Method for killing gram-negative bacteria through combination of blue light, far ultraviolet light and antibacterial peptide Thanatin

Abstract

The invention discloses a method for killing gram-negative bacteria through combination of blue light, far ultraviolet light and antibacterial peptide Thanatin, and belongs to the field of gram-negative bacteria sterilization. The method comprises the following steps: (1) adding an antibacterial peptide Thanatin or a mutant of the antibacterial peptide Thanatin in a sterilization environment; and (2) carrying out blue light and far ultraviolet irradiation on the sterilization environment. According to the method, the synergistic effect is achieved, 5.4 gCFU / ml gram-negative bacteria can be killed within 32 minutes, the sterilization effect is excellent, the effect is fast, the sterilization environment is not limited, and the harm of the gram-negative bacteria can be inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of Gram-negative bacteria sterilization, and particularly relates to a method for killing Gram-negative bacteria by combining blue light, far ultraviolet light and antibacterial peptide Thanatin. Background Art

[0002] Gram-negative bacteria (GNB) pose a serious threat to the host immune system and conventional antibacterial agents due to their outer membrane structure (containing lipopolysaccharide LPS), and specifically include: 1) Medical field: GNB can cause nosocomial infections. For example, Klebsiella pneumoniae and Acinetobacter baumannii are the main pathogens infecting ICU patients, which can cause pneumonia and bloodstream infections, and their mortality rate is as high as 30%-50%. Moreover, GNB also triggers a drug resistance crisis. For example, GNB producing carbapenemases (such as NDM and KPC) renders carbapenem antibiotics ineffective, and some strains are even resistant to polymyxin, the "last line of defense". 2) Food safety field: GNB is the main cause of foodborne diseases. For example, Salmonella, Escherichia coli O157:H7, and Listeria can contaminate meat, dairy products and water sources, thereby causing diseases such as diarrhea and hemolytic uremic syndrome. Another example is that Enterobacter sakazakii can contaminate infant formula milk powder, and its fatality rate is as high as 40%-80%. 3) Public health field: GNB can cause community transmission. Drug-resistant Escherichia coli can be transmitted through water sources or contact, exacerbating the global burden of antibiotic resistance, and forming environmental persistence. Moreover, GNB can survive in soil and water for a long time and spread drug-resistant genes through horizontal gene transfer. Therefore, there is an urgent need to develop a method for killing Gram-negative bacteria with a broad bactericidal spectrum and environmental friendliness.

[0003] Up to now, a variety of bactericidal technologies have been reported for GNB, mainly including high-pressure steam (121°C), pasteurization (72°C, 15 seconds), ultraviolet light (254 nm), antibacterial peptides and various chemical bactericidal methods (oxidants, quaternary ammonium salts, aldehydes, phenols, etc.). These methods each have their applicable scenarios and their own advantages and disadvantages. Among them, antibacterial peptide Thanatin (Tn) is a new type of antibacterial peptide reported in recent years, and its sequence is GSKKPVPIIYCNRRTGKCQRM, as reported in patent CN 118240026 A.

[0004] Tn is a cationic antimicrobial peptide isolated from the hemipteran insect Podisus maculiventris, which has broad-spectrum activities against Gram-negative, Gram-positive bacteria and various fungi, and can also strongly inhibit the growth of bacteria and fungi at quite low concentrations. Although Tn is considered to be one of the ways to solve antibiotic resistance, the present invention finds that in the in vitro bactericidal simulation of Gram-negative bacteria, Tn does not show excellent effects, with a long bactericidal onset time and low efficiency, and is insufficient to meet the requirements of killing Gram-negative bacteria in vitro. Therefore, it is necessary to develop a method to improve the bactericidal efficiency of the antimicrobial peptide Tn. Summary of the Invention

[0005] Technical Problem

[0006] When using the antimicrobial peptide Thanatin alone for Gram-negative bacteria killing, its bactericidal onset time is long and the efficiency is relatively low. Therefore, it is necessary to provide a method to improve the bactericidal efficiency of the antimicrobial peptide Tn.

