Study On The Synthesis Of Tannin From Pomegranate Peel /Nano-silver Composites And Their Antibacterial Activity

Objective: Nano silver is a new type of antimicrobial material based on nanotechnology,which has high antimicrobial activity and drug resistance that other antimicrobial agents can not match.The biosynthesis of nano-silver has the characteristics of safety,low cost,green and environmental protection,and has broad application prospects in the field of synthesis of nano-silver.In this study,the optimum preparation technology of tannins from pomegranate peel/nano-silver composites(PPT-AgNPs)is studied by biosynthetic method,and the broad-spectrum antimicrobial activity of the synthesized PPT-AgNPs against common clinical pathogens in vitro and the antimicrobial mechanism of S.aureus and E.coli are discussed.It provides a reference for the subsequent development of nano-silver antibacterial materials synthesized by traditional Chinese medicine,and also provides a theoretical basis for the further application of nano-silver in bacteriostasis.Method: 1)PPT is used as reductant and dispersant for the first time in this experiment.Based on the absorbance of PPT-AgNPs,the effects of PPT content,AgNO3 concentration,synthesis temperature and time on the formation of PPTAgNPs particles are investigated,and the optimum synthesis process are selected.The silver nanoparticles are characterized by ultraviolet spectrophotometer and transmission electron microscopy.2)The antimicrobial activity of PPT-AgNPs against several common clinical pathogenic bacteria is determined by double dilution test tube method.Gram-positive bacteria-S.aureus and Gram-negative bacteria-E.Coli with higher sensitivity are selected as test bacteria.The growth curve of PPT-AgNPs before and after the interaction with bacteria is determined by ultraviolet absorbance.The ultrastructural changes of bacteria before and after administration are observed by transmission electron microscopy(TEM)? scanning electron microscopy(SEM)and X-ray diffraction(XRD).The contents of AKP,K+,protein nucleic acid and the activities of ATPase and respiratory chain dehydrogenase are determined by the corresponding kits before and after administration.RT-PCR assay is used to determine the expression of recA and recN genes in bacteria before and after administration.The bacteriostatic mechanism of PPT-AgNPs on bacteria is preliminarily elucidated Results: 1)The optimum conditions for preparation of PPT-AgNPs are as follows:the volume ratio of PPT(1 g/L)to 2 mmol/L AgNO3 solution is 1:4 and the reaction time is 4 h at 25?.The obtained composites are stable and reproducible.Most of thecomposites are spherical in shape,with an average particle size of about 42 nm.2)PPT-AgNPs have the same high antimicrobial activity against Gram-negative bacteria and gram-positive bacteria.Of course,the MIC of PPT-AgNPs is-slightly different for different strains.Among them,E.Coli and S.aureus had the most obvious effect.The MIC and MBC of PPT-AgNPs against Gram-negative bacteria E.Coli were 0.625 mg/ml and 0.625 mg/ml respectively.The MIC and MBC of S.aureus were 0.625 mg/ml and 1.25 mg/ml respectively.The growth curve experiment showed that the growth curve of the drug group had been significantly inhibited in the first three hours of bacterial growth,which was different from the S-type growth cycle of the normal bacteria.With the increase of drug concentration,bacteria may be killed directly,leading to no growth curve.At the same time,the extracellular AKP,K + and nucleic acid contents of the bacteria were significantly higher than those of the control group,while the intracellular ATPase and respiratory chain dehydrogenase contents were significantly lower,which were positively correlated with the drug concentration.These changes indicate that PPT-AgNPs can increase the permeability of cell membrane and cell wall,inhibit the expression of protein nucleic acid,and inactivate respiratory chain dehydrogenase.TEM and SEM show that the cell integrity of the bacteria was destroyed,resulting in abnormal deformation and abnormal growth of the cells.The results of PCR showes that the expression of recA and recN genes in bacteria increased and then decreased after PPT-AgNPs treatment.This suggests that PPT-AgNPs may destroy the structure of DNA,forcing the organism to start SOS repair in a short time.However,with the prolongation of drug action time,SOS repair system no longer works,and bacteria may mutate or even die.Conclusion: The preparation method is green,stable and feasible.PPT-AgNPs has broad-spectrum antimicrobial effect on common clinical pathogenic bacteria.By destroying the integrity of bacterial cell membrane,cell wall and DNA structure,bacterial metabolism is disordered and bacterial death is ultimately caused.