The Separation And Purification Of Intestine Antimicrobial Substances Of Grass Carp And Its Identification

In order to improve the value of grass carp intestine, and provide theoretical basis for the efficient purification and application of the grass carp intestinal antimicrobial peptide, this subject optimized the grass carp intestinal antimicrobial substances extraction process. Compared the methods of ammonium sulfate graded precipitation, ethanol graded precipitation and ultrafiltration centrifuge, and combined with Sephadex G-25 column and half preparative RP-HPLC to separate the grass carp intestinal antimicrobial substances. Chromatographic conditions of C₁₈ analytical RP-HPLC, structure, stability as well as antibacterial effect of grass carp intestinal antimicrobial peptide were also analyzed.1) Used grass carp intestine as raw material, the influence of volume fraction of acetic acid, ratio of material to liquor, ultrasonic time on preliminary extraction of grass carp intestinal antimicrobial substances was determined by single factor and orthogonal experimental study. The results show that the optimal preliminary extracting condition for the grass carp intestinal antimicrobial substances are as follows:the volume fraction of acetic acid is 10%, ratio of material to liquor is 1:3.5?g:mL?, ultrasonic time is 10 min, socking 24 h at 4 ?, under this condition, the antimicrobial activity is effective, antibacterial circle diameter of S.aureus is 3.106 cm, antibacterial circle diameter of E.coli is 2.028 cm.2) Compared the methods of ammonium sulfate graded precipitation, ethanol graded precipitation and ultrafiltration centrifuge, and combined with Sephadex G-25 column and half preparative RP-HPLC to separate the grass carp intestinal antimicrobial substances. The results show that the separation efficiency of the extracted grass carp intestinal antibacterial substances centrifuged with the ultrafiltration centrifuge tube is perfect. After the centrifugal liquid separating by Sephadex G-25 column, the second fluid peak which has antibacterial effect to E.coli and S.aureus was collected, and separated by half preparative RP-HPLC, a single antibacterial ingredients was obtained, antibacterial circle diameter of E.coli is 1.023 cm, antibacterial circle diameter of S.aureus is 1.106 cm.3)The C₁₈ analytical R-HPLC chromatographic conditions for identifying the grass carp intestinal antimicrobial substances was optimized, its primary structure and secondary structure are analyzed with mass spectrometry technology. The suitable chromatographic conditions are as follows:Acetonitrile volume fraction is 50%, the flow rate is 0.6 mL/min, the column temperature is 20 ?, or acetonitrile volume fraction is 60%, the flow rate is 1.0 mL/min, column temperature is 35 ?. The antibacterial peptide extracted from this experiment are neutral polypeptide, total net charge is zero, the proline content is 25%, relative molecular weight is 752 Da, amino acid sequence is ACAYSPPG. The highest homology sequence is red insect antibacterial peptide, rana antibacterial peptide Tigerinins, Ranacyclin-B-RN2, Ranacyclin-B-LK2 and Ranacyclin-B-AL1.4) To study the bacteriostatic effect of grass carp intestinal antimicrobial peptide, the solid phase synthetic grass carp intestinal antimicrobial peptides and ampicillin, Nisin, sodium metabisulfite, sodium benzoate, potassium sorbate are compared. The results show that, to inhibit S.aureus and E.coli, the minimum solubility of grass carp intestinal antimicrobial peptide can be as low as 0.16 ?g/mL. Within range of 0.16?100 ?g/mL, it's ability to inhibit E.coli is better than that of Nisin, potassium sorbate, sodium benzoate and sodium metabisulfite, to inhibit S.aureus is better than that of ampicillin. Within range of 0.16?1000 ?g/mL, it's ability to inhibit S.aureus is better than that of Nisin, potassium sorbate, sodium benzoate and sodium metabisulfite. Within range of 0.16?20 ?g/mL, it's ability to inhibit E.coli is better than that of ampicillin.5) The solid phase synthetic grass carp intestinal antimicrobial peptide was treated with different temperature, pH, freezing-thawing, protease digestion and metal salt for studying it's stability. The results show that, the peptide has good antibacterial activity at low temperature of 0?20 ?. Within 100 ?, has certain ability of heat-resistant. In acid environment, its antimicrobial activity is best. After repeat freezing and thawing, the stability of the peptide's ability to inhibit E.coli and S.aureus is decreased obviously. Stomach protease and neutral protease reduced it's ability to inhibit E.coli, but raised it's ability to inhibit S.aureus. Glutamine transaminase presents the opposite effect. MgCl₂, ZnSO₄, KCl and CuSO₄ could increase it's ability to inhibit S.aureus, while Al₂?SO₄?₃ and FeSO₄ decrease it's ability to inhibit S.aureus. MgCl₂ could increase it's ability to inhibit E.coli, while ZnSO₄, KCl, CaCl₂, NaCl and CuSO₄ could decrease it's ability to inhibit E.coli.