[0007] Technical Solution

[0008] To solve the above problems, the present invention combines specific wavelength blue light (400 - 480 nm), far ultraviolet light of 222 nm and the antimicrobial peptide Thanatin (including Thanatin mutants), which greatly shortens the bactericidal time and significantly improves the bactericidal efficiency. Antimicrobial blue light (405 - 470 nm) achieves the bactericidal effect by generating reactive oxygen species (ROS) through photochemical reactions to damage cell membrane lipids, proteins and intracellular metabolic enzymes. Far ultraviolet light Far-UVC (222 nm) is a newer optical bactericidal method, which causes cell death by damaging nucleic acids and proteins. When using antimicrobial blue light or 222 nm far ultraviolet light alone for GNB killing, a certain irradiation time is required, which increases the bactericidal timeliness and also increases the energy consumption cost. Therefore, this patent combines antimicrobial blue light, 222 nm far ultraviolet light and the antimicrobial peptide Thanatin, which not only significantly shortens the optical bactericidal time and improves the bactericidal effect, but also reduces the energy consumption.

[0009] The present invention provides a mutant of the antimicrobial peptide Thanatin, and the sequence is shown as SEQ ID NO.1, specifically GKKKPVPIIYCNRRKGKCQRM.

[0010] Furthermore, the mutant of the antimicrobial peptide Thanatin is obtained by mutating the 15th threonine in the antimicrobial peptide Thanatin to lysine.

[0011] The present invention also provides a method for killing Gram-negative bacteria, and the method includes the following steps:

[0012] (1) Add an antimicrobial peptide in a sterilization environment;

[0013] (2) Irradiate the sterilization environment with blue light and far - ultraviolet light.

[0014] Further, the Gram - negative bacteria include Cronobacter sakazakii or Salmonella typhimurium SL1344.

[0015] Further, the sterilization environment includes a solid surface or a liquid environment.

[0016] Further, the solid surface includes the human skin, food surface, packaging surface, ground, surface of tables and chairs, medicine surface, surface of medical tools or surface of medical instruments.

[0017] Further, the solid surface includes one or more of the top, side, and bottom of the solid.

[0018] Further, the liquid environment includes water body or dairy products.

[0019] Further, the liquid in the liquid environment needs to be stored in a transparent container or an opaque container with an opening.

[0020] Further, when the sterilization environment is a solid surface, the addition amount of the antimicrobial peptide is not less than 1 mg / cm 2 .

[0021] Further, when the sterilization environment is a solid surface, the addition amount of the antimicrobial peptide is 1 - 10 mg / cm 2 .

[0022] Further, when the sterilization environment is a liquid environment, the addition amount of the antimicrobial peptide is not less than 1 mg / L.

[0023] Further, when the sterilization environment is a liquid environment, the addition amount of the antimicrobial peptide is 1 - 10 mg / L.

[0024] Further, when the sterilization environment is a solid surface, the addition operation includes spraying, brushing or dipping.

[0025] Further, when the sterilization environment is a liquid environment, the addition operation includes dropping or pouring.

[0026] Further, the antimicrobial peptide is antimicrobial peptide Thanatin or a mutant of antimicrobial peptide Thanatin.

[0027] Further, the sequence of antimicrobial peptide Thanatin is GSKKPVPIIYCNRRTGKCQRM.

[0028] Further, the sequence of the mutant of the antibacterial peptide Thanatin is GKKKPVPIIYCNRRKGKCQRM.

[0029] Further, the wavelength of the blue light is 405 - 465 nm.

[0030] Further, the irradiation dose of the blue light is not less than 0.5 J / cm 2 ·min.

[0031] Further, the irradiation dose of the blue light is 0.5 - 5 J / cm 2 ·min.

[0032] Further, the wavelength of the far - ultraviolet light is 200 - 250 nm.

[0033] Further, the wavelength of the far - ultraviolet light is 210 - 230 nm.

[0034] Further, the wavelength of the far - ultraviolet light is 222 nm.

