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Study on Isolation, Purification of Apple Snail (Ampullaria Gigas) Antianicrobial Peptides and Their Antibacterial Activities

Author: TangShuMing
Tutor: HuangZhiJian
School: Fujian Agriculture and Forestry University
Course: Clinical Veterinary Medicine
Keywords: Apple snails Hang nisin Purification Active
CLC: Q51
Type: Master's thesis
Year: 2008
Downloads: 277
Quote: 1
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Abstract

In this experiment, freshwater snails as the research object, its various body tissues and tissue fluid extraction and peptide components were separated, and the in vitro antibacterial activity was studied. The main research is divided into the following sections: Test 1: snails antimicrobial substance isolated Method Objective: snails in vivo antibacterial substances isolated preliminary exploration. Methods: This test uses ammonium sulfate extraction, methanol extraction method, Tris-HCL extraction method, improved acid extraction of four different methods snails intestinal tissue fluid, liver and muscle tissue separating peptides studied snails the body's ability to produce antimicrobial peptides and antibacterial effects. Results: Escherichia coli and Staphylococcus aureus as indicator bacteria, acetic acid extraction method with antibacterial activity can be isolated from the crude extract, both snails and intestinal tissue fluid extract antibacterial effect is obvious, while the other three methods are not vivo antimicrobial substances isolated from apple snails. Conclusion: acetic acid extraction method of tissue fluid from snails and intestinal tissue isolated antibacterial substances. Test 2: Purification of antimicrobial peptides snails Objective: To study the best way snails purified peptides. Methods: sephadexG-100 gel filtration chromatography column snails antimicrobial peptides, column specifications for the 2.5 × 60cm, eluting with 0.2mol / L sodium chloride solution at a flow rate of 0.6 ml / min, 8 min / Tube . Results: The separation effect is more obvious and very clear distinction between the peaks, wherein the active peak Ⅱ peak 27 to 32. Test 3: Identification of snails and properties of antimicrobial peptides Objective: snails purified antimicrobial substances were identified. Methods: polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, temperature, pH, antimicrobial substances to be identified snails. Results: The electrophoretic patterns of antimicrobial peptides isolated as a single band with a molecular weight of about 5000 ~ 7000 Da; 40 ℃ ~ 80 ℃ for 30min its antimicrobial activity unchanged, 90 ℃ for 30min antibacterial activity declined slightly; at pH6. 5 to 7.5 near neutral environment, snails strongest antibacterial activity of antimicrobial peptides, acidic (pH <6.0) or alkaline (pH> 8.0) solvent environment can lead to decreased antibacterial activity of antimicrobial peptides snails. Conclusion: The purified antimicrobial substance is a single, heat-resistant, small peptide molecules. Test 4: Determination of antibacterial spectrum antimicrobial peptides snails and Antimicrobial Mechanism Objective: To study snails antibacterial spectrum antimicrobial and antibacterial mechanisms. Methods: Determination of antibacterial agarose diffusion Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Bacillus subtilis minimum inhibitory concentration; observed by scanning electron microscopy morphology antimicrobial effect against E. coli. Results: The peptide Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Bacillus subtilis minimum inhibitory concentrations were 12.5μg/ml, 25μg/ml, 100μg/ml, 25μg/ml. Scanning electron microscopy, adding snails antimicrobial surface morphology of E. coli atrophy, rough, contents outflow. Conclusion: The antimicrobial peptide Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Bacillus subtilis has some antibacterial effect.

Study on Isolation, Purification of Apple Snail (Ampullaria Gigas) Antianicrobial Peptides and Their Antibacterial Activities

Abstract

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