Preparation Of Ovotransferrin Antimicrobial Peptide And Application Of Nanoparticles

Ovotransferrin（OVT）belongs to the transferrin family,and it is well known that it exhibits antibacterial activities.This activity is mainly displayed by the peptides derived from partial hydrolysis of ovotransferrin,it had not been known.In order to develop OVT antimicrobial peptides and improve its application value,in this study,OVT was used as the raw material for enzymatic hydrolysis,and the antibacterial activity was selected as the screening condition to obtain the best enzymatic hydrolysis product.The peptides with antibacterial activities were obtained by separation and purification technology,and the antibacterial mechanism was further studied.An efficient design of peptide-coated silica nanoparticles is reported which specifically target bacteria cells.In addition,the antibacterial activity,cell uptake,targeted therapy and biosafety of the nanoparticles were studyed in vivo.（1）OVT was obtained by two-step ethanol extraction method from egg white,and then the protein purity of OVT was analyzed by SDS-PAGE（>95%）.In order to obtain a protein hydrolysate with antibacterial activity under the optimal hydrolysis conditions of pepsin.We explored the pepsin hydrolysis process conditions for research,and the inhibitory rate was used as an indicator to obtain the optimal enzymatic hydrolysis conditions by orthogonal experiments.The substrate concentration is 4%,enzyme dosage is 1.5%,and hydrolysis time is 200 min.The obtained OVT hydrolysate was first passed through an ultrafiltration membrane of 3000 Da,and then an antimicrobial peptide was successfully separated from the pepsin OVT hydrolysate by ion exchange chromatography,G-15 gel filtration column or RP-HPLC.Finally,we used LC-MS/MS to identify its sequence as AGLAPYKLKPIA（"OVTp12"）.OVTp12 is a novel dodecapeptide,especially it is positively charged and hydrophobic,and its spatial structure prediction is consistent with the prediction results of the antimicrobial peptide database APD.In addition,OVTp12 also showed that it had antibacterial effect against Escherichia coli and Staphylococcus aureus.（2）The antibacterial activity of OVTp12 against Gram-positive bacteria and Gram-negative bacteria was investigated,especially Escherichia coli and Staphylococcus aureus.In addition,the results of OVTp12 showed that the peptide had obvious membrane permeability to Escherichia coli.CLSM and flow cytometry analysis showed that OVTp12 could destroy the integrity of cell membrane,increase the permeability of pathogen membrane.This further reveal its antibacterial mechanism.Scanning electron microscope analysis showed that OVTp12 could cause morphological changes of Escherichia coli and Staphylococcus aureus.These results suggest that OVTp12 may be a promising food preservative or antimicrobial agent.（3）The antibacterial peptide OVTp12 was modified on the drug loaded mesoporous silica by the interaction between OVTp12 and the bacterial cell membrane,so as to prepare a new nanoplatform with bacterial cell membrane targeting.Firstly,MSNs were synthesized by covalent condensation method.Then,the peptides were covalently coupled to MSN-NH₂ via-COOH group by EDC and NHS.Then,MSNs and MSNs@OVTp12 was mixed with gentamicin（Gen）to obtain the peptide MSNs@Gen and MSNs@OVTp12@Gen.The morphology and microstructure of MSNs,MSNs@Gen and MSNs@OVTp12@Gen were analyzed by microscopic technology.The results indicated that the average diameter of the particles increased gradually.EDS analysis showed that the MSNs were successfully modified by OVTp12.In addition,XRD,nitrogen adsorption desorption and thermogravimetric analysis also proved that OVTp12was successfully modified on MSNs.The drug release of Gen from MSNs@Gen and MSNs@OVTp12@Gen was studied in phosphate buffer solution（p H 6.5）.And the release rate was significant within 8 h.The antibacterial activity showed that the peptide modified nanoparticles showed better antibacterial activity in vitro.The characterization of scanning electron microscopy and laser confocal microscopy also showed that the nanoparticles had better antibacterial activity.MSNs@OVTp12 showed excellent interaction with bacterial cell membrane.（4）The nanosystem prepared by targeting the bacterial targeting peptide（OVTp12）to Escherichia coli to study its targeted drug delivery ability.On the other hand,surface modified nanoparticles could change the uptake capacity of cells,which provided a promising choice for the treatment of intracellular Escherichia coli infection.It was worth noting that the effective Escherichia coli targeting ability of nanoparticles ensured the accumulation of antibiotics in the infected site,so as to eliminate bacteria and effectively avoid the abuse of antibiotics.In addition,compared with free drugs,antibiotic loaded nanosystems could prevent drug inactivation,treat bacterial infection,and reduce inflammation in vivo.At the same time,the nanosystem has the characteristics of high drug loading,good drug release and good biocompatibility,which could apply to the release of a variety of antibiotics,and provides a new idea for the treatment of bacterial infection.