In Situ Synthesis Of Nano Selenium Particles By Reducing Selenite From Biomass And Its Inhibition Mechanism Against Vibrio Alginolyticus

Selenium is an essential trace element for the health of human body and other life bodies.It is also one of the elements lacking in human body.Compared with inorganic and organic selenium,nano-selenium(SeNPs)has many advantages as selenium supplement due to its high activity and low toxicity.So,its synthesis and biological effects have attracted wide attention.There are many preparation methods for SeNPs.Compared with physical and chemical preparation methods,plant method has more advantages.However,there are many kinds of plants in the natural world.The types and the quantities of reducing substances in different plants are quite different.Consequently,the speed,efficiency,structure,and properties of the products in the process of the preparation of SeNPs are different.In order to efficiently prepare SeNPs with excellent performance,it is necessary to screen plant raw materials.But there are few reports in this regard.It is well known that the properties of nanomaterials are closely related to their particle size,morphology and crystal structure.However,the synthesis of small sized SeNPs by plant method has not been solved yet,which hinders the wide application of SeNPs.The structural properties of SeNPs are also closely related to their preparation processes.The antioxidant,antibacterial and application of SeNPs synthesized by plant method have been deeply concerned by researchers and society.Vibrio alginolyticus(V.alginolyticus)is the main pathogen of marine aquaculture worldwide,causing serious economic losses to aquaculture.At present,many antibiotics and natural antimicrobial agents are used to prevent and control V.alginolyticus,but antibiotics are prone to drug resistance and cause environmental pollution.The cost of natural antimicrobial agents is high and the efficacy is slow,and the raw materials for preparation of V.alginolyticus are restricted by seasons.Since the SeNPs synthesized by plant method has good antibacterial activity,the problem of V.alginolyticus infection can be solved.It is a hot topic for researchers to talk about the function of V.alginolyticus as an efficient inhibitor against pathogenic microorganisms.With this in mind,the raw materials for the synthesis of SeNPs by plant method were screened in this paper.The plant raw materials were treated by different methods to explore the synthesis process of small-sized SeNPs.The antioxidant activity of SeNPs and the activity of inhibiting V.alginolyticus were detected.To further improve the research level of SeNPs,the mechanism of SeNPs synthesis by plant method and the molecular mechanism of SeNPs inhibiting V.alginolyticus were studied.The main contents and the conclusions achieved are as follows:1.Evaluation on the performance SeNPs synthesized from biomass materials.SeNPs particles were prepared by using plant juice as a reducing agent and stabilizer.The redness,particle size,?-potential,dispersion,X-ray diffraction,infrared spectroscopy,antioxidant activity and anti-V.alginolyticus activity of SeNPs prepared by nine different kinds of plant juice were detected.The optimal raw materials were screened.Results showed that the spherical amorphous SeNPs with particle size of 95.5 nm was prepared by lemon juice.The synthesized SeNPs had antioxidant activity and certain antibacterial activity against V.alginolyticus.Compared with other eight plants,the performance index of the lemon juice was better.Therefore,lemon was selected as the optimal plant raw material for the preparation of SeNPs.2.Rapid synthesis of SeNPs based on lemon juice.With lemon fruit as the raw material,lemon juice was obtained by squeezing,centrifugation,micro membrane filtration.SeNPs particles were synthesized at room temperature.The synthesis process of SeNPs was optimized by controlling the concentration of selenite,synthesis time and pH value.The spherical highly dispersed amorphous SeNPs with the size of 60 nm was prepared at 50 mmol/L selenite,pH=9,and reaction time for 1 h.pH value was the key factor that affected the synthesis rate and particle size of SeNPs.In the synthesis of SeNPs,the strong reduction of chemical substances in plants is the chemical basis for the formation of SeNPs.The complexation of polyphenol structure,conjugated system formation and properties of chemical functional groups are the theoretical basis for the formation of biomacromolecule protective film on the surface of SeNPs,the stable existence of SeNPs and the prevention of further agglomeration and growth of SeNPs.The surface free energy of SeNPs and its changes in the synthesis process are the basis for aggregation,formation and stability of surface protective film in the synthesis of SeNPs,and also an important reason for the composition of SeNPs by many small SeNPs grains.The antioxidant activity and antibacterial activity of SeNPs against V.alginolyticus were tested.The results showed that the free radical scavenging rate of SeNPs followed the order of DPPH free radical>superoxide anion free radical>hydroxyl free radical,which was consistent with the order by thermodynamic,kinetic and chemical reaction collision theory analyses.Compared with large-sized SeNPs,small-sized SeNPs showed stronger inhibitory activity against V.alginolyticus.3.Synthesis of small-sized SeNPs based on Tris assisted lemon juice.The preparation of lemon extract by Tris-HCl buffer treatment(Tris-HCl lemon juice)is a key step in the synthesis of small sized crystal SeNPs.Single factor experiment and response surface optimization were used to determine the optimum condition for the synthesis of small-sized crystalline SeNPs.When the concentration of selenite was 17 mmol/L,the solid-liquid ratio was 1:6.4,and the volume ratio was 1:10.5,the spherical highly dispersed crystalline SeNPs with the size of 8.5 nm was prepared.The study on the synthesis mechanism of SeNPs showed that,in the extract prepared by Tris-HCl buffer treatment of lemon,the coordination and complementarity of protein molecules with different molecular weights and with other biological molecules in the surface complexation and adsorption of SeNPs were essential for the formation of small-sized SeNPs.The electrostatic interaction between the dipoles on the surface of SeNPs and inside it caused by the electron absorption of high electronegativity N and O atoms in Tris-HCl and protein molecules was mainly responsible for the synthesis of crystalline SeNPs.The antioxidant activity and V.alginolyticus inhibitory activity of SeNPs were analyzed.The results showed that the free radical scavenging ability of SeNPs was higher than that of Na2SeO3.Compared with amorphous SeNPs,the antibacterial activity of crystalline SeNPs was somewhat weakened.4.The inhibitory effect of SeNPs on the activity of V.alginolyticus.By detecting the growth curve of V.alginolyticus after SeNPs treatment,the inhibitory effect of SeNPs on V.alginolyticus was evaluated.The results showed that SeNPs had a significant inhibitory effect on the growth of V.alginolyticus.The minimum inhibitory concentration and minimum bactericidal concentration of SeNPs against V.alginolyticus were determined to be 0.1 mg/mL and 0.2 mg/mL.0.05 mg/mL and 0.025 mg/mL were selected as sub-inhibitory concentrations.The changes of V.alginolyticus motility and biofilm formation after SeNPs treatment were investigated.It was found that SeNPs had a significant inhibitory effect on V.alginolyticus motility and biofilm formation(P<0.01).After SