Preparation And Antibacterial Effects Of A Novel Antibacterial Agent-Glucomannan Nanosilver Sol

Vibrio spp.is one of the main pathogens in aquaculture.The increasingly serious disease not only causes huge economic losses for aquaculture farmers,but also poses a great threat to the environment and human health.At present,traditional anti-Vibrio drugs have certain defects to varying degrees in bactericidal performance,safety,drug resistance and compatibility,and cannot meet the requirements.Polysaccharide nanosilver sol(AgNC)is a new bactericide made from natural polysaccharides using modern nanotechnology.It has broad-spectrum,strong effects of antibacterial activity,high biosafety and environmental friendliness.It has no problem of bacterial resistence,and thus has the potential to replace the traditional chemical synthetic bactericides.AgNC has now received extensive attention in the field of aquaculture.In this study,konjac glucomannan(KGM)was degraded by 60Co γ-ray irradiation at doses of 0,8,10,20,50,100kGy to obtain 6 KGMs.These 6 KGMs were used as reducing agents and stabilizers,and silver nitrate was used as a reaction silver source,all the silver ions were completely reduced by ultraviolet irradiation without the use of other reducing agents,and the glucomannan nanosilver sol(KGM-AgNPs)was prepared by using the photocatalytic properties of nano-silver(AgNPs).In this study,the synthesis of six KGM-AgNPs were characterized,and the dispersion stability was determined.The common pathogenic vibrio spp.of aquatic products(Vibrio parahaemolyticus,Vibrio anguillarum,Vibrio alginolyticus,Vibrio harveyi),common indicator strains(Escherichia coli,Staphylococcus aureus)and intestinal probiotics(Lactobacillus rhamnosus,Bifidobacterium longum,Bifidobacterium breve)were used for bacteriostatic test to analysis the bactericidal ability of AgNC.The results will lay a theoretical basis and technical guidance in the prevention and treatment of common vibriosis in aquatic products.The contents and results of this study are as follows:(1)In this study,0kGy-KGM was used as the reducing agent and stabilizer,and silver nitrate was used as the reaction silver source.The influences of UV irradiation time,silver nitrate concentration and 0kGy-KGM concentration on the synthesis of KGM were investigated.The reduction ability of 0kGy-KGM was also investigated under the optimal reaction concentration,room temperature(25℃)and stirring in dark conditions.The experimental results show that,when the temperature is 15℃,humidity is 72%,total irradiation power is 196W,and the concentrations of 0kGy-KGM,silver nitrate are 10mg/mL and 0.8mM,respectively,after 510min UV radiation at 254nm wavelength,the nanosilver sol are prepared with a silver ion reaction rate of more than 99.9%,and no obvious agglomerations were observed.However,the silver ions cannot be reduced to form nanosilver without 0kGy-KGM in the above reaction.(2)According to the optimal concentration of 0kGy-KGM for preparing KGM-AgNPs,different doses of 60Co γ-ray were used to irradiate the 0kGy-KGM to obtain the 8kGy-KGM,10kGy-KGM,20kGy-KGM,50kGy-KGM,100kGy-KGM.These irradiated KGMs and unirradiated 0kGy-KGM were used for preparation of different KGM-AgNPs at the same concentration(the concentration of different KGM was 10mg/mL,and the concentration of silver nitrate was 0.8mM).In the meantime,the influence of temperature changes of the reaction solution during the preparation,and the irradiation time,on the synthesis of different KGM-AgNPs,were also investigated.The stabilities of the six kinds of KGM-AgNPs were analyzed using multi-light scattering instrument,and the physical properties were further characterized by XRD,XPS,FTIR,ICP-OES and TEM.The results show that,when the irradiation times were 300 min,360 min,420 min,450 min,480 min and 