[0035] Further, the irradiation dose of the far - ultraviolet light is not less than 10 J / cm 2 ·min.

[0036] Further, the irradiation dose of the far - ultraviolet light is 10 - 50 J / cm 2 ·min.

[0037] Further, the irradiation is to arrange the blue - light source and the far - ultraviolet - light source above, on the side or at the bottom of the sterilization environment, so that the blue light and the far - ultraviolet light can jointly cover the sterilization environment.

[0038] Further, when the sterilization environment is a solid surface, the blue light and the far - ultraviolet light can jointly cover one or more surfaces of the solid surface to be sterilized.

[0039] Further, when the sterilization environment is a liquid environment, the blue light and the far - ultraviolet light can jointly cover the surface of the liquid environment or cover a certain side of the liquid environment through a transparent container.

[0040] Further, the irradiation time is not less than 24 minutes.

[0041] Further, the irradiation time is not less than 30 minutes.

[0042] Further, the irradiation time is 30 - 60 minutes.

[0043] The present invention also provides a method for killing Gram - negative bacteria for non - therapeutic purposes, and the method includes the following steps:

[0044] (1) Adding an antibacterial peptide in the sterilization environment;

[0045] (2) Irradiate the sterilization environment with blue light and far ultraviolet light.

[0046] Furthermore, the sterilization environment includes a solid surface or a liquid environment.

[0047] Furthermore, the solid surface includes a food surface, a packaging surface, a floor surface, a table and chair surface, a drug surface, a medical tool surface, or a medical instrument surface.

[0048] Furthermore, the solid surface is a food surface, a packaging surface, a floor surface, a table and chair surface, a drug surface, a medical tool surface, or a medical instrument surface.

[0049] Furthermore, the solid surface includes one or more of the top, side, and bottom of the solid.

[0050] Furthermore, the liquid environment includes a water body or dairy products.

[0051] Furthermore, the liquid in the liquid environment needs to be stored in a transparent container or an opaque container with an opening.

[0052] Furthermore, when the sterilization environment is a solid surface, the addition amount of the antimicrobial peptide is not less than 1 mg / cm 2 .

[0053] Furthermore, when the sterilization environment is a solid surface, the addition amount of the antimicrobial peptide is 1 - 10 mg / cm 2 .

[0054] Furthermore, when the sterilization environment is a liquid environment, the addition amount of the antimicrobial peptide is not less than 1 mg / L.

[0055] Furthermore, when the sterilization environment is a liquid environment, the addition amount of the antimicrobial peptide is 1 - 10 mg / L.

[0056] Furthermore, when the sterilization environment is a solid surface, the addition operation includes spraying, brushing, or dipping.

[0057] Furthermore, when the sterilization environment is a liquid environment, the addition operation includes dropping or pouring.

[0058] Furthermore, the antimicrobial peptide is antimicrobial peptide Thanatin or a mutant of antimicrobial peptide Thanatin.

[0059] Furthermore, the sequence of antimicrobial peptide Thanatin is GSKKPVPIIYCNRRTGKCQRM.

[0060] Furthermore, the sequence of the mutant of antimicrobial peptide Thanatin is GKKKPVPIIYCNRRKGKCQRM.

[0061] Further, the wavelength of the blue light is 405 - 465 nm.

[0062] Further, the irradiation dose of the blue light is not less than 0.5 J / cm 2 ·min.

[0063] Further, the irradiation dose of the blue light is 0.5 - 5 J / cm 2 ·min.

[0064] Further, the wavelength of the far ultraviolet light is 200 - 250 nm.

[0065] Further, the wavelength of the far ultraviolet light is 210 - 230 nm.

[0066] Further, the wavelength of the far ultraviolet light is 222 nm.

[0067] Further, the irradiation dose of the far ultraviolet light is not less than 10 J / cm 2 ·min.

[0068] Further, the irradiation dose of the far ultraviolet light is 10 - 50 J / cm 2 ·min.

