Preparation Of Silver Nanoparticles Using Abalone Viscera Hydrolysate And Their Biologic Effects On Zebrafish

Bacterial diseases are the most common diseases in aquaculture.However,traditional antimicrobial agents have many problems,such as drug residue,drug resistance,and safety which have adversely affected human health and ecological environment.Silver nanoparticles(AgNPs)is a new antibacterial agent developed based on nano technology,it has been widely concerned by researchers in recent years.In this study,the abalone visceral hydrolysates were used as reducing agent and stabilizer for green synthesis of AgNPs.The preparated AgNPs were further characterized and the antibacterial properties were studied.Different concentrations of AVH-AgNPs were added to the zebrafish culture water,and the effects of AVH-AgNPs on growth index,antioxidant enzyme activity,immune gene expression and intestinal microbial community structure were studied.The results were as follows:(1)AVH-AgNPs was synthesized using abalone viscera hydrolysate.The optimal experimental conditions for the preparation of AVH-AgNPs were as follows:AgNO3 concentration was 1.3 mmol,the abalone viscera hydrolysate concentration was 0.7 g/L,the reaction temperature was 100℃,pH was 7,the reaction time was 6 h.The synthesized AVH-AgNPs are spherical,with an average particle size of 54.57 ± 12.96 nm,and a PDI of 0.029.The results of XRD and XPS showed that the preparated AVH-AgNPs was elemental silver and there was no other silver complex.The AVH-AgNPs had good stability.(2)The minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC)and growth curve were used to evaluate the antibacterial effect of AVH-AgNPs.The common indicator strains(Escherichia coli,Staphylococcus aureus)and common aquatic pathogenic bacteria(Vibrio Parahemolyticus,Vibrio anguillarum,Aeromonas hydrophila,Vibrio harveyi)were used for analysis.MIC and MBC results show that AVH-AgNPs had bacteriostatic and bactericidal activity to all of the 6 strains.The order of antibacterial activity was Escherichia coli,Vibrio harveyi>Vibrio Parahemolyticus,Vibrio anguillarum,Aeromonas hydrophila>Staphylococcus aureus.Growth curves show that AVH-AgNPs can affect the growth cycle of the 6 kinds of bacteria at 1/2MIC and MIC concentration,and inhibit the proliferation of bacteria.(3)The effects of different concentrations(6-18 μg/L)of AVH-AgNPs on zebrafish growth indexes,and liver antioxidant indexes were studied.Furthermore,real-time PCR was used to investigate the expression changes of immune-related genes.The results show that,within the concentration range of this experiment,AVH-AgNPs have a certain promoting effect on the weight and length of zebrafish,and the promotion effects in low and medium concentration are more obvious.At the same time,AVH-AgNPs could induce the activity of antioxidant enzymes(SOD and CAT),increase the GSH content,and upregulate the expression of immune related genes(TNF-α,IL-1β,IL-10,IL-12 and DEFB1).Different concentrations of AVH-AgNPs had different effects,in the medium and high concentration treatment groups,the stimulation effects were more obvious,while in the low concentration group treatment group,the effects were not obvious.(4)The effects of AVH-AgNPs on the microbial community structure in fish intestines and culture water were studied by Illumina miseq sequencing technology.The results showed that,the microbial community structure in zebra fish was significantly different from that in the culture water,and the diversity of microorganisms in the water was significantly higher than that in the intestines.AVH-AgNPs treatment reduced the microbial diversity in the aquaculture water,but the effect of AVH-AgNPs on the intestinal microbial diversity showed no regular pattern.The results of beta diversity showed that,the high concentration of AVH-AgNPs had significant influences on the composition of intestinal and aquaculture water microbiota from class to OTU levels,and the influence showed a concentration dependent trend,the higher concentration had a more obvious influence.The influence on the intestinal bacteria:At the genus level,in the high(H)and medium(M)concentration of AVH-AgNPs treatment groups,the abundance of Gemmobacter and Rhodococcus in zebrafish intestines increased,while the abundance of Uliginosibacterium,Pseudomonas,Halomonas and Cytophaga decreased.On the species level,the abundance of Flavobacterium_cucumis decreased significantly in the H and M groups,and the abundance of Plesiomonas_shigelloides decreased significantly only in the H group.The influence on the bacteria in aquaculture water:At the genus level,in the low(L),medium(M),high(H)concentration of AVH-AgNPs treatment groups,the abundance of Candidatus_Finniella,norank_f_Burkholderiaceae and Sediminibacterium in zebrafish aquaculture waters increased,while the abundance of norank_f_norank_o_RBG-13-54-9 decreased.On the species level,the abundance of Polynucleobacter_asymbioticus and metagenome_g_norank_f_LiUU-11-161 increased significantly only in M group,and the abundance of metagenome_g_Devosia decreased significantly only in M group.