Induction, Antibacterial Activity, Separation And Purification Of Antimicrobial Peptides Of Tenebrio Molitor

Antimicrobial peptides are small molecules peptides produced by the biological defense system which against exogenous pathogens, antimicrobial peptides used as a class of body defense effectors wide spread in the biosphere. They have characteristics of broad spectrum, unique mechanism of action, not easy to produce drug resistance, thermal stability, good water solubility, extensive source of raw material. They have broad application prospects in the field of health care, agricultural production, food, health care and so on. Tenebrio molitor has characteristics of miscellaneous eating habits, strong, disease resistance, short growth cycle, breeding investment small and simple. It’s a good test material for insect antimicrobial peptides induction and study. For exploring good Induction methods of Tenebrio molitor’s, antimicrobial peptides, initially clear the differences in type and nature of antimicrobial peptides between Tenebrio molitor that of different color patterns. In this study, we use Tenebrio molitor’s larvae of yellow pattern and black pattern as the test material, insects for test,use pathogens, ultraviolet light, pesticides, polyinosinic injection as inducements, treat the Tenebrio molitor’s larvae in single and compound injection way. Each induced set different induction dose and different induction time. By detecting and analysising the antibacterial activity of crude extracts of Tenebrio molitor’s antimicrobial peptides of different induction treatments, we get optimum induction treatment groups. Then we measure the thermal stability, pH stability, inhibitory spectrum of the Crude extracts; At last we used Sephadex G-75gel filtration chromatography to preliminary separation and purification of it, collected the composition of antibacteria activity, and used Tricine-SDS-PAGE to estimate the type and molecular weight of the antimicrobial peptides. The main results obtained in this study are as follows:1. Effects induced by the different induction on the two color patterns Tenebrio molitor’s larva are significant different. Induced effects are vary because of different source, induction dose (or intensity) and induction time. Effects of antimicrobial peptides induced by compound induction have no significant difference to that of single factor induction. In all the induction,120d larvae of yellow color pattern Tenebrio molitor soaked in the1500fold dilution of dimethoate for5s, normal feeding72h, the crude extracts of their antimicrobial peptides have the best antibacterial activity.2. There are significant differences on the effect caused by the same induction between two color patterns Tenebrio molitor’s larvae. But the antibacterial activity size of the larvae antimicrobial peptides crude extracts does not have a clear correlation with its body wall color. 3. The thermal stability test results show that the Tenebrio molitor antimicrobial peptides have better thermal stability. In the60℃water bath for10min they still have good antibacterial activity. When the water bath temperature reaches to80℃and water bath time reaches to20min, the antibacterial activity declines rapidly with the rise of the processing temperature and time.4. By measuring the antimicrobial peptides crude extracts’pH stability, we found that Tenebrio molitor antimicrobial peptides have the greatest antibacterial activity in the pH about5.2. pH5.2as the axis, the antibacterial activity of antimicrobial peptides declines rapidly with the decrease of pH and declines slowly with the decrease of pH.5. The inhibitory spectrum test results show that antimicrobial peptides crude extracts of Tenebrio molitor have antibacterial activity to both G+bacterial and G-bacterial, such as Staphyloccocus aureus Rosenbach, Bacillus subtilis Cohn, E.coli, Pseudomonas aeruginosa and Aeromonas hydrophila. They have no antibacterial activity to fungi, such as Arthrinium phaeospermum and Fusrium oxysporum.-6. Sephadex G-75gel filtration chromatography results show that there are only two UV absorption peaks. Only collection of fluid in58min-76min UV absorption peak district has antibacterial activity. This shows that the separation of Sephadex G-75to the crude extracts of Tenebrio molitor antimicrobial peptides is not ideal.7. Tricine-SDS-PAGE discontinuous electrophoresis results show that crude extracts of Tenebrio molitor antimicrobial peptides contain polypeptides of molecular weight62kD,45.5kD,37kD,26.4kD,12kD,9kD,1.3kD, in which molecular weight of12kD,9kD,1.3kD antibacterial peptides composition have the relative content of more. After Sephadex G-75gel filtration chromatography. Freeze-dried concentrate the collection of fluid in the UV absorption peak distriction. Tricine-SDS-PAGE discontinuous electrophoresis shows that antibacterial peptides of1.3kD may have the more abundant in Tenebrio molitor antimicrobial peptides composition.