[0069] Further, the irradiation is to arrange the blue light source and the far ultraviolet light source above, on the side or at the bottom of the sterilization environment, so that the blue light and the far ultraviolet light can jointly cover the sterilization environment.

[0070] Further, when the sterilization environment is a solid surface, the blue light and the far ultraviolet light can jointly cover one or more surfaces of the solid surface to be sterilized.

[0071] Further, when the sterilization environment is a liquid environment, the blue light and the far ultraviolet light can jointly cover the surface of the liquid environment or cover a certain surface of the liquid environment through a transparent container.

[0072] Further, the irradiation time is not less than 30 minutes.

[0073] Further, the irradiation time is 30 - 60 minutes.

[0074] Beneficial effects

[0075] (1) The present invention provides a method for killing Gram-negative bacteria by combining antibacterial blue light, 222 nm far ultraviolet light and antibacterial peptides (Tn, Tn-K). This method can achieve a synergistic effect and can kill Gram-negative bacteria at 5.4 lgCFU / ml in 32 minutes, which is greater than the sum of the combined bactericidal effects of antibacterial blue light and 222 nm far ultraviolet light and the bactericidal effect of using antibacterial peptides alone.

[0076] (2) The method for killing Gram-negative bacteria provided by the present invention has excellent bactericidal effect, rapid onset, does not limit the bactericidal environment, is a bactericidal method with low energy consumption and wide application scenarios, and helps to inhibit the harm of Gram-negative bacteria. Description of the Drawings

[0077] Figure 1 The bactericidal curves of different combinations of 415 nm blue light, 222 nm far ultraviolet light, and the antibacterial peptide Thanatin against Enterobacter sakazakii.

[0078] Figure 2 The bactericidal curves of different combinations of 460 nm blue light, 222 nm far ultraviolet light, and the antibacterial peptide Thanatin against Salmonella.

[0079] Figure 3 The HPLC chromatogram of the antibacterial peptide Tn-K.

[0080] Figure 4 The MS spectrum of the antibacterial peptide Tn-K. Detailed Embodiments

[0081] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0082] Source of Raw Materials

[0083] Preparation of liquid LB medium: Take 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, add 1 L of deionized water, adjust the pH to 7.2, and then sterilize at 121 °C for 30 min.

[0084] Preparation of LB solid agar medium: Take 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar powder, add 1 L of deionized water, adjust the pH to 7.2, and then sterilize at 121 °C for 30 min.

[0085] BMGY fermentation medium (1 L): 10 g of yeast powder, 20 g of peptone, 10 g of glycerol, 100 mL of potassium phosphate buffer (pH 6.0), 13.4 g of YNB, 400 μg of biotin, adjust the pH to 7.2, and then sterilize at 121 °C for 30 min.

[0086] BMMY fermentation medium (1 L): 10 g of yeast powder, 20 g of peptone, 10 g of methanol, 100 mL of potassium phosphate buffer (pH 6.0), 13.4 g of YNB, 400 μg of biotin, adjust the pH to 7.2, and then sterilize at 121 °C for 30 min.

[0087] The Cronobacter sakazakii BAA-894 bacterial solution was purchased from ATCC; Salmonella typhimurium SL1344 was purchased from ATCC; nisin was purchased from Sangon Biotech (Shanghai); bacitracin was purchased from Solarbio Science & Technology Co., Ltd. in Beijing; polymyxin was purchased from Solarbio Science & Technology Co., Ltd. in Beijing; thanatin (Tn) was purchased from Yuanye Bio-Technology Co., Ltd. in Shanghai.

[0088] Instrument use

[0089] In the examples, the blue LED light was PN1600 for optical therapy in Canada; the 222 nm far ultraviolet light was emitted by a UV lamp module, and this instrument was purchased from Ohtailight Co., Ltd. in Japan.

