CN115710305B - Fermented large yellow croaker succinic acid dehydrogenated protein antibacterial peptide SDH73 and application thereof - Google Patents

Abstract

The invention discloses a fermentation large yellow croaker succinic acid dehydrogenation protein antibacterial peptide SDH73, the amino acid sequence of which is KRGMLENCILLSLFAK, and the molecular weight of the antibacterial peptide SDH73 is 1836 daltons. Experiments prove that the antibacterial peptide SDH73 can effectively inhibit food spoilage bacteria such as nitrifying bacteria, bacillus aryabhattai, bacillus stratospheric and the like. The invention lays a foundation for further researching the use of the fermented large yellow croaker succinic acid dehydrogenated protein in food preservative, biological medicine and feed additive.

Description

A fermented large yellow croaker succinate dehydrogenase protein antimicrobial peptide SDH73 and its application

Technical Field

The invention relates to the field of biotechnology, and in particular to a fermented large yellow croaker succinate dehydrogenase protein antimicrobial peptide SDH73 and an application thereof.

Background Art

Microbial activity is the main cause of spoilage of aquatic products. Through extensive research on fish bacteriology during capture, storage and circulation, it has gradually become clear that although fish bodies are initially contaminated by a variety of microorganisms, only some of these bacteria are involved in the spoilage process. These bacteria that are suitable for survival and reproduction and produce metabolites of spoilage odor and foreign taste are the specific spoilage bacteria of the fish. Specific spoilage bacteria grow faster than other microorganisms in storage and have strong spoilage activity. Therefore, controlling the growth of specific spoilage bacteria, inhibiting their production of harmful substances and preventing product quality degradation has important social and economic value.

Nitrobacter, Bacillus agglomerans, and Bacillus stratospermidis have been proven to be spoilage bacteria that can degrade nutrients such as proteins, fats, and nucleotides during their growth and metabolism, produce small molecules such as volatile basic nitrogen and trimethylamine, release unpleasant odors, and ultimately cause the fish to spoil.

Antimicrobial peptides are a class of polypeptide substances widely found in natural organisms. They have good inhibitory effects on bacteria and are highly safe. They meet the requirements of today's green production and can be used as a substitute for traditional antibiotics and feed additives. Fish belong to the phylum Chordata. The nonspecific immune system is the first line of defense for fish against various pathogens. Antimicrobial peptides are an important part of the nonspecific immune system of fish. Fish live in an aquatic environment containing various microorganisms. When they are invaded by pathogenic microorganisms or their bodies are damaged, they can quickly produce immune molecules mainly composed of antimicrobial peptides to defend against the invasion of pathogenic microorganisms or kill pathogenic microorganisms.

Therefore, the small molecule peptides isolated from fermented large yellow croaker have very promising application prospects as antibacterial drugs for inhibiting specific food spoilage bacteria (such as Nitrobacter, Bacillus aeruginosa, and Bacillus stratospermum), aquatic feed additives, and food preservatives.

Summary of the invention

The purpose of the present invention is to provide a fermented large yellow croaker succinate dehydrogenase protein antimicrobial peptide SDH73 and its application, and to provide an experimental basis for finding new food preservatives, biopharmaceuticals and aquatic feed additives through the study of the antibacterial activity of the fermented large yellow croaker succinate dehydrogenase protein antimicrobial peptide SDH73 on food spoilage bacteria, so as to promote the healthy and sustainable development of my country's food, medicine and aquaculture industries.

One of the technical solutions adopted by the present invention to solve the technical problem is: providing a fermented large yellow croaker succinate dehydrogenase protein antimicrobial peptide SDH73, whose amino acid sequence is KRGMLENCILLSLFAK, as shown in SEQ ID NO:1.

The molecular weight of the antimicrobial peptide SDH73 is 1836 Daltons, the charge is +2, and the overall hydrophobicity ratio is 56%.

The antimicrobial peptide SDH73 destroys bacteria based on the following principles:

(i) The positive charge of the antimicrobial peptide SDH73 can interact with the bacterial cell membrane and increase the pore-forming activity of the bacterial cell membrane;

(ii) destroying the cell membrane and changing the permeability of the bacterial cell membrane, causing the intracellular substances to leak out and thus leading to bacterial death;

(iii) It combines with DNA in an intercalation manner, destroying the bacterial DNA structure and causing bacterial death.

The second technical solution adopted by the present invention to solve its technical problem is: to provide an application of fermented large yellow croaker succinate dehydrogenase protein antimicrobial peptide SDH73 in the preparation of antimicrobial drugs, which are used to inhibit and/or kill food spoilage bacteria.

