peptides belong to the walnut protein.

Results: β-1,3-glucan synthase is responsible for the synthesis of fungal cell wall component β-1,3-glucan, which consists of at least two subunits. The regulatory subunit Rho1 GTPase has two switches, both of which are involved in the activation of β-1,3-glucan synthase. Therefore, the Rho1 GTPase is critical for fungal cell wall biosynthesis, and this subunit is often a target for the development of new antifungal drugs.

The 122 polypeptide sequences with ≤10 amino acids obtained after LC-MS/MS were subjected to virtual screening with Rho1 GTPase, and the small molecular polypeptides were sorted according to the docking energy. The docking energy is usually a negative number, and the smaller the value, the more stable the binding between the peptide and Rho1 GTPase, that is, the more likely it has antibacterial activity. Because the fermentation product of B. subtilis itself contains cyclolipopeptides with certain antibacterial effects, including iturin and Fengyuansu. Therefore, it is necessary to compare the polypeptide and walnut protein sequences by Geneious software to determine whether the polypeptide belongs to the walnut protein sequence. After sequence comparison, it was found that among the top 10 peptides in docking ability, 4 peptides belonged to the walnut protein sequence, namely FGGDSTHP, ALGGGY, YVVPW and PLLRW (Table 4).

Table 4 The top 10 peptides that can be docked and belong to the walnut protein sequence

(4) Artificially synthesized peptides

FGGDSTHP, ALGGGY, YVVPW and PLLRW peptides were artificially synthesized. The synthesis of peptides adopts the Fmoc solid-phase synthesis method, and peptides are synthesized according to the amino acid sequence, and powdered peptides are obtained after cleavage, precipitation, and purification. The synthesis process was entrusted to Nanjing Yuanpeptide Biotechnology Co., Ltd., and the purity was greater than 95%.

(5) Antibacterial activity of synthetic peptides

Results: The antibacterial activity of the synthetic peptide was evaluated by the diameter of the inhibition zone (Table 5). The study found that the four peptides had no antibacterial activity against E.coli, which was consistent with the aforementioned results that fermented walnut meal and its lyophilized powder had no antibacterial activity against E.coli. For P.victoriae, 16.0mg/mL of 4 kinds of polypeptides were used to treat it sequentially. It was found that the antibacterial activity of polypeptide YVVPW was the best, and the MIC of YVVPW on P.victoriae was 16.0mg/mL. The antibacterial activity of ALGGGY and FGGDSTHP was next, and PLLRW had no antibacterial activity against P.victoriae. For S.aureus, it was treated sequentially with 16.0 mg/mL of 4 peptides, and no antibacterial activity was found. Increase the concentration of peptides, and use 20.0mg/mL peptides to treat S.aureus sequentially. It is found that the antibacterial activity of ALGGGY is greater than that of YVVPW and PLLRW, and FGGDSTHP has no antibacterial activity. Since the microorganisms that cause fruit and vegetable spoilage are mainly fungi, and the dominant rot-causing bacteria of Rosa roxburghii is P.victoriae, the follow-up experiments will study the polypeptide YVVPW. The secondary mass spectrum and chemical structure of YVVPW are shown in Figure 4.

Antibacterial activity of different peptides in table 5

Note: Different lowercase letters indicate a significant difference in the antibacterial activity of different polypeptides at 16.0mg/mL against P. victoriae (p<0.05), and different uppercase letters indicate a significant difference in the antibacterial activity of different polypeptides at 20.0mg/mL against S.aureus (p<0.05). Sterile water and natamycin were used as negative and positive controls, respectively.

(6) Molecular docking peptide YVVPW and Rho1 GTPase

Maestro软件的Glide模块分子对接Rho1GTPase和肽段。利用ProteinPreparation Wizard模块对Rho1 GTPase进行预处理、优化和最小化。导入Rho1 GTPase，确定坐标和盒子大小，选取Rho1 GTPase的预测活性位点作为

盒子的质心，使用SP方法分子对接。The peptide was used as a ligand and imported into Schrodinger software for hydrogenation, structure optimization, and energy minimization. Rho1GTPase was used as the acceptor, processed on the Maestro11.9 platform, and Schrodinger software was used to remove crystal water, add hydrogen, and minimize energy. use

Molecular docking of Rho1GTPase and peptides in the Glide module of Maestro software. Rho1 GTPase was preconditioned, optimized and minimized using the ProteinPreparation Wizard module. Import Rho1 GTPase, determine the coordinates and box size, and select the predicted active site of Rho1 GTPase as

The centroid of the box, molecular docking using the SP method.