[0090] Example 1: Bactericidal effect of the combination of 415 nm blue light, 222 nm far ultraviolet light and the antimicrobial peptide Thanatin (Tn) on Cronobacter sakazakii

[0091] The operation is as follows:

[0092] (1) Preparation of bacterial suspension: The activated Cronobacter sakazakii BAA-894 bacterial solution was inoculated into 5 mL of liquid LB medium respectively, and the initial OD 600 value was controlled to be 0.02. The three strains were cultured to the logarithmic growth phase at 37 °C with a rotation speed of 200 r / min. 1.0 mL of each of the above-mentioned bacterial solutions was taken into a centrifuge tube and centrifuged at 12,000 r / min for 1 min, and the supernatant was discarded. Then the precipitate was resuspended with the same volume of PBS with a pH value of 7.4, and this operation was repeated 2 times. Finally, the bacterial cells were resuspended with 1 mL of PBS buffer solution, and their OD 600 value was measured.

[0093] (2) Dual-light irradiation with 415 or 460 + 222 nm: 18 mL of PBS buffer solution with a pH value of 7.4 was evenly divided into 3 wells of a 6-well plate, with 6 mL in each well. According to the OD 600 value of the bacterial suspension measured above, the amount of bacterial solution to be added to the 6-well plate was calculated, and the initial OD 600 was controlled to be 5, and 1 mg / L of Tn was added and incubated for 10 min. The blue LEDs (415 nm, 460 nm) and the 222 nm light source were fixed on an iron stand, and the height was adjusted so that the small magnetic stirrer was 10 cm directly below the dual-light lamps. The blue LED lights and the 222 nm light source were turned on to start timing. All the above operations were carried out in a sterile laminar flow hood, and the environmental temperature was 20 ± 2 °C.

[0094] (3) Sampling and serial dilution: Add 180 μL of PBS buffer solution to each well of a 96-well plate. Samples were taken at the 0 min, 8 min, 12 min, and 45 min of dual-light irradiation. Three parallel samples were set at each time point. Each time, 20 μL of the sample was taken from each well of the experimental group in the 6-well plate and transferred to the first column of the 96-well plate. By pipetting up and down, ensure that the taken sample is fully mixed with other samples in the first column. Then, 20 μL of the mixed solution was taken from the first column and transferred to the second column of the well plate for further dilution. This process was repeated to obtain a mixed solution of samples with dilution factors of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 10 -6 . According to the light irradiation measurement of the illuminometer, the light dose of the blue LED lamp on the sample surface is 1.824 J / cm 2 ·min, that is, the irradiation dose corresponding to the 15th min is 27.360 J / cm 2 , 54.720 J / cm 2 at 30 min, and 82.080 J / cm 2 at 45 min. The light dose of the far-ultraviolet 222 nm lamp on the sample surface is 29.184 J / cm 2 ·min, that is, the irradiation dose corresponding to the 15th min is 437.76 J / cm 2 .

[0095] (4) Plate counting and bactericidal curve plotting: For the above-mentioned strains, 5 μL of the bacterial solution from each of the 6 dilution gradients at different time points was spotted on the LB solid agar medium for subsequent colony counting. Then, the inoculated plates were transferred to an incubator at 37 °C for more than 10 h. During the incubation process, the number and morphology of colonies on the plates were observed regularly. After all single colonies had grown, the number of gradient colonies on each plate was counted. According to the colony counting results, the bactericidal curve of blue light against C. sakazakii with different lipopolysaccharide structures was plotted, and the bactericidal efficiency at different time points of blue light irradiation was calculated.

[0096] According to the calculated bactericidal efficiency data, the bactericidal curve was plotted. In addition, to set different controls, referring to the above operations, the bactericidal efficiency tests of using antibacterial peptide Tn alone, 415 nm blue light alone, 222 nm far-ultraviolet light alone, and the combination of 415 nm blue light and 222 nm far-ultraviolet light were carried out respectively. The bactericidal efficiency of each group was calculated, and the results are shown in Table 1 below and Figure 1 .

