Antimicrobial Mechanism Of Glycinin Basic Peptide Against E.coli And Its Application Effect

The consistent use of chemical synthetic preservatives in the food industry may cause various hazards to human health.Therefore,safe natural food preservatives have garnered interest and have become a priority for the food industries and public consumers because of their safety,such as glycinin basic peptides.In this study,antibacterial activity of purified glycinin basic peptides(GBP)against Escherichia coli(E.coli)was investigated.The effect of GBP on cell membrane and its component also were researched.In addition,GBP were applied to food preservative,and investigated that its effect on sensory properties and physicochemical indexes of milk.The main experimental results were as follows:(1)According to the method of colony-counting and Oxford cup,GBP had significant antibacterial activity against E.coli,and the antibacterial activity increased with increasing GBP concentration.The minimum inhibitory concentration of GBP for E.coli was determined to be 200 ?g/ml.Atomic force microscope(AFM)results indicated that GBP could destroy the morphology of E.coli,and cells exhibited irregularly wrinkled outer surfaces,with grooves and aggregations of the damaged cells.After 8 h,the structure of the E.coli cells nearly collapsed and became fragmented.(2)To reveal the effect of GBP on membrane of E.coli treated with GBP,hydrophobicity of cell surface,the leakage of alkaline phosphatase,the permeability of the inner and outer membrane of E.coli were researched.Hydrophobic grouping of E.coli cells treated with GBP could be exposed,and hydrophobicity of cell surface increased.Becise,when the E.coli cells were treated with different concentration of GBP,the leakage of alkaline phosphatase incresase with treatment time,these results indicated that GBP had significant destruction to E.coli cell membrane.Subsequently,GBP disrupted the intact outer membrane of E.coli and increased the permeability of the outer membrane,consequently enhancing the sensitivity of E.coli cells to erythromycin and rifampicin.GBP also could permeate the inner membrane of E.coli,ONPG immediately reacts with ?-galactosidase and hydrolyzes ONPG into yellow ortho-nitrophenol(ONP).The permeability of the outer membrane depended on GBP concentration.(3)The bacterial outer membrane,existing in Gram-negative bacteria,presents a barrier to the passage of macromolecules,and plays a protective role in hindering the attack to cell.So the protection role of cell walls was revealed according to the colony-counting of E.coli spheroplast.This result indicated that E.coli spheroplast was more sensitive than intact E.coli cell to GBP.To reveal the effect of GBP on membrane component of E.coli treated with GBP,the leakage of intracellular reducing sugar was measured,and GBP could increase significantly leakage of reducing sugar of E.coli cell with the increase of treatment time by destroying the bacterial cell membrane.GBP also could decompose macromolecule of LPS into small molecules,therefore,leading to damage of bacterial outer membrane,and promote the extraction membrane proteins in the presence of Triton X-114.The higher concentration GBP caused more significant damage to the cell membrane of E.coli.The effect of GBP on Phe residue fluorescence spectra of E.coli membrane protein demonstrated that GBP could affect bacterial membrane protein,and expose the Phe residue located on the surface of membrane,resulting in the change of the membrane structure.200 ?g/ml of GBP could expose adequately the Phe residue of E.coli membrane.(4)In the research of the effect of GBP on sensory properties and physicochemical indexes of milk,compared to control,GBP had a significantly impact on sensory properties,acidity,protein content and total bacterial count of milk with the increase of concentration of GBP.GBP could significantly inhibit the growth of bacteria in milk,and also could inhibition of protein decomposition and milk rancidity.These results revealed that GBP had the potential to be a nature preservative to increase shelf life of milk.But according to the requirements of food additive dosages as low as possible,the 200 ug/ml GBP was the optimal concentration to extend the shelf life of milk.