| Antibacterial peptides have broad application prospects in medicine and food field because of its unique antibacterial mechanism and broad-spectrum antibacterial activity,however,the low yield has become the bottleneck of the commercialization of antimicrobial peptide.In this article,protein from Tenebrio Molitor was hydrolyzed by alkaline protease to prepare antibacterial peptides.Use Central composite design to optimize the preparation process.Then the hydrolysate was separated and purified.The antibacterial activity of different constituent were also investigated.At last,we determined the thermostability,minimal inhibitory concentration(MIC)and molecular weight of the antibacterial peptide.The main results were listed as the followings:(1)The protein,extracted from Tenebrio molitor by the method of alkaline extraction and acid precipitation,was hydrolyzed with trypsin,alkaline protease,papain,neutral proteinase and pepsin,respectively.The antimicrobial activity of alkaline protease hydrolysate was the most strong.The peptide content was 73.38mg/mL and the diameter of inhibiting bacteria circle was 14.16mm.(2)The single test chose 5 factors,substrate concentration,enzyme concentration,reaction time,temperature and pH value.Central composite test selected 3 factors which was significant to the o antimicrobial activity from those factors.The optimal enzymatic condition was as follows:Substrate concentration 10%,reaction time 4.4h,enzyme concentration 440U/g,temperature 54℃,pH value 9.5.(3)Hydrolysate of Tenebrio Molitor protein was separated by DA201-C macroporous resin and fractionated into five portions.Fraction of Anhydrous ethanol elution showed the strongest antimicrobial activity.And then the Fraction of Anhydrous ethanol elution was separated into two ingredients(H-1,H-2)by preparative HPLC.The H-2 show stronger antimicrobial activity on E.coli,Salmonella and Staphylococcus aureus.(4)The MIC of H-2 on E.coli,Salmonella and Staphylococcus aureus,measured by broth microdilution method,was 0.512 mg/mL.Thermostability tests showed that H-2 was heat stable and it can keep antimicrobial activity after heating at 121℃ for 20 minutes.The mass spectrogram indicated the molecular weight of the H-2 was 756.82... |