[0097] Table 1

[0098]

[0099]

[0100] As Figure 1 shown in Table 1, when 415 nm blue light, 222 nm far ultraviolet light, and the antimicrobial peptide Thanatin are used alone, the bactericidal effect of Tn is slow to take effect. At 40 min, its bactericidal rate is close to 0.1 lgCFU / ml. The bactericidal rate of 415 nm irradiation is slightly higher, and at 40 min, the bactericidal rate is close to 2 lgCFU / ml. The bactericidal rate of 222 nm is the highest, reaching 3.5 lgCFU / ml at 40 min.

[0101] When 415 nm and 222 nm are used in combination, the bactericidal rate is further increased compared with that of 222 nm, exceeding 4.7 lgCFU / ml at 40 min.

[0102] When the three are used in combination, the bactericidal rate is increased most significantly. All Cronobacter sakazakii have been killed at 40 min, and it can reach 5.4 lgCFU / ml at 32 min. The bactericidal efficiency exceeds the sum of the bactericidal rates of 415 + 222 nm (2.9 lgCFU / ml) and Tn (0.11 lgCFU / ml) at this time, showing a synergistic effect; and the bactericidal energy consumption is reduced by 20% compared with that of 415 + 222 nm at 40 min, so it has significant advantages.

[0103] Set different controls, and refer to the above operations to detect the bactericidal efficiency of using the antimicrobial peptide Tn alone, using 460 nm blue light alone, using 222 nm far ultraviolet light alone, and using 460 nm blue light and 222 nm far ultraviolet light together, respectively. Calculate the bactericidal efficiency of each group, and the results are shown in Table 2 below.

[0104] Table 2

[0105]

[0106] It can be seen that 460 nm blue light, 222 nm far ultraviolet light, and the antimicrobial peptide Thanatin can also have a synergistic effect on killing Gram-negative bacteria.

[0107] Example 2 Bactericidal effect of the combination of 460 nm blue light, 222 nm far ultraviolet light, and the mutant of the antimicrobial peptide Thanatin (Tn-k) on Salmonella

[0108] The operation steps are the same as those in Example 1, except for the following changes:

[0109] 1) The indicator bacterium is changed from Cronobacter sakazakii to Salmonella typhimurium SL1344;

[0110] 2) Replace Tn with Tn-K of equal concentration. The sequence after the change of Tn-K is GKKKPVPIIYCNRRKGKCQRM.

[0111] The preparation process of the antimicrobial peptide Tn-K is as follows:

[0112] Design site-directed mutagenesis primers, and use the recombinant plasmid pPICZαA as a template for site-directed mutagenesis to obtain the mutant Tn-K.

[0113] The primer sequences involved are as follows:

[0114] The primer for introducing the Tn-K mutation is:

[0115] AOXT2-F: 5’-CATCTACTGCAATAGGAGAAAGGGCAAATGTCAAAGAATGTGAGTTTGTAGCCTTAGACATGACTG-3’;

[0116] AOXT2-R: 5’-TCTCCTATTGCAGTAGATGATTGGTACAGGCTTTTTCTTACCAGCTTCAGCCTCTCTTTTCTCGAGA-3’;

[0117] The primers for introducing the enterokinase recognition sequence (DDDDK) and the 6×His tag are:

[0118] AOXT2-HF: 5’-CAAAGAATGGATGATGATGATAAACACCACCACCACCACCACTGAGTTTGTAGCCTTAGACATGAC-3’;

[0119] AOXT2-HF: 5’-GCTACAAACTCAGTGGTGGTGGTGGTGGTGTTTAGTCAGTCAGTCAGCATTCTTTGACATTTGCCCTTTC-3’;

[0120] The PCR reaction system is as follows: The operation is carried out according to the 2×Super Pfx Master Mix instruction manual. The total volume of the PCR reaction system is 50 μL, including 25 μL of enzyme, 2.5 μL of each of the upstream and downstream primers, 100 ng of plasmid, and finally filled up to 50 μL with ddH2O.

[0121] The PCR reaction conditions are as follows: Pre-denaturation at 98 °C for 30 s; Denaturation at 98 °C for 10 s, annealing for 20 s according to the primer temperature, extension at 72 °C at 4 kb / min, and the reaction is set for 30 - 35 cycles (denaturation, annealing, and extension are one cycle); Renaturation at 72 °C for 5 min.

