Study On Characterization And Biological Effects Of Silver Nanoparticles,Biosynthesized Using Abalone Visceral Glycoprotein

Bacterial disease is one of the main causes of a large number of aquatic organisms’death,which seriously endangers the sustainable development of aquaculture.Silver nanoparticles（AgNPs）have the characteristics of strong bactericidal effect,broad-spectrum sterilization and no drug resistance,and thus have gained wide attention.AgNPs would provide new ideas for controling bacterial diseases in aquaculture.Fujian is the major province of abalone farming.Most of the abalone viscera produced during processing was discarded directly.This not only causes resources waste,but also pollutes the environment.In this study,the glycoprotein extracted from abalone’s viscera（Avg）was used as a reducing agent and stabilizer to prepare silver nanoparticles（Avg-AgNPs）.The optimal conditions for Avg-AgNPs synthesis were explored,and the characterizations of antibacterial properties,cytotoxicity and stability of Avg-AgNPs were investigated.The main contents and results of this study were as follows:（1）The effects of different reaction concentrations and reaction times on the preparation of Avg-AgNPs were studied.The results showed that the prepared Avg-AgNPs were the best when the concentrations of Avg and silver nitrate were 0.5 mg/m L and 3.25 mM,respectively,and the reaction time was 240 min.The characterizations of the Avg-AgNPs prepared at the optimal condition showed that the nanoparticles contain not only AgNPs but also AgCl crystals.The AgNPs were coated with a layer of Avg,and the system contained chlorine.The morphology of nanoparticles was similar to spherical and triangular,and particle sizes range from 5 to 25 nm.（2）In order to remove the AgCl crystals in the system,the effects of three different conditions for preparing Avg-AgNPs were investigated.The results showed that more AgNPs were synthesised by the optimized Avg.The nanoparticles prepared by centrifugation and optimized Avg only contained AgNPs,and no AgCl crystals were detected.The nanoparticle sols still contained AgCl crystals when prepared without pH adjusting.In summary,the effect of AgNPs prepared by the 0.5 mg/m L optimized Avg and 1.625 mM silver nitrate was the best.The morphology analysis showed that the AgNPs was spherical,the main size was between 5-15 nm,and the dispersion was good.The-OH,-NH₂,-COO^-and glucosidic linkages in the Avg were related to the reduction and stabilization process of AgNPs.（3）The antibacterial properties of Avg-AgNPs against four kinds of common pathogenic Vibrio of Penaeus vannamei,Staphylococcus aureus and Escherichia coli were studied using minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）methods.The results showed that Avg-AgNPs had good antibacterial activities against the six tested strains.By comparing the antibacterial properties with Chinese traditional medicines using inhibition zone method,the Avg-AgNPs were more stable than Chinese traditional medicines and had a broad-spectrum antibacterial effect.（4）The cytotoxicity of Avg-AgNPs was studied using MTT assay,cell morphology observation and oxidative damage assay.The results showed that when the concentration of Avg-AgNPs was above 50μg/m L,the survival rate of LO2 cells and the activity of SOD,GSH-Px and LDH decreased,the morphology changed,and the content of MDA increased,indicating the oxidative stress and cell damage.When the concentration of Avg-AgNPs was below 25μg/m L,there was no effect on cell viability and morphology.And no obvious oxidative stress reaction or cytotoxicity was found.（5）The stability analysis showed that the dispersion stability of Avg-AgNPs was good after the treatment of 4°C,18°C,25°C,32°C,high temperature sterilization and pasteurization,no obvious aggregation phenomenon was observed.The stability of Avg-AgNPs was good under pH values of 4,7.5,9.5,and 11.However,Avg-AgNPs were not stable in seawater and were easy to aggregate.