Results: Molecular docking was used to simulate the interaction between conformation YVVPW and Rho1 GTPase, and the docking results are shown in Figure 5. The antimicrobial peptide YVVPW was tightly combined with Rho1 GTPase and had a high degree of matching. Hydrogen bonds, hydrophobic interactions and π-π stacking were the main interaction forces between the antimicrobial peptide YVVPW and Rho1 GTPase. The complex formed by antibacterial peptide YVVPW and Rho1 GTPase after docking was visualized using Pymol2.1 software to obtain the binding mode of antimicrobial peptide YVVPW and Rho1 GTPase. According to the binding mode, it can be clearly seen that the amino acid residues interacting between YVVPW and Rho1 GTPase active site are PRO-36, CYS-25, VAL-38, TYR-39, ALA-20, LYS-123, PHE-35, LYS-167, LEU-126, etc. The antimicrobial peptide YVVPW forms four hydrogen bonds with the active site amino acids of Rho1 GTPase, including Tyr residues and LYS-167, Pro residues and LYS-123, Trp residues and PRO-36, Trp residues and VAL- 38. The hydrogen bond distance is shorter and the binding force is stronger, and the hydrogen bond plays an important role in the YVVPW in the cavity of the anchor protein Rho1 GTPase. In addition, the phenyl ring of the antimicrobial peptide YVVPW can form π-π conjugate interactions with TYR-39, CYS-25 and PHE-35, and interact with LYS-167, LYS-123, ALA-20, TYR-39, PRO-36 It forms a hydrophobic interaction force (two kinds of alkyl bond and π-alkyl bond) with CYS-25, which also plays an important role in stabilizing the complex. In summary, the molecular docking simulation conformation clarifies the binding mode of the two from the molecular level and theoretical point of view, and the antibacterial peptide YVVPW binds closely to Rho1GTPase with a high degree of matching.

Embodiment 4YVVPW and SA joint antibacterial activity

In this example, the antimicrobial peptide was combined with SA, and the antibacterial activity of the mixture against P. victoriae was determined, and the structural interaction between the two was explored by using fluorescence spectroscopy, Fourier transform infrared spectroscopy, and molecular docking.

(1) The minimum inhibitory concentration (MIC) of SA and YVVPW against P. victoriae was determined by double dilution method. After drawing 100 μL of P. victoriae spore suspension with a concentration of about 10 ⁵ CFU/mL and spreading it on the PDA medium, punch holes with a sterile Oxford cup, add 100 μL of a certain concentration of SA to the first group, and add 1/2 of the second group to the second group. One concentration of SA, the third group was added with 1/2 of the concentration of SA in the second group, and in turn, a total of 5 groups were measured. Sterile water was used as a negative control, and natamycin was used as a positive control. Place the plate in a 28°C incubator and incubate for 5 days, observe the results, and take the concentration at which no visible colonies appear as the MIC of SA for P.victoriae. The MIC of YVVPW was determined in the same way.

The dilution concentration of antibacterial agent was determined according to the MIC of SA and YVVPW alone. The highest concentration of antibacterial agent was MIC, which was sequentially diluted with sterile water to 1/2, 1/4, 1/8, and 1/16 MIC for 5×5 combination. Place the plate in a 28°C incubator and incubate for 5 days, observe the results, and take the concentration at which no visible colonies appear as the combined MIC of SA and YVVPW for P.victoriae. The joint antibacterial experiment uses the graded inhibitory concentration index (FIC) as the judgment basis:

FIC≤0.5, synergistic effect, the smaller the FIC, the better the synergistic effect; 0.5<FIC≤1, additive effect; 1<FIC≤2, irrelevant effect; FIC>2, antagonistic effect.