[0122] Construction of expression plasmid: After gel extraction of the target fragment, it can be transformed into JM109 chemically competent cells by chemical transformation method. The transformants are spread on solid LB resistant plates containing bleomycin (25 mg / L) and incubated statically in a 37 °C incubator for 12 - 16 h. After colonies grow out, single colonies are picked into liquid LB medium containing bleomycin (25 mg / L) and cultured overnight at 37 °C with 200 rpm shaking. The bacterial solution is sent to Suzhou Anshengda Biotechnology Co., Ltd. for determination. The plasmid containing the correct mutant is obtained: pPICZαA-Tn-K.

[0123] Construction of expression strain: The pPICZαA plasmid with correct gene sequencing is digested with SacI. It is introduced into P. pastoris GS115 competent cells by electroporation method and spread on YPD plates containing 100 mg / L bleomycin resistance. After colonies grow out, single colonies are picked into liquid YPD medium containing bleomycin (100 mg / L) and cultured overnight at 30 °C with 200 rpm shaking. The bacterial solution is sent to Suzhou Anshengda Biotechnology Co., Ltd. for determination. The successfully transformed strain GS115 / pPICZαA-Tn-K is obtained.

[0124] Flask fermentation production of Tn-K: Pick the correct single colony and inoculate it into a YPD test tube and culture it overnight at 30 °C and 220 rpm shaking to obtain the seed culture solution for flask fermentation. Inoculate 1% of the seed culture solution into 50 mL of BMGY medium and culture it at 30 °C and 220 rpm for 48 h. Centrifuge to harvest the cells, resuspend them in 50 mL of BMMY medium, and add 0.25 mL of inducer methanol every 12 h to maintain its final concentration at 0.5%. After 120 h of fermentation, centrifuge to collect the supernatant.

[0125] Purification of Tn-K: The fermentation supernatant is filtered through a 0.22 μM filter membrane. Operate according to the instructions of the His-tag protein purification kit. The collected eluate is the His-tagged Tn-K. Operate according to the instructions of the Biovision enterokinase cleavage kit and use enterokinase (EK) to cleave and remove the His tag. The purified Tn-K can be obtained.

[0126] The prepared antibacterial peptide Tn-K is detected by HPLC and MS, and the results are shown in Figure 3 and Figure 4 .

[0127] Refer to the operations in Example 1, and perform the bactericidal efficiency detection of using antibacterial peptide Tn-K alone, using 460 nm blue light alone, using 222 nm far ultraviolet light alone, and using 460 nm blue light and 222 nm far ultraviolet light together respectively. Calculate the bactericidal efficiency of each group, and the obtained bactericidal data are shown in Table 3 below and Figure 2 .

[0128] Table 3

[0129]

[0130] From Figure 2 and Table 3, it can be seen that when 460 nm blue light, 222 nm far ultraviolet light, and antibacterial peptide Tn-K are used alone, their bactericidal effects from low to high are: Tn-k, 460 nm, 222 nm. When 460 nm and 222 nm are used in combination, the bactericidal rate is further increased, reaching 4.9 lgCFU / ml at 40 min.

[0131] When the three are used in combination, a bactericidal synergistic effect similar to that of Enterobacter sakazakii is shown. All Salmonella have been killed at 40 min, and 5.7 lgCFU / ml can be reached at 32 min. The bactericidal efficiency exceeds the sum of the bactericidal rates of 415 + 222 nm (3.1 lgCFU / ml) and Tn-K (0.2 lgCFU / ml) at this time, showing a synergistic effect.

[0132] The bactericidal efficiencies of using antibacterial peptide Tn-K alone, 415 nm blue light alone, 222 nm far ultraviolet light alone, and 415 nm blue light and 222 nm far ultraviolet light together were detected respectively, and the bactericidal efficiencies of each group were calculated. The results are shown in Table 4 below.

