In Vitro Functional Identification Of Recombiant Antimicrobial Peptide Hispid Alin And Its Application In Cold Storage Of Pork As A Biopreservative

Antimicrobial peptide Hispidalin isolated from the seeds of Benincasa hispida inhibits several bacteria as tested in 2014.Hispidalin is consisted of 49 amino acids and its molecular weight is 5.7 KDa.The natural Hispidalin has a good inhibitory effect on bacterial and fungal pathogens and anti-oxidation ability.Hispidalin has great value for application and research.This study used Pichia pastoris to express recombinant Hispidalin(rHispidalin).The expression conditions of Pichia pastoris were optimized.Finally,it was determined that the optimal induction time was 96 h and the methanol concentration was 1.5%,The rHispidalin reached a yield of as high as approximately 99 mg/L.After nickel column affinity purification,3.9 mg of target protein with a purity of 92.2%was obtained from 500 mL supernatant of cell culture medium.Further minimal inhibition concentration(MIC)assay,inhibition zone assay,bactericidal kinetics assay,hemolytic and cytotoxicity activity assays,cell membrane permeability assay and antioxidant activity assay were conduced.The results showed that rHispidalin showed a board antibacterial ability with a MIC value ranging between 40 and 60 ?g/mL when tested on both Gram-positive and Gram-negative bacteria.And rHispidalin was highly resistant to temperature and pH and had a selective resistance to proteases.The rHispidalin at a concentration of 4×MIC can rapidly kill 1×104 CFU/mL of Staphylococcus aureus(ATCC 25923)within 40 min.The rHispidalin displayed antioxidant activity against ABTS+,superoxide anions,DPPH and hydroyl free radicals.The rHispidalin had extremely low hemolysis on human and rat erythrocytes but was cytotoxic to human embryonic kidney cells and hamster kidney cells.To further explore the mechanism of rHispidalin,the membrane disruption assays were performed.The rHispidalin destroyed the integrity of the membrane,and caused the death of bacteria.Finally,rHispidalin was efficient in inhibiting bacterial growth and preventing food spoilage in a meat model system.Treatment with 100 ?g/mL Hispidalin significantly decreased the total viable counts,total volatile basic nitrogen and the lipid oxidation,and it also maintained good sensory characteristics and cell structure in pork meat during storage at 4?.In summary,rHispidalin has great potential to develop into a new food anti-corrosion and biopreservative,as well as an alternative to antibiotics.