Results: The MIC of antimicrobial peptide YVVPW to P. victoriae alone was 16.0 mg/mL, and the MIC of SA to P. victoriae alone was 4.40 mg/mL. The concentration range of the antimicrobial peptide YVVPW in the composition is 1.0 mg/mL-4.0 mg/mL, and the concentration range of SA is 0.27 mg/mL-1.10 mg/mL, when the two have synergistic antibacterial activity (Table 6). When the concentration of antimicrobial peptide YVVPW in the composition is 2.0 mg/mL, and the concentration of SA is 0.55 mg/mL, the FIC value is 0.25, and the antibacterial activity against P. victoriae is the highest, and the inhibition zone reaches 11.50±0.41mm. When the concentration of antibacterial peptide YVVPW in the composition is 1.0 mg/mL, and the concentration of SA is 0.27 mg/mL, the inhibition zone against P. victoriae reaches 7.35±0.20 mm, the FIC value is the lowest, and the synergistic effect is the best.

Table 6 Synergistic antibacterial activity of SA and YVVPW against P. victoriae

Note: Different lowercase letters (a～h) indicate significant differences in the antibacterial activity of SA and YVVPW at different concentrations of synergistic work against P.victoriae (p<0.05). Different capital letters (A～G) indicate significant differences in graded inhibitory concentration indices of different synergistic working concentrations SA and YVVPW (p<0.05).

(2) Fluorescence spectroscopy to explore the interaction between YVVPW and SA

A certain concentration of peptide YVVPW was selected to be mixed with SA, and the excitation wavelength was determined by excitation and emission steady-state spectroscopy. Then 1.0mg/mL of YVVPW was mixed with different concentrations (0, 0.27, 0.55, 1.10, 2.20, 4.40mg/mL) of SA, and the emission spectrum from 270nm to 500nm was recorded.

Results: Fluorescence spectroscopy can be used to explore the quenching behavior and binding constants between active substances, and has been widely used to study the interaction between small molecules and proteins. The antimicrobial peptide YVVPW has intrinsic fluorescence because it contains Trp and Tyr residues, so the interaction between YVVPW and SA can be studied through intrinsic fluorescence quenching. A certain concentration of YVVPW was mixed with SA, and the excitation wavelength was determined to be 261nm by excitation plus emission steady-state spectrum. The changes in the fluorescence spectrum of YVVPW in the presence of different concentrations of SA (0, 0.27, 0.55, 1.10, 2.20, 4.40 mg/mL) were measured, as shown in FIG. 6 . When no SA was added, the maximum absorption wavelength of YVVPW appeared at 368nm. With the increase of SA concentration, the fluorescence intensity of YVVPW gradually decreased, indicating that the fluorescence quenching occurred, and the maximum absorption wavelength of YVVPW was red-shifted. When the added concentration of SA was 4.40mg/mL, the maximum wavelength red shifted to 397nm. These results confirmed that the interaction with SA may change the molecular conformation of the antimicrobial peptide YVVPW, and the Trp and Tyr residues of YVVPW are involved in the interaction.

(3) Fourier transform infrared spectroscopy to explore the interaction between YVVPW and SA

In order to identify the functional groups of YVVPW, SA and YVVPW-SA mixture, the FTIR-ATR measurement mode was used for analysis. The spectra were recorded in the transmittance mode of 400-4000 cm ^-1 , the number of scans was 32, and the resolution was 4 cm ^-1 .

Results: In order to further explore the interaction between the antimicrobial peptide YVVPW and SA, the FTIR results of YVVPW, SA and YVVPW-SA are shown in Figure 7. The absorption peak of YVVPW at 3294.57cm ^-1 is attributed to the -OH stretching vibration peak, the absorption peak at 2968.92cm ^-1 corresponds to the CH stretching vibration on saturated carbon, and the absorption peak at 1658.18cm ^-1 corresponds to the amide I band, It is mainly caused by C=O stretching vibration, and the absorption peak at 1518.26cm ^-1 corresponds to the amide Ⅱ band structure, which is mainly caused by NH bending vibration. The absorption peak of SA at 3231.97 cm ^-1 is mainly related to the stretching vibration of -OH, the absorption peak at 2851.82 cm- ¹ is mainly related to the stretching vibration of CH on saturated carbon, and the absorption peak at 1657.04 cm ^-1 is mainly caused by C = O caused by the stretching vibration, the absorption peaks at 1609.05cm ^-1 and 1557.0cm ^-1 are mainly caused by the skeleton vibration of the benzene ring, and the absorption peaks at 755.54cm ^-1 and 690.77cm ^-1 are mainly related to the CH out-of-plane of the benzene ring related to bending vibration. Compared with YVVPW, YVVPW-SA has a narrow peak at 3403.26cm ^-1 , indicating that there is a hydrogen bond between the two. The absorption peaks of the amide I band and amide II band of the complex were slightly red-shifted, and the relative intensity increased with the addition of SA. Compared with SA, the absorption peaks of YVVPW-SA at 1609.05cm ^-1 , 755.54cm ^-1 and 690.77cm ^-1 were red-shifted to 1613.02cm ^-1 , 756.44cm ^-1 and 698.59cm ^-1 , respectively, and the relative intensities varied with The addition of YVVPW decreased, indicating that there was an intermolecular interaction between YVVPW-SA. Therefore, the results of FTIR indicated that there were hydrogen bonds and intermolecular forces between YVVPW and SA.