[0133] Table 4

[0134]

[0135] It can be seen that 415 nm blue light, 222 nm far ultraviolet light, and antibacterial peptide Tn-K can also have a synergistic effect on killing Gram-negative bacteria.

[0136] Comparative Example 1

[0137] The operation steps are the same as those in Example 1, except that Tn is replaced with an equal concentration of nisin. The obtained bactericidal data are shown in Table 5 below.

[0138] Table 5

[0139]

[0140] Comparative Example 2

[0141] The operation steps are the same as those in Example 1, except that Tn is replaced with an equal concentration of bacitracin.

[0142] The obtained bactericidal data are shown in Table 6 below.

[0143] Table 6

[0144]

[0145] Comparative Example 3

[0146] The operating steps are the same as those in Example 1, except that Tn is replaced with polymyxin at the same concentration.

[0147] The obtained bactericidal data are shown in Table 7 below.

[0148] Table 7

[0149]

[0150] Comparative Example 4

[0151] The operating steps are the same as those in Example 1, except that Tn is replaced with melittin at the same concentration.

[0152] The obtained bactericidal data are shown in Table 8 below.

[0153] Table 8

[0154]

[0155]

[0156] As can be seen from Tables 3 to 5, when Tn is replaced with other antibacterial peptides at the same concentration (nisin, bacitracin polymyxin, melittin), the final bactericidal efficiency does not achieve synergy, and its highest bactericidal effect is far lower than the bactericidal effect of the combination of blue light, far ultraviolet light and antibacterial peptides (Tn, Tn-K) in Example 1 and Example 2. This shows that blue light and far ultraviolet light do not synergize with any antibacterial peptide, and only in combination with Tn and Tn-K can the synergistic effect of 1+1>2 be achieved.

[0157] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit their execution order. Obvious improvements made by those skilled in the art in combination with the existing common general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A mutant of the antimicrobial peptide Thanatin, characterized in that, The mutant contains the sequence shown in SEQ ID NO.

1.

2. A method for killing Gram-negative bacteria, characterized in that, The method comprises the following steps: (1) Adding an antimicrobial peptide in a sterilization environment; (2) Irradiating the sterilization environment with blue light and far ultraviolet light; The antimicrobial peptide is antimicrobial peptide Thanatin or the mutant described in claim 1; The wavelength of the blue light is 405 - 465 nm; the blue light irradiation dose is not less than 0.5 J / cm 2 ·min; The wavelength of the far ultraviolet light is 200 - 250 nm; the irradiation dose of the far ultraviolet light is not less than 10 J / cm 2 ·min.

3. The method according to claim 2, characterized in that, The sterilization environment includes a solid surface or a liquid environment.

4. The method according to claim 3, characterized in that, The solid surface includes the human skin, food surface, ground, surface of desks and chairs, medicine surface, surface of medical tools or surface of medical instruments.

5. The method according to claim 3, characterized in that, The liquid environment includes water body or dairy products; the liquid in the liquid environment is stored in a transparent container or an opaque container with an opening.

6. The method according to claim 2, wherein When the sterilization environment is a solid surface, the addition amount of the antimicrobial peptide is not less than 1 mg / cm 2 ; when the sterilization environment is a liquid environment, the addition amount of the antimicrobial peptide is not less than 1 mg / L.

7. The method according to claim 2, wherein When the sterilization environment is a solid surface, the addition amount of the antimicrobial peptide is 1-10 mg / cm 2 ; when the sterilization environment is a liquid environment, the addition amount of the antimicrobial peptide is 1-10 mg / L.

8. The method according to claim 2, wherein The blue light irradiation dose is 0.5 - 5 J / cm 2 ·min; the far ultraviolet irradiation dose is 10 - 50 J / cm 2 ·min.

9. The method according to claim 2, characterized in that, The irradiation is to arrange a blue light source and a far ultraviolet light source above, on the side or at the bottom of the sterilization environment, so that the blue light and the far ultraviolet light can jointly cover the sterilization environment.

10. The method according to claim 2, wherein The irradiation time is not less than 30 minutes.

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