A large yellow croaker whey acidic protein antibacterial peptide and its application
A technology of whey acidic protein and antimicrobial peptides, which can be used in applications, protein-containing food ingredients, peptides, etc., and can solve problems such as difficult purification, easy inactivation, research and application difficulties, etc.
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Embodiment 1
[0035] Example 1: Screening of antimicrobial peptides derived from large yellow croaker whey acidic protein
[0036] The antibacterial peptide prediction online server AntiBP, APD3 and CAMP were used to predict the possible antibacterial sequences in the acidic protein sequence of large yellow croaker whey, and the charge, hydrophobicity and reliability of the possible antibacterial sequences were analyzed, and finally the amino acid sequence was screened FTKPGVCPRRRWGAG was chemically synthesized, and its antibacterial activity was verified.
Embodiment 2
[0037] Embodiment 2: Minimum Inhibitory Concentration (MIC) Determination
[0038] Staphylococcus aureus, Vibrio alginolyticus and Aeromonas hydrophila were cultured at 37°C for 12 hours to logarithmic growth phase, and diluted to 10 in 0.01M pH 7.2 phosphate buffer 6-7 CFU / mL. Dissolve the peptide in phosphate buffer, and mix it with bacteria in an equal volume at 37°C for 2 hours. The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial peptide at which no bacterial growth is visible from the microtiter plate after overnight incubation at 37°C. like figure 1 , figure 2 and image 3As shown, the minimum inhibitory concentration (MIC) of LCWAP to Staphylococcus aureus was 62.5 μg / mL, the minimum inhibitory concentration (MIC) to alginolytic Vibrio was 15.6 μg / mL, and the minimum inhibitory concentration (MIC) to Aeromonas hydrophila The minimum inhibitory concentration (MIC) is 31.2μg / mL ( figure 1 , figure 2 and image 3 ).
Embodiment 3
[0039] Embodiment 3: transmission electron microscope analysis
[0040] to 10 6-7 CFU / mL bacteria were treated with 2×MIC LCWAP for 2 h at 37° C., then centrifuged at 2700 g for 10 min, and washed twice with phosphate buffer (pH 7.2). After fixing with 1% osmic acid, dehydrate with 95% ethanol, and then treat with acetone for 20 min. The samples were baked at 70°C for 24 h, and thin slices of 70–90 nm were prepared on copper grids, which were then stained with lead citrate and uranyl acetate. The ultrastructure was observed and captured with a H-7650 transmission electron microscope.
[0041] Take Staphylococcus aureus as an example, such as Figure 4 As shown, for the untreated bacteria, the intracellular structure of the bacteria is dense, the organization is intact, and the cell surface is not damaged. However, after 0.5 h of treatment with peptide LCWAP, it can be clearly observed that the bacterial cells become transparent, and the edges of the cell membrane begin to ...
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