A bacteriocin mutant BM-1 and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a bacteriocin mutant BM-1 and its application. The amino acid sequence of the bacteriocin mutant BM-1 is shown in SEQ ID NO.1. The bacteriocin mutant BM-1 is obtained by mutating the polypeptide with the amino acid sequence shown in SEQ ID NO.2 in the dipeptide bacteriocin Crustoricin X. Compared with the polypeptide, the bacteriocin mutant BM-1 has higher antibacterial activity against a variety of foodborne pathogens, and also has high bactericidal activity against a variety of foodborne pathogens. The bacteriocin mutant BM-1 has a simple structure and can be synthesized by conventional polypeptide solid-phase synthesis method. It is easy to produce and prepare, and can be used as an active ingredient in antibacterial and / or antiseptic products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a bacteriocin mutant BM-1 and an application thereof. Background Art

[0002] Preservatives and antibiotics are commonly used food additives to inhibit or kill foodborne pathogens and reduce contamination. However, preservatives are mostly synthetic chemicals and pose certain health risks, while antibiotics can lead to drug resistance. Neither meets consumers' increasing demand for natural and healthy foods. Therefore, it is necessary to explore new substances that can be used for green food preservation.

[0003] Bacteriocins are small peptides with antibacterial activity that are synthesized by bacteria through the ribosome during metabolism. They are natural antibacterial substances, safe and non-toxic, and are ideal alternatives to chemical preservatives and antibiotics. However, the antibacterial activity of bacteriocins is not always ideal. Bacteriocins with low antibacterial activity require higher dosages to produce the expected antibacterial effect. However, the natural resources of microorganisms are limited, and bacteriocins have problems such as difficulty in separation and purification due to their small molecular weight, cumbersome extraction steps, and low yields, which restrict their large-scale production and, in turn, their widespread use as active ingredients in antibacterial / preservative additives. Artificial design and modification of bacteriocins is a fast and effective way to change their activity, but bacteriocins after artificial design and modification do not necessarily produce ideal antibacterial activity, and bacteriocins with complex structures also increase the difficulty of preparation and increase production costs. Summary of the Invention

[0004] To address the above issues, the present invention provides a bacteriocin mutant BM-1 and its applications. Experimental studies have shown that the bacteriocin mutant BM-1 provided by the present invention has high inhibitory and killing activity against a variety of common foodborne pathogens. It also has a simple structure and is easy to prepare, making it suitable as an active ingredient in antibacterial and / or antiseptic products.

[0005] To achieve the above-mentioned purpose, the embodiment of the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a bacteriocin mutant BM-1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The mutant BM-1 is obtained by mutating the polypeptide of the dipeptide bacteriocin Crustoricin X, whose amino acid sequence is shown in SEQ ID NO. 2. The specific mutation method is as follows: in the polypeptide of the dipeptide bacteriocin Crustoricin X, whose amino acid sequence is shown in SEQ ID NO. 2, the glycine at position 1 is mutated to lysine, the glutamic acid at position 4 is mutated to arginine, the glycine at position 5 is mutated to lysine, the glycine at position 7 is mutated to tryptophan, the aspartic acid at position 11 is mutated to arginine, the glutamine at position 12 is mutated to lysine, and the histidine at position 15 is mutated to tryptophan.

[0008] Compared to the dipeptide bacteriocin Crustoricin X, whose amino acid sequence is shown in SEQ ID NO. 2, this mutant BM-1 exhibits higher antibacterial activity against a variety of foodborne pathogens and can exert good antibacterial activity at a lower dosage. Furthermore, this bacteriocin mutant BM-1 exhibits high bactericidal activity against a variety of foodborne pathogens, effectively reducing the number of foodborne pathogens.

[0009] The bacteriocin mutant BM-1 has a simple structure, can be synthesized by conventional polypeptide solid-phase synthesis method, and is easy to produce and prepare.

[0010] In a second aspect, the present invention provides the use of the bacteriocin mutant BM-1 in the preparation of antibacterial and / or antiseptic products.

[0011] The bacteriocin mutant BM-1 has a broad antibacterial spectrum and high inhibitory activity against Gram-positive and Gram-negative bacteria, and can therefore be used to prepare antibacterial and / or preservative products that inhibit the above-mentioned pathogens.

[0012] In combination with the second aspect, the antibacterial and / or preservative product is a product that inhibits and / or kills any one or more pathogenic bacteria among Escherichia coli, Staphylococcus aureus, Bacillus cereus, Enterobacter sakazakii, Salmonella typhimurium, Klebsiella pneumoniae, Enterococcus faecium, Micrococcus luteus and Listeria monocytogenes.

[0013] Preferably, the antibacterial and / or preservative product inhibits and / or kills any one or more pathogens selected from the group consisting of Escherichia coli, Staphylococcus aureus, Bacillus cereus, Enterobacter sakazakii, Salmonella typhimurium, Klebsiella pneumoniae, Enterococcus faecium, and Listeria monocytogenes. The bacteriocin mutant BM-1 exhibits significantly higher antibacterial activity against the aforementioned pathogens than the dipeptide bacteriocin Crustoricin X.

[0014] In a third aspect, the present invention further provides an antibacterial and / or antiseptic product, the ingredients of which include the above-mentioned bacteriocin mutant BM-1.

[0015] In combination with the third aspect, the antibacterial and / or preservative products include food preservatives, feed additives and antibacterial drugs.

[0016] The present invention has the following beneficial effects: the bacteriocin mutant BM-1 provided by the present invention has a simple structure and can be synthesized by conventional solid-phase synthesis methods and other methods, making it easy to prepare. The bacteriocin mutant BM-1 can effectively inhibit and kill Gram-positive bacteria such as Staphylococcus aureus, Bacillus cereus, Enterococcus faecium, Listeria monocytogenes, and Gram-negative bacteria such as Enterobacter sakazakii, Salmonella typhimurium, Klebsiella pneumoniae, Micrococcus luteus, and Escherichia coli. Its antibacterial and bactericidal activities are significantly higher than those of other mutants obtained by mutating the polypeptide with the amino acid sequence shown in SEQ ID NO. 2 in the dipeptide bacteriocin Crustoricin X. In addition, the bacteriocin mutant BM-1 has significantly higher antibacterial and bactericidal activities than the dipeptide bacteriocin Crustoricin X against the other pathogens mentioned above except Micrococcus luteus. In addition, the bacteriocin mutant BM-1 can also destroy the cell membrane and cell morphology of pathogenic bacteria, thereby reducing the drug resistance of pathogens. Therefore, the bacteriocin mutant BM-1 provided by the present invention can be used to prepare antibacterial and / or antiseptic products such as food preservatives, feed additives, and antibacterial drugs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The test results of the effect of BM-1 on the cell membrane integrity of Escherichia coli ATCC 25922 in Example 4 are as follows;

[0018] Figure 2 These are the test results of the effect of BM-1 on the cell morphology of Escherichia coli ATCC 25922 in Example 5. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0020] Bacteriocins are natural antibacterial substances and are ideal alternatives to chemical preservatives and antibiotics. However, the antibacterial activity of bacteriocins is not always ideal. Bacteriocins with low antibacterial activity require higher dosages to produce the desired antibacterial effect. However, natural microbial resources are limited, and bacteriocins, due to their small molecular weight, are difficult to isolate and purify, with cumbersome extraction steps and low yields.

[0021] To address this issue, the inventors artificially designed and modified a natural bacteriocin, obtaining a bacteriocin mutant, BM-1, with the amino acid sequence shown in SEQ ID NO. 1. Testing revealed that these bacteriocin mutants, BM-1, exhibited a broad antimicrobial spectrum and, compared to the dipeptide bacteriocin Crustoricin X, exhibited higher inhibitory activity against a variety of Gram-positive and Gram-negative bacteria.

[0022] Based on the antibacterial and bactericidal effects of the bacteriocin mutant BM-1, the present invention provides an embodiment of the use of the bacteriocin mutant BM-1 in the preparation of antibacterial and / or antiseptic products, including but not limited to food preservatives, feed additives, antibacterial drugs, etc.

[0023] The embodiments of the present invention also provide an antibacterial and / or antiseptic product containing the bacteriocin mutant BM-1.