510min,respectively,more than 99.9%Ag+were reduced to form 100KGM-AgNPs,50KGM-AgNPs,20KGM-AgNPs,10KGM-AgNPs,8KGM-AgNPs,and 0KGM-AgNPs,respectively,and the temperature of each reaction solution varies between 20～21℃.The results of UV-Vis absorption spectrum(UV-ViS)showed that,the 100KGM-AgNP prepared by 100kGy-KGM has the highest UV absorption peak intensity.The hydrodynamic radius of the six KGM-AgNPs measured by the dynamic light scattering(DLS)did not show a certain decrease or increase with the ultraviolet irradiation time.According to the multiple light scattering analysis,the stability sequence was 100KGM-AgNPs>50KGM-AgNPs>0KGM-AgNPs>10KGM-AgNPs>20KGM-AgNPs>8KGM-AgNPs.XRD and XPS results showed that,Ag0 exists in all of the 6 different KGM-AgNPs,and the outer layer of the nano-silver particles is encapsulated by different KGM.FTIR results showed that,the functional groups-OH,-CH,C=O,and C-O-C,etc,corresponding to mobile absorption peaks of 6 different KGM,participate in the synthesis and stabilization process of KGM-AgNPs.The morphology of AgNPs observed by transmission electron microscopy(TEM)indicated that,the synthesized 100KGM-AgNPs,20KGM-AgNPs,10KGM-AgNPs,8KGM-AgNPs and 0KGM-AgNPs were spherical,and the particle sizes were 9～28nm,20～32nm,12～30nm,25～30nm,and 42～54nm,respectively.The 50KGM-AgNPs were irregularly shaped particles with an average particle size of 72～87 nm.Inductively coupled plasma emission spectroscopy(ICP-OES)results indicated that,the silver contents in the 100KGM-AgNPs,50KGM-AgNPs,20KGM-AgNPs,10KGM-AgNPs,8KGM-AgNPs and 0KGM-AgNPs were 83.69±0.34mg/L,83.30±0.45mg/L,80.38±0.33mg/L,80.03±0.32mg/L,80.78±0.17mg/L and 81.70±0.17mg/L,respectively.In the lyophilized powder,the Ag content is 1.310±0.008%,1.236±0.005%,1.211 ±0.015%,1.208 ± 0.008%,1.215± 0.009%and 1.230 ± 0.008%,respectively.(3)In order to evaluate the antibacterial activity of 6 different KGM-AgNPs,the common aquatic pathogenic bacteria(Vibrio parahaemolyticus,Vibrio anguillarum,Vibrio alginolyticus,Vibrio harveyi),common indicator strains(E.coli,Staphylococcus aureus)and intestinal probiotics(Lactobacillus rhamnosus,Bifidobacterium longum,Bifidobacterium breve)were used for analysis.The results showed that,all of the KGM-AgNPs have bactericidal effects on the 6 pathogenic strains,and the bactericidal actity against Escherichia coli was stronger than that of Staphylococcus aureus.The bactericidal abilities to 4 kinds of aquatic pathogenic Vibrio spp.were different.Six kinds of KGM-AgNPs had the same MBC for Vibrio anguillarum and Vibrio harveyi.50KGM-AgNPs had the best bactericidal effect on Vibrio alginolyticus,and 100KGM-AgNPs and 50KGM-AgNPs had the best bactericidal effects against Vibrio parahaemolyticus.KGM-AgNPs have no harmful effects on three kinds of intestinal probiotics under the concentration which could irreversibly damage the Vibrio spp.The effects of 100KGM-AgNPs and 50KGM-AgNPs on the growth curves of four pathogenic Vibrio spp were also investigated.The results showed that all the two KGM-AgNPs had the ability to delay the logarithmic growth phase of aquatic pathogens at low concentrations,and the results from growth curve test were in consistent with the results of MIC and MBC.The antibacterial activity of the KGM-AgNPs were also evaluated by the inhibition zone method.The results showed that the bactericidal abilities of the six KGM-AgNPs against Escherichia coli were stronger than those to Staphylococcus aureus,which was in consistent with the results of MBC.However,the inhibition zones of the four aquatic pathogens were different.The sensitivity of Vibrio cholerae to the same kind of KGM-AgNPs was different.100KGM-AgNPs had the best bactericidal activity against Vibrio harveyi,Vibrio parahaemolyticus and Vibrio anguillarum,and 50KGM-AgNPs had the best bactericidal effect on Vibrio alginolyticus.