The third technical solution adopted by the present invention to solve its technical problem is: to provide an antibacterial drug, whose active ingredient includes fermented large yellow croaker succinate dehydrogenase protein antibacterial peptide SDH73, and the amino acid sequence of the antibacterial peptide SDH73 is SEQ ID NO: 1.

In a preferred embodiment of the present invention, the active ingredient of the antibacterial drug is the fermented large yellow croaker succinate dehydrogenase protein antibacterial peptide SDH73, and the amino acid sequence of the antibacterial peptide SDH73 is SEQ ID NO: 1.

In a preferred embodiment of the present invention, the antimicrobial drug is used to inhibit and/or kill spoilage bacteria of by-products.

The fourth technical solution adopted by the present invention to solve its technical problem is: to provide a feed additive, whose effective ingredient includes fermented large yellow croaker succinate dehydrogenase protein antimicrobial peptide SDH73, and the amino acid sequence of the antimicrobial peptide SDH73 is SEQID NO: 1.

In a preferred embodiment of the present invention, the active ingredient of the feed additive is the fermented large yellow croaker succinate dehydrogenase protein antimicrobial peptide SDH73, and the amino acid sequence of the antimicrobial peptide SDH73 is SEQ ID NO: 1.

In a preferred embodiment of the present invention, the feed additive is used to inhibit and/or kill food spoilage bacteria.

The fifth technical solution adopted by the present invention to solve its technical problem is: to provide a food preservative, whose effective ingredient is the antimicrobial peptide SDH73 of fermented large yellow croaker succinate dehydrogenase protein, and the amino acid sequence of the antimicrobial peptide SDH73 is SEQ ID NO: 1.

In a preferred embodiment of the present invention, the food preservative is used to inhibit and/or kill food spoilage bacteria.

The antimicrobial peptides of the present invention can be synthesized by methods known to those skilled in the art, such as solid phase synthesis, and purified by methods known to those skilled in the art, such as high performance liquid chromatography.

The implementation of the present invention has the following beneficial effects:

The present invention uses fermented yellow croaker as the research object, and uses LCMS mass spectrometry results to perform bioinformatics prediction on the peptide segments, and discovers a peptide SDH73 with a completely new amino acid sequence. Taking Nitrobacter as an example, the antibacterial activity of the peptide SDH73 is studied; the experimental results show that the peptide has a strong inhibitory effect on Nitrobacter. Its antibacterial mechanism is to first adsorb on the bacterial surface, then destroy the bacterial cell membrane, and combine with bacterial DNA to destroy the structure of DNA, thereby achieving the effect of inactivating the bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a mass spectrum of the antimicrobial peptide SDH73.

FIG. 2 is a schematic diagram of the structure of the antimicrobial peptide SDH73.

Figure 3 is a comparison chart of the minimum inhibitory concentration (MIC) of the antimicrobial peptide SDH73 against Nitrobacter.

A: antimicrobial peptide concentration 0 μg/mL;

B: antimicrobial peptide concentration 250 μg/mL;

C: antimicrobial peptide concentration 125 μg/mL;

D: antimicrobial peptide concentration 62.5 μg/mL;

E: antimicrobial peptide concentration 31.25 μg/mL;

F: Antimicrobial peptide concentration: 15.625 μg/mL.

FIG. 4 is a time-kill curve analysis diagram of the antimicrobial peptide SDH73 against Nitrobacter.

FIG. 5 is a graph showing the determination and analysis of the effect of the antimicrobial peptide SDH73 on the cell membrane permeability of Nitrobacter.

FIG. 6 is a fluorescence spectrum diagram of the competitive binding of antimicrobial peptide SDH73 and ethidium bromide (EB) to DNA in Nitrobacter.

FIG. 7 is a fluorescence spectrum of the antimicrobial peptide SDH73 binding to DNA in Nitrobacter.

FIG. 8 is a graph showing the results of determining the nucleic acid content in the bacterial solution.

FIG. 9 is a graph showing the results of determining the protein content in the bacterial solution.

FIG. 10 is an electrophoretic diagram of the binding of the antimicrobial peptide SDH73 to Nitrobacter DNA.

in,

Strip l: blank control;

Bands a-k: the mass ratios of SDH73 to DNA are 100/1, 100/2, 100/4, 100/6, 100/8, 100/10, 100/20, 100/40, 100/60, 100/80, and 100/100, respectively.