[0024] The solutions of the present invention are described below through specific embodiments.

[0025] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0026] Example 1

[0027] This example provides a bacteriocin mutant BM-1 having an amino acid sequence as shown in SEQ ID NO. 1 and a process for obtaining the same.

[0028] The bacteriocin mutant BM-1 is obtained by mutating the glycine at position 1 to lysine, the glutamic acid at position 4 to arginine, the glycine at position 5 to lysine, the glycine at position 7 to tryptophan, the aspartic acid at position 11 to arginine, the glutamine at position 12 to lysine, and the histidine at position 15 to tryptophan in the bacteriocin Crustoricin X with an amino acid sequence as shown in SEQ ID NO. 2, thereby obtaining the bacteriocin mutant BM-1 with an amino acid sequence as shown in SEQ ID NO. 1.

[0029] The bacteriocin mutant BM-1 used in the following Examples 2 to 5 was prepared by Qiangyao Biotechnology Co., Ltd. (Shanghai) through conventional solid-phase synthesis, with a purity of >90% (the content of the synthesized target peptide in the total peptide).

[0030] Example 2

[0031] This example provides the antibacterial activity of the bacteriocin mutant BM-1, whose amino acid sequence is shown in SEQ ID NO. 1, against various foodborne pathogens.

[0032] A series of gradient BM-1 solutions (8, 16, 31, 62, 125, 250, 500, 1000, 2000 μM) were prepared by the two-fold dilution method using 5% DMSO as the diluent. Indicator bacteria such as Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes were cultured overnight and diluted with LB medium to a final bacterial density of 5.0 × 10 5 CFU / mL. 10 μL of diluted antibacterial peptides at each concentration was added to a 96-well plate containing 90 μL of bacterial solution. Growth and sterility controls were set up and cultured at 37°C for 20 h. The minimum inhibitory concentration (MIC) was determined based on the turbidity of the bacterial solution in the wells. The results are shown in Table 1.

[0033] Table 1 Determination of minimum inhibitory concentrations for various foodborne pathogens

[0034]

[0035] Example 3

[0036] This example provides the bactericidal activity of the bacteriocin mutant BM-1, whose amino acid sequence is shown in SEQ ID NO. 1, against various foodborne pathogens.

[0037] A series of gradient BM-1 solutions (8, 16, 31, 62, 125, 250, 500, 1000, 2000 μM) were prepared by the two-fold dilution method using 5% DMSO as the diluent. Indicator bacteria such as Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes were cultured overnight and diluted with LB medium to a final bacterial density of 5.0 × 10 5 CFU / mL. 10 μL of the diluted antibacterial peptide at each concentration was added to a 96-well plate containing 90 μL of bacterial solution. A growth control and a sterile control were set up and incubated at 37°C for 20 hours. 10 μL of the contents without obvious turbidity was spread on an LB agar plate and incubated at 37°C for 18-24 hours. The culture medium was observed for bacterial growth. The minimum concentration corresponding to the culture medium without bacterial growth was the minimum bactericidal concentration (MBC) of the bacteriocin. The results are shown in Table 2.

[0038] Table 2 Determination of minimum bactericidal concentration for various foodborne pathogens

[0039]

[0040] Example 4

[0041] This example provides the effect of the bacteriocin mutant BM-1, whose amino acid sequence is shown in SEQ ID NO. 1, on the cell membrane integrity of pathogenic bacteria.

[0042] The indicator bacteria (Escherichia coli ATCC 25922) cultured to the logarithmic phase were centrifuged at 8000×g at 4°C for 5 min, the cells were collected and resuspended in LB medium to a concentration of 10 8 CFU / mL. BM-1 was added to the bacterial suspension to a final concentration of 0, 1, 2, and 4× MIC, and the culture was continued at 37°C for 1 hour. The cells were collected by centrifugation at 8000×g for 5 minutes at 4°C, washed three times with 0.01M PBS (pH 7.2), and resuspended in 1× buffer from the PI staining kit to 10 7 CFU / mL. 10 μL of PI stain was added to 190 μL of bacterial suspension and incubated at room temperature in the dark for 30 min. PI absorption for each treatment was measured using a FACS Calibur flow cytometer (Becton Dickinson, USA).

