Isolation And Antibacterial Activity Of Peptide From Body Wall Of Apostichopus Japonicas

Fresh Apostichopus japonicas was selected as material in this article, with antibacterial activity as an index, two extraction methods including acetic acid extraction and organic solvent extraction were used to extract peptides of Apostichopus japonicas body wall. Single factor test and response surface test analysis were chosen to optimize extraction technology. Extracts were divided into 3 components with different molecular weights with ultrafiltration. The antibacterial activity of each sample was tested against Escherichia coli, Salmonella eternitidis, Staphylococcus aureus and Bacillus subtilis. The best active component was purified in the next step by using macroporous resin. The component with better activity was taken into the next purification step with high-speed counter-current chromatography(HSCCC). Finally, the antimicrobial peptide was analyzed by high performance liquid chromatography(HPLC). The main research projects were as follows: 1. Optimization of extraction technology of peptides from the body wall of Apostichopus japonicas.The response surface method was used to optimize extraction method with an index of yield, considering concentration of liquid, the ratio of material to liquid and centrifugal rotational speed. The best conditions were determined: acetic acid extraction: concentration of acetic acid 6.28%, the ratio of material to solvent 1:1(v/v), centrifugal rotational speed 8540r/min. And the highest yield in theory was 2.803%. Actual yield under the optimum conditions was 2.78%. Organic solvent extraction: concentration of methanol 80%, the ratio of material to solvent 5.46:1(v/v), centrifugal rotational speed 8081r/min. And the highest yield in theory was 1.87%. Actual yield under the above conditions was 1.82%. The peptide contents of crude extracts were tested by using the biuret method. And the results were 2.28mg/m L and 1.50 mg/m L respectively。 2. Two extracts were separated by ultrafiltration method. Then antibacterial activitives of different cascade peptides were measured.The crude extracts from acetic acid and organic solvent after ultrafiltration were divided into three peptides with different molecular weights of 5~10kD, 1~5kD and <1kD. Different cascade peptides were separated thoroughly after 4 times ultrafiltration. Turbidimetric method was applied to test activity of each sample against bacteria which including Escherichia coli, Salmonella eternitidis, Staphylococcus aureus and Bacillus subtilis. And the growth curves of each bacterium were studied. The result showed that crude extract with moleacular weight of < 1kD from acetic acid exhibited the best antibacterial activity against four strain bacteria. 3. Antimicrobial peptide was purified and its antibacterial activity was measured by turbidimetric method.Separated crude extract with moleacular weight of < 1kD from acetic acid with macroporous adsorption resin, and then four elution peaks were obtained with 25%, 50%, 75% and 95% ethanol as elution agents, which were marked as F1、F2、F3、F4 respectively. Turbidimetric method was applied to test the antibacterial activity of F1、F2、F3、F4. The results showed that F3 owned the best antibacterial activity. The molecular weight of antibacterial peptide was detected by using electrophoresis of Tricine-SDS-PAGE. The component(F3) with the highest antibacterial activity was achieved and its molecule weight was tested about 4.35 kD. Through high-speed countercurrent chromatography separation, four independent spectrum peaks of component F3 were got. The solvent system was hexane, ethyl acetate, methanoland and water with volume ratio 3:7:5:5. The experimental conditions were that sampling amount 25 mg, flow rate of mobile phase 1mL/min, rotate speed 850r/min and the temperature 25℃. T1 was the main component of F3. The result of HPLC showed that component T1 had five substances under 280 nm wavelength.