DETAILED DESCRIPTION

In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example 1: Liquid chromatography/mass spectrometry (LCMS) of fermented large yellow croaker

Weigh 100-200g of fermented large yellow croaker sample meat into a beaker, add appropriate amount of ultrapure water and crush it with a blender for 3-5 minutes, then boil the crushed tissue in a 100℃ water bath for 2-3 hours. Prepare the sample by centrifugation at 11000g centrifugal force for 20 minutes at 4℃. Then pass the centrifuged supernatant through a 0-3000 mesh sieve and freeze it in a refrigerator overnight before freeze-drying. The freeze-dried sample is then subjected to LCMS experiment.

Chromatographic conditions: Injection volume: 5.0 μL

Chromatographic column: C18 analytical column, length 25cm, inner diameter 75μm

Mobile Phase:

A: 0.1% methanol aqueous solution

B: Acetonitrile

The obtained peptide mass spectra were distinguished and identified by combining the search software: MAXQUANTv1.6.5.0 and the database: uniprot large yellow croaker protein library, and 85 peptides were obtained. The mass spectrometry results of the antimicrobial peptide SDH73 are shown in Figure 1.

Example 2: Screening of antimicrobial peptide SDH73 from fermented large yellow croaker succinate dehydrogenase protein

The 20 amino acid sequences obtained from the LCMS results were used to predict the possible antibacterial sequences in the fermented large yellow croaker protein sequence using the antimicrobial peptide prediction online server APD3 and CAMP, and the charge and hydrophobicity of the possible antibacterial sequences were analyzed. Finally, the amino acid sequence KRGMLENCILLSLFAK was chemically synthesized (synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd.) and the antibacterial activity was verified. The screening results are shown in the following table.

Table 1 Prediction of potential antimicrobial peptides from fermented large yellow croaker

As can be seen from Table 1, the sequence KRGMLENCILLSLFAK has the characteristics of a typical antimicrobial peptide, namely the number of amino acids (<50), total positive charge (+2 to +9) and percentage of hydrophobic amino acids (>30%). Two peptides (KRGMLENCILLSLFAK and MAALQLMQQKANK) that meet the characteristics were synthesized and subjected to antibacterial experiments against Nitrobacter. The results showed that the peptide sequence KRGMLENCILLSLFAK had the strongest antibacterial effect.

Example 3: 3D structure prediction of antimicrobial peptide SDH73

The structures of fermented large yellow croaker succinate dehydrogenase protein and its derived antimicrobial peptide SDH73 were predicted using the online structure prediction server Swiss-model, and edited and modified using Pymol software to obtain the structure of the antimicrobial peptide SDH73, as shown in Figure 2.

Example 4: Determination of the minimum inhibitory concentration (MIC) of the antimicrobial peptide SDH73

Nitrobacter was cultured at 37°C for 12 hours until the logarithmic growth phase, and diluted to 10 ^4-5 CFU/mL in 0.01M pH 7.2 phosphate buffer. The peptide was dissolved in phosphate buffer and mixed with bacteria in equal volumes at 37°C for 2 hours. The minimum inhibitory concentration (MIC) refers to the lowest concentration of antimicrobial peptide at which no bacterial growth can be seen on the microtiter plate after incubation at 37°C overnight. As shown in Figure 3, the minimum inhibitory concentration (MIC) of the antimicrobial peptide SDH73 against Nitrobacter was 62.5μg/mL.

Example 5: Time-Kill determination of antimicrobial peptide SDH73

Nitrobacter was cultured at 37°C for 12h to the logarithmic growth phase and diluted to 10 ^4-5 CFU/mL in 0.01M pH 7.2 phosphate buffer. 1×MIC and 2×MIC concentration peptides were mixed with bacteria in equal volumes at 37°C and incubated separately. Samples were taken every 30 minutes and plated. The total number of colonies was recorded after culturing at 37°C overnight. The results show that the antimicrobial peptide SDH73 has a significant effect on Nitrobacter starting at 1h. Under the action of the antimicrobial peptide SDH73, the number of bacteria decreased faster. This shows that the antimicrobial peptide SDH73 has a significant inhibitory effect on Nitrobacter as the action time increases (as shown in Figure 4).

Example 6: Determination of the effect of antimicrobial peptide SDH73 on cell membrane permeability

Nitrobacter was cultured at 37℃ for 12h to the logarithmic growth phase, centrifuged at 10000r/min for 1min, washed three times with PBS, resuspended in 10mL sterile M9 lactose induction medium, and cultured at 37℃ and 130r/min for 2h. 100μL of 1.5mmol/mL ONPG solution was added to each group, incubated at 37℃, and the absorbance was measured at 420nm on a microplate reader every 1h. Bacteria can induce the production of β-galactosidase when lactose is the only carbon source. After the cell membrane is damaged, extracellular ONPG can penetrate into the cell and be hydrolyzed into galactose and o-nitrophenol (ONP); ONP has characteristic ultraviolet absorption at 420nm. Within a certain concentration range, the absorbance of ONP is proportional to the cell membrane permeability, which can be used as an important indicator for evaluating cell membrane permeability. As shown in Figure 5, as the concentration of the antimicrobial peptide SDH73 increased, the cell membrane permeability gradually increased over time.

Example 7: Fluorescence spectrum experiment of competitive binding of antimicrobial peptide SDH73 and EB to DNA

The fluorescence spectrum experiment of the competitive binding of antimicrobial peptide SDH73 and ethidium bromide (EB) to DNA was used to analyze the action mode of antimicrobial peptide SDH73 and genomic DNA of Nitrobacter: Nitrobacter genomic DNA was diluted to 50 μg/mL with TE buffer. The reaction was carried out in a 96-well plate. First, 50 μL of DNA solution and 0.75 μL of EB solution with a concentration of 100 μg/mL were added to each well, mixed and placed in a biochemical incubator at 37°C in the dark for 10 minutes. Then 50 μL of peptide solution of different concentrations was added, and the blank control group was replaced with 50 μL of distilled water. After mixing, it was placed in a biochemical incubator at 37°C in the dark for 30 minutes. After the incubation, the fluorescence spectrum of the sample was measured with a multifunctional microplate reader at an excitation wavelength of 535 nm and an emission wavelength of 570-710 nm. In aqueous solution, the fluorescence of EB is very weak, but when it binds to double-stranded DNA by high affinity and embedding, its fluorescence intensity is greatly enhanced. If there is a substance in the EB-DNA complex system that can have a similar effect with DNA and compete with EB bound to DNA, the fluorescence intensity of the system will decrease, indicating that the competitor binds to DNA in the same intercalation mode as EB. Therefore, by measuring the changes in the fluorescence spectrum of the DNA-EB complex system and the competitor, it is determined whether the competitor also binds to DNA in the same intercalation manner as EB. As shown in Figure 6, with the increase of the concentration of the antimicrobial peptide SDH73, the fluorescence intensity of the EB-DNA complex also decreases significantly, indicating that the antimicrobial peptide SDH73 has intercalated with the DNA of Nitrobacter, competing with the EB previously bound to the DNA base pair, replacing EB to bind to DNA in an intercalation manner, and reducing the fluorescence intensity of the entire system.

Example 8: Fluorescence spectrum of antimicrobial peptide SDH73 binding to DNA in Nitrobacter

Nitrobacter was inoculated into LB broth and cultured at 37℃ and 200r/min for 12h to grow to the logarithmic phase. 1mL of the bacterial solution in the logarithmic phase was centrifuged and the supernatant was discarded. The collected bacteria were washed three times with 0.1M PBS and the bacterial solution concentration was adjusted to 10 ⁷ CFU/mL. The antimicrobial peptide SDH73 was added to the bacterial solution to make the final concentrations of 1/4×MIC, 1/2×MIC, 1×MIC, and 2×MIC. No antimicrobial peptide SDH73 was added as a blank control. The sample was cultured at 37℃ and 120r/min for 3h, 100μL was taken out and 100μL PI (propidium iodide) dye was added, vortexed at 25℃, and incubated in the dark for 15min. The fluorescence intensity was measured on an ELISA reader. The excitation wavelength was set to 535nm, and the emission wavelength was measured in the range of 570nm-830nm. PI (Propidium lodide) is a nucleic acid dye with an excitation wavelength of 535nm and the strongest fluorescence absorption at an emission wavelength of 615nm. It cannot pass through normal cell membranes, but when the cell membrane is damaged or ruptured, PI can enter the cell membrane and bind to DNA to show red fluorescence. The degree of cell damage can be determined based on the intensity of fluorescence. The binding of PI to DNA of Nitrobacter treated with different concentrations of antimicrobial peptides is shown in Figure 7. The fluorescence intensity of Nitrobacter treated with different concentrations of antimicrobial peptide SDH73 is significantly higher than that of the blank control group, indicating that the antimicrobial peptide treatment damaged the bacterial cell membrane and increased the permeability of the cell membrane, and high doses of antimicrobial peptides greatly increased the permeability of the bacterial cell membrane, resulting in more PI entering the cell and binding to DNA.