[0043] Effects of BM-1 on the integrity of Escherichia coli cell membranes Figure 1 As shown. Figure 1 It can be seen that BM-1 has different degrees of destructive effect on the cell membrane of Escherichia coli, and the destructive effect is enhanced with the increase of treatment concentration.

[0044] Example 5

[0045] This example provides the effect of the bacteriocin mutant BM-1, whose amino acid sequence is shown in SEQ ID NO. 1, on the cell morphology of pathogenic bacteria.

[0046] The indicator bacteria (Escherichia coli ATCC 25922) cultured to the logarithmic phase were centrifuged at 8000×g at 4°C for 5 min, the cells were collected and resuspended in LB medium to a concentration of 10 8 CFU / mL. BM-1 was added to the bacterial suspension to a final concentration of 4×MIC, and the culture was continued at 37°C for 1 hour. The bacteria were washed three times with sterile 0.01M PBS (pH 7.2) to remove residual peptides. The bacteria were fixed with 2.5% glutaraldehyde overnight, and the indicator bacteria were washed again with PBS 2-3 times, each time for 10 minutes. Subsequently, different concentrations of ethanol were used for gradient dehydration, and each concentration was dehydrated for 10 minutes. After 10 minutes of isoamyl acetate substitution, the bacteria were placed in a fully automatic critical point dryer for dehydration and drying, sprayed with gold powder, and the bacterial morphology was observed on a high resolution Nova NanoSEM450 scanning electron microscope (FEI, USA).

[0047] Effects of BM-1 on the integrity of Escherichia coli cell membranes Figure 2 As shown. Figure 2 It can be seen that after BM-1 treatment, E. coli cells showed visible pore formation and cell membrane wrinkling.

[0048] Comparative Example 1

[0049] This comparative example provides a bacteriocin mutant BM-2, whose amino acid sequence is: GFFEGIGDAIDQDAD, which is obtained by changing the arginine at position 8 of the amino acid sequence of the bacteriocin CruB shown in SEQ ID NO.2 to aspartic acid, the tryptophan at position 9 to alanine, the arginine at position 13 to aspartic acid, the tryptophan at position 14 to alanine, and the histidine at position 15 to aspartic acid in the dipeptide bacteriocin Crustocin X.

[0050] After testing, the net charge of the bacteriocin mutant BM-2 was -5, which was not conducive to electrostatic attraction with the negatively charged lipids on the bacterial surface, and therefore it was difficult to aggregate on the bacterial membrane surface to exert an antibacterial effect.

[0051] SOPMA predicted the secondary structure of the bacteriocin mutant BM-2 protein: the overall structure is an irregular coil, which is not conducive to causing bacterial membrane perforation, making it difficult to exert antibacterial activity.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A bacteriocin mutant BM-1, characterized in that The amino acid sequence of the bacteriocin mutant BM-1 is shown in SEQ ID NO.

1.

2. Use of the bacteriocin mutant BM-1 according to claim 1 in the preparation of antibacterial and / or antiseptic products, characterized in that: The antibacterial and / or preservative product is a product that inhibits and / or kills any one or more pathogenic bacteria selected from the group consisting of Escherichia coli, Staphylococcus aureus, Bacillus cereus, Enterobacter sakazakii, Salmonella typhimurium, Klebsiella pneumoniae, Enterococcus faecium, Micrococcus luteus and Listeria monocytogenes.

3. The use according to claim 2, characterized in that The antibacterial and / or preservative product is a product that inhibits and / or kills any one or more pathogenic bacteria selected from the group consisting of Escherichia coli, Staphylococcus aureus, Bacillus cereus, Enterobacter sakazakii, Salmonella typhimurium, Klebsiella pneumoniae, Enterococcus faecium, and Listeria monocytogenes.

4. An antibacterial and / or antiseptic product, characterized in that: The composition thereof comprises the bacteriocin mutant BM-1 according to claim 1.

5. The antibacterial and / or antiseptic product according to claim 4, characterized in that: The antimicrobial and / or antiseptic products include food preservatives, feed additives and antimicrobial drugs.

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