(4) Molecular docking to explore the interaction between YVVPW and SA

的氢键，与抗菌肽N端的第2个和第3个Val残基形成键长都为

的氢键。氢键距离短，远小于传统氢键的

表明SA与YVVPW之间的氢键结合力强，氢键对抗菌肽YVVPW锚定SA有着重要作用。另外，SA的苯环可以与YVVPW的Trp残基的五元环形成很强的π-π共轭相互作用，对稳定复合物也有着重要贡献。综上，SA与YVVPW靶点结合紧密，能够形成稳定的复合物，这与本研究的荧光光谱和FTIR结果一致。Molecular docking was used to further verify the interaction mechanism between YVVPW and SA. The docking results are shown in Figure 8. Hydrogen bonds, hydrophobic interactions, and π-π stacking are the main interaction forces between the antimicrobial peptide YVVPW and SA. The antimicrobial peptide YVVPW binds closely to SA with a high matching degree, and the binding energy is -5.27kcal/mol. Using Pymol2.1 software to visualize the complex formed by antimicrobial peptide YVVPW and SA after docking, it was found that SA formed three strong hydrogen bonds with YVVPW, including the bonds with Tyr residues with a length of

The hydrogen bond with the 2nd and 3rd Val residues at the N-terminus of the antimicrobial peptide is both long

of hydrogen bonds. The hydrogen bond distance is short, much smaller than that of traditional hydrogen bonds

It shows that the hydrogen bond between SA and YVVPW is strong, and hydrogen bond plays an important role in the anchoring of antibacterial peptide YVVPW to SA. In addition, the benzene ring of SA can form a strong π-π conjugated interaction with the five-membered ring of Trp residue of YVVPW, which also makes an important contribution to the stability of the complex. In summary, SA binds tightly to the YVVPW target and can form a stable complex, which is consistent with the results of fluorescence spectroscopy and FTIR in this study.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, without departing from the concept of the patent, several modifications, combinations and improvements can be made to the above-mentioned embodiments, all of which belong to the protection scope of the patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims (9)

1. An antimicrobial peptide, characterized in that, the amino acid sequence of the antimicrobial peptide is respectively Phe-Gly-Gly-Asp-Ser-Thr-His-Pro, referred to as FGGDSTHP; or Ala-Leu-Gly-Gly-Gly- Tyr, ALGGGY for short; or Tyr-Val-Val-Pro-Trp, YVVPW for short; or Pro-Leu-Leu-Arg-Trp, PLLRW for short. 2. An antibacterial composition, characterized in that the antibacterial composition comprises antimicrobial peptide YVVPW and salicylic acid SA. 3. antibacterial composition as claimed in claim 2, is characterized in that, the concentration of antimicrobial peptide YVVPW is 1.0mg/mL～4.0mg/mL in the described antibacterial composition, and the concentration of SA is 0.27mg/mL～1.10mg /mL. 4. antibacterial composition as claimed in claim 3, is characterized in that, the concentration of antibacterial peptide YVVPW is 1.0mg/mL in the composition, and the concentration of SA is 0.27mg/mL. 5. antibacterial composition as claimed in claim 3, is characterized in that, the concentration of antimicrobial peptide YVVPW is 2.0mg/mL in composition, and the concentration of SA is 0.55mg/mL. 6. the application of the described antimicrobial peptide of claim 1 or the described antibacterial composition of claim 2. 7. The application according to claim 6, characterized in that it is applied in food preservation. 8. The application as claimed in claim 7, characterized in that, it is applied in fresh-keeping of vegetables and fruits. 9. application as claimed in claim 8, is characterized in that, is applied in the fresh-keeping of Rosa roxburghii.

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