Example 9: Determination of nucleic acid and protein content in bacterial liquid

Nitrobacter was inoculated into LB broth and cultured at 37°C and 200r/min for 12h to grow to the logarithmic phase. Washed with sterile saline three times, and the concentration of the bacterial suspension was adjusted to 10 ³ CFU/mL. Similarly, the antimicrobial peptide SDH73 was diluted with sterile saline to a final concentration of 1×MIC, 2×MIC, and 4×MIC, respectively. The bacterial suspension and the antimicrobial peptide solution were mixed at a volume ratio of 1:1, and sterile saline without antimicrobial peptide was mixed with the bacterial suspension in equal amounts as a control group. Cultured at 37°C with constant temperature shaking, the contents of nucleic acids and proteins in the bacterial solution were measured at 260nm and 280nm every 10min using a UV-visible spectrophotometer. The leakage of intracellular substances is one of the important indicators of changes in cell membrane permeability or damage. Therefore, the degree of cell membrane damage can be indirectly reflected by detecting the leakage of macromolecular substances such as nucleic acids and proteins in the cell. The leakage of intracellular nucleic acids and proteins in Nitrobacter after treatment with antimicrobial peptides is shown in Figures 8 and 9. As time goes by, the release of nucleic acid and protein in the treatment group is higher than that in the control group, indicating that the antimicrobial peptide can change the permeability of bacterial cell membrane, causing the leakage of membrane substances to the outside of the cell, thereby inducing the death of bacteria.

Example 10: Interaction between antimicrobial peptide SDH73 and Nitrobacter DNA

The interaction between the antimicrobial peptide SDH73 and the DNA of Nitrobacter was studied by the DNA gel retardation method. Nitrobacter was cultured in 50 mL of nutrient broth (NB) at 37°C for 12 h, and the bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit. The purity of the extracted genomic DNA was evaluated by the optical density ratio at 260 and 280 nm (OD260/OD280 ≥ 1.90). Next, DNA (168 ng/μL) was mixed with peptide SDH73 at different mass ratios at 37°C for 60 min, and the mixture was electrophoresed on a 0.8% agarose gel. Gel retardation was observed under ultraviolet irradiation using the GelDoc XR gel imaging system (Bio-Rad, USA), as shown in Figure 10.

In summary, the present invention provides a new antimicrobial peptide SDH73 with a minimum inhibitory concentration MIC of 62.5 μg/mL against Nitrobacter, which can inhibit the growth of Nitrobacter. The antimicrobial peptide SDH73 of the present invention first adsorbs on the surface of bacteria, then destroys the cell membrane of bacteria, and combines with bacterial DNA to destroy the structure of DNA, thereby achieving the effect of inactivating bacteria.

Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims (8)

1. A fermented large yellow croaker succinic acid dehydrogenated protein antibacterial peptide SDH73, the amino acid sequence of which is shown in SEQ ID NO: 1.

2. The use of the fermented large yellow croaker succinic acid dehydrogenase protein antibacterial peptide SDH73 according to claim 1 for the preparation of antibacterial drugs, characterized in that: the antibacterial drug is used for inhibiting and/or killing nitrifying bacilli.

3. An antibacterial agent characterized in that: the active ingredient of the anti-bacterial peptide is fermented large yellow croaker succinic acid dehydrogenation protein SDH73, and the amino acid sequence of the anti-bacterial peptide SDH73 is SEQ ID NO:1.

4. An antimicrobial agent as claimed in claim 3 wherein: the antibacterial drug is used for inhibiting and/or killing nitrifying bacilli.

5. A feed additive, characterized in that: the active ingredient of the anti-bacterial peptide is fermented large yellow croaker succinic acid dehydrogenation protein SDH73, and the amino acid sequence of the anti-bacterial peptide SDH73 is SEQ ID NO:1.

6. A feed additive as claimed in claim 5, wherein: the feed additive is used for inhibiting and/or killing nitrifying bacilli.

7. A food preservative characterized by: the active ingredient of the anti-bacterial peptide is fermented large yellow croaker succinic acid dehydrogenation protein SDH73, and the amino acid sequence of the anti-bacterial peptide SDH73 is SEQ ID NO:1.

8. The food preservative according to claim 7, characterized in that: the food preservative is used for inhibiting and/or killing nitrifying bacilli.