Antibacterial peptide induced by sulfate reducing bacteria and application thereof

Abstract

The invention discloses an antibacterial peptide induced by sulfate reducing bacteria and application of the antibacterial peptide, and belongs to the technical field of marine corrosion protection. The amino acid sequence of the SRB-induced antibacterial peptide is VAHKRLVVGTR, and the SRB-induced antibacterial peptide has efficient antibacterial activity on corrosive bacteria such as SRB and has the advantages of being environmentally friendly and not prone to inducing the bacteria to generate drug resistance, and the problems that an existing antibacterial peptide is insufficient in corrosive bacteria specificity, poor in environmental stability and the like are solved; meanwhile, peptide chains are short, synthesis is easy, and the production cost is low; the compound corrosion inhibition bactericide formed by combining the compound with a chemical bactericide can significantly improve the antibacterial and antiseptic effects, saves the dosage of the chemical bactericide, effectively solves the problems of bacterial drug resistance, high cost, environmental pollution and other pain points caused by abuse of the existing bactericide, and is suitable for the fields of marine microbial corrosion protection and the like.

Description

Technical Field

[0001] This invention relates to the field of marine corrosion protection technology, and in particular to an antimicrobial peptide induced by sulfate-reducing bacteria and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Microbial corrosion poses a wide-ranging threat, becoming a significant problem affecting key industries such as marine engineering equipment and facilities, oil and gas infrastructure, and nuclear waste storage facilities. It directly accounts for over 20% of total corrosion losses. Among numerous corrosive bacteria, sulfate-reducing bacteria (SRB) are the most prevalent. Widely present in anaerobic / anoxic environments, they accelerate corrosion by forming biofilms on metallic surfaces, severely threatening the safe and reliable operation of marine engineering equipment and facilities.

[0004] To address corrosion problems caused by microorganisms such as SRB, adding corrosion inhibitors and bactericides is a common treatment method in practical engineering. However, the protective effect of biofilms makes it difficult to completely eliminate attached bacteria, requiring repeated addition of bactericides. The large-scale use of bactericides not only induces bacterial resistance and reduces long-term bactericidal effects, but also increases economic costs and causes environmental pollution due to chemical residues.

[0005] To address the problems caused by the overuse of bactericides, utilizing small-molecule antimicrobial peptides as synergists to enhance the efficacy of existing bactericides is considered a promising solution. Antimicrobial peptides are an important component of the body's innate immune defense system, achieving bactericidal effects through membrane disruption mechanisms. They possess advantages such as broad-spectrum and highly effective antimicrobial activity, low biotoxicity, and low likelihood of inducing bacterial resistance. However, the application of antimicrobial peptides in this field still faces significant limitations: existing antimicrobial peptides are mostly derived from clinical medicine, food, and animal husbandry, exhibiting insufficient antimicrobial activity against corrosive bacteria such as SRB; simultaneously, the relatively long peptide chain length leads to high synthesis costs and poor environmental stability, greatly limiting their practical application in marine antimicrobial and preservative fields. Furthermore, the synergistic effect of antimicrobial peptides on bactericides provided in existing studies has not yet reached an ideal level.

[0006] Therefore, it is an urgent problem to solve how to provide an antimicrobial peptide that has strong antibacterial activity against corrosive bacteria such as SRB, short peptide chain length, low synthesis cost, good environmental stability, and can significantly enhance the synergistic effect of bactericides. Summary of the Invention

[0007] In view of this, the present invention provides an SRB-induced antimicrobial peptide and its application. The antimicrobial peptide provided by the present invention has significant antimicrobial activity and can be used as a bactericidal synergist, significantly reducing the amount of bactericide required, and can effectively inhibit the adhesion and corrosion of corrosive biofilms.

[0008] In a first aspect, the present invention provides an SRB-induced antimicrobial peptide having the amino acid sequence VAHKRLVVGTR.

[0009] Secondly, the present invention provides the application of the above-mentioned antimicrobial peptides in inhibiting the growth of corrosive bacteria or the adhesion of biofilms.

[0010] Thirdly, the present invention provides the use of the above-mentioned antimicrobial peptide as a bactericidal synergist.

[0011] Fourthly, the present invention provides the application of the above-mentioned antimicrobial peptides in the preparation of bactericides or bacteriostatic agents.

[0012] Fifthly, the present invention provides a composite corrosion inhibitor and bactericide, comprising the above-mentioned antimicrobial peptide and chemical bactericide.

[0013] In a sixth aspect, the present invention provides the application of the above-mentioned antimicrobial peptide or the above-mentioned composite corrosion inhibitor and bactericide in marine microbial corrosion protection.

[0014] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The SRB-induced antimicrobial peptide VAHKRLVVGTR (named DVHI) provided by the present invention has high antimicrobial activity against corrosive bacteria such as SRB. It can effectively inhibit their growth and biofilm adhesion at a concentration of 20 ppm. It also has the advantages of being green and environmentally friendly and not easily inducing bacteria to develop drug resistance. It solves the problems of insufficient antimicrobial activity and poor environmental stability of existing antimicrobial peptides against corrosive bacteria. At the same time, the peptide chain is relatively short, easy to synthesize, and has low production cost.

[0015] (2) The antimicrobial peptide DVHI of the present invention can be used as a bactericidal synergist. When used in combination with chemical bactericides such as quaternary ammonium salts, organic guanidines or aldehydes, it forms a composite corrosion inhibitor bactericide, which can significantly improve the antibacterial and anti-corrosion effect, reduce the number of attached bacteria, reduce the corrosion rate, and save the amount of chemical bactericides used. It effectively solves the pain points of bacterial resistance, high cost and environmental pollution caused by the current abuse of bactericides, and is suitable for marine microbial corrosion protection and other fields. Attached Figure Description

[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0017] Figure 1 This refers to the number of immobilized desulfurized Vibrio cells on the surface of carbon steel in culture media containing different concentrations of DVHI and bactericide THPS after soaking for 14 days in Example 3 of the present invention. Figure 2 These are the weight loss data of carbon steel in culture media containing different concentrations of DVHI and different types of bactericides after soaking for 14 days in Example 4 of this invention. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] This invention provides an SRB-induced antimicrobial peptide with the amino acid sequence VAHKRLVVGTR.

[0020] The antimicrobial peptide DVHI in this invention is obtained through a biological induction method: using SRB as the inducing source, a specific host (such as insect larvae) is stimulated to produce an immune response, thereby expressing a specific antimicrobial peptide against SRB. The amino acid sequence of the differentially expressed peptide in the larvae is analyzed using peptidomics technology, and the physicochemical properties and antimicrobial activity of the differentially expressed peptide are predicted using machine learning algorithms in the antimicrobial peptide database. Further verification is carried out through experiments, and it is found that the antimicrobial peptide DVHI provided by this invention has highly efficient antimicrobial activity against SRB.

[0021] The present invention also provides the application of the above-mentioned antimicrobial peptides in inhibiting the growth of corrosive bacteria or the formation of biofilms.

[0022] Bacterial biofilms are three-dimensional structures formed by bacteria secreting extracellular matrix (such as polysaccharides and proteins) and attaching to the surface of materials, protecting bacteria from bactericides and environmental stress. The antimicrobial peptides of this invention inhibit biofilm formation through two mechanisms: firstly, they directly disrupt the cell membrane of SRBs, preventing them from attaching to the material surface; secondly, they interfere with biofilm signal transduction or extracellular matrix synthesis, dissociating the already formed biofilm structure.

[0023] In this invention, the corrosive bacteria are typical marine corrosive bacteria such as SRB and nitrate-reducing bacteria (NRB). Experimental verification in this invention has shown that the antimicrobial peptide DVHI at a concentration of 20 ppm can effectively inhibit the growth of SRB and biofilm adhesion.

[0024] This invention provides the use of the above-mentioned antimicrobial peptide as a bactericidal synergist.

[0025] The antimicrobial peptide DVHI of the present invention can enhance the effect of other bactericides through synergistic action. When used as a bactericidal synergist, its concentration can be as low as 1~120 nM, more preferably 80~120 nM.

[0026] This invention provides the application of the above-mentioned antimicrobial peptides in the preparation of bactericides or bacteriostatic agents.

[0027] The present invention can achieve a broad-spectrum bactericidal and bacteriostatic effect. The bactericide or bacteriostatic agent can inhibit or kill at least one of Escherichia coli, Salmonella, Staphylococcus aureus, Desulfovibrio (a common marine SRB), or Pseudomonas aeruginosa (a common marine NRB).

[0028] The present invention also provides a composite corrosion inhibitor and bactericide, comprising the above-mentioned antimicrobial peptide and chemical bactericide.

[0029] In one or more embodiments of the present invention, the chemical bactericide includes one or more of quaternary ammonium salt bactericides, organic guanidine bactericides, quaternary phosphate bactericides, or aldehyde bactericides. Quaternary ammonium salt bactericides include benzalkonium chloride, benzalkonium bromide, benzalkonium chloride, dodecyltrimethylammonium chloride, or dodecyldimethylbenzylammonium chloride, etc.; organic guanidine bactericides include chlorhexidine, polyhexamethylene biguanide hydrochloride, polyhexamethylene guanidine hydrochloride, dodecyl guanidine acetate, biguanide octyl acetate, etc.; quaternary phosphate bactericides include tetramethylolphosphine sulfate (THPS), etc.; aldehyde bactericides include glutaraldehyde, formaldehyde, o-phthalaldehyde (OPA), or glyoxal, etc.

[0030] In one or more embodiments of the present invention, the ratio of the antimicrobial peptide to the chemical bactericide is (1~120)nM:(10~80)ppm, more preferably (80~120)nM:(20~60)ppm.

[0031] The present invention also provides the application of the above-mentioned antimicrobial peptide or the above-mentioned composite corrosion inhibitor and bactericide in marine microbial corrosion protection.

[0032] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0033] Example 1 This embodiment provides the method for obtaining the antimicrobial peptide DVHI.

[0034] 600 live 3-year-old black soldier flies ( H. illucens After cleaning the larvae, an equal volume of SRB bacterial solution was injected into 300 black soldier fly larvae, while the remaining larvae were left untreated. After 48 hours of routine rearing, peptide samples were extracted from all larvae for peptidomics analysis. First, ultra-high performance liquid chromatography (UPLC) was used to dissolve and separate the peptide samples, followed by mass spectrometry analysis to construct a database of differentially expressed peptides and analyze their amino acid sequences. Finally, an antimicrobial peptide prediction tool based on machine learning algorithms was used to predict the antimicrobial activity of all differentially expressed peptides. The four peptides with the highest predicted antimicrobial activity scores were artificially synthesized using a solid-phase synthesis method. Further antimicrobial experiments verified the antimicrobial activity of these four peptides. It was found that the antimicrobial peptide VAHKRLVVGTR, composed of only 11 amino acids, exhibited the highest antimicrobial activity, effectively inhibiting SRB growth at a concentration of 20 ppm. This peptide was named DVHI.

[0035] Example 2 This embodiment provides a broad-spectrum antibacterial test of the antimicrobial peptide DVHI.

[0036] The antimicrobial peptide DVHI was determined to have significant inhibitory activity against Escherichia coli, Salmonella, Staphylococcus aureus, Desulfovibrio, and Pseudomonas aeruginosa, with minimum inhibitory concentrations ranging from 4 to 32 μg / mL. At a concentration of 20 μg / mL, DVHI showed an inhibition rate of over 85% against all tested strains within 24 hours, demonstrating excellent broad-spectrum antimicrobial properties.

[0037] Example 3 This embodiment provides the determination of antimicrobial activity when the antimicrobial peptide DVHI is used in combination with the bactericide THPS.

[0038] (1) System: Each 500 mL anaerobic bottle contained 300 mL of culture medium, 3 mL of *Vibrio desulfurans* bacterial suspension, and 3 parallel carbon steel samples. The culture medium was treated differently: a. Untreated group; b. 100 nM DVHI; c. 40 ppm THPS; d. 100 nM DVHI + 40 ppm THPS; e. 200 ppm THPS. Each treatment group was placed in a constant temperature incubator and incubated at 37 °C for 14 days.

[0039] (2) Antibacterial activity verification: After taking out the samples, they were first rinsed with PBS buffer at pH 7.4 to remove airborne bacteria from the sample surface. Then, the biofilm on the surface of each sample was scraped into 10 mL of PBS solution at pH 7.4 using a sterile brush. Finally, the sample, brush and PBS solution were placed in a test tube and vortexed for 30 s to disperse the cells evenly before counting them using a cell counting chamber.

[0040] The results of the antibacterial activity test on bacteria attached to biofilms on carbon steel surfaces are as follows: Figure 1 As shown, under the treatment conditions of 100nM DVHI + 40ppm THPS, the number of bacteria on the carbon steel surface was even lower than that under the treatment conditions of 200ppm THPS. This indicates that the combination of antimicrobial peptide DVHI and bactericide THPS has a significant synergistic effect. The trace amount of antimicrobial peptide DVHI has a significant bactericidal and bactericidal effect, reducing the amount of bactericide THPS by more than 80%.

[0041] Example 4 This embodiment provides the application of the antimicrobial peptide DVHI in the protection of carbon steel against microbial corrosion when it is used in combination with different types of corrosion inhibitors and bactericides in actual oilfield produced water.

[0042] 1. Pre-weighed carbon steel samples were immersed in produced water from eutrophic oilfields treated with different methods and incubated at 37°C for 14 days. Different treatment groups: a. Untreated group; b. 100 nM DVHI + 50 ppm benzalkonium chloride; c. 100 nM DVHI + 40 ppm polyhexamethylene biguanide; d. 100 nM DVHI + 30 ppm glutaraldehyde; e. 260 ppm benzalkonium chloride; f. 200 ppm polyhexamethylene biguanide; g. 150 ppm glutaraldehyde.

[0043] 2. After cultivation, the carbon steel samples were cleaned with acid pickling solution to remove surface corrosion products and biofilm, dried, and weighed. The weight loss data of samples from different treatment groups were calculated and compared.

[0044] Experimental results are as follows Figure 2 As shown, the untreated group had the highest weight loss rate; while 260 ppm benzalkonium chloride, 200 ppm polyhexamethylene biguanide, and 150 ppm glutaraldehyde, when used alone, all significantly reduced the weight loss rate. It is noteworthy that when the above corrosion inhibitors and bactericides were used in combination with 100 nM DVHI, the amount of chemical corrosion inhibitors and bactericides required was significantly reduced, which helps to reduce the risk of bacterial resistance.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An SRB-induced antimicrobial peptide, characterized in that, Its amino acid sequence is VAAGRLVVGTR.

2. The application of the antimicrobial peptide as described in claim 1 in inhibiting the growth of corrosive bacteria or the formation of biofilms.

3. The application as described in claim 2, characterized in that, The corrosive bacteria are SRB or NRB.

4. The use of the antimicrobial peptide as described in claim 1 as a bactericidal synergist.

5. The use of the antimicrobial peptide as described in claim 1 in the preparation of bactericides or bacteriostatic agents.

6. The application as described in claim 5, characterized in that, The bactericide or bacteriostatic agent can inhibit or kill at least one of Escherichia coli, Salmonella, Staphylococcus aureus, Desulfovibrio, and Pseudomonas aeruginosa.

7. A composite corrosion inhibitor and bactericide, characterized in that, Includes the antimicrobial peptide and chemical bactericide as described in claim 1.

8. The composite corrosion inhibitor and bactericide as described in claim 7, characterized in that, The chemical bactericides include one or more of the following: quaternary ammonium salt bactericides, quaternary phosphate salt bactericides, organic guanidine bactericides, or aldehyde bactericides.

9. The composite corrosion inhibitor and bactericide as described in claim 7, characterized in that, The ratio of the antimicrobial peptide to the chemical bactericide is (1~120)nM:(10~80)ppm.

10. The application of the antimicrobial peptide as described in claim 1 or the composite corrosion inhibitor and bactericide as described in claims 7-9 in marine microbial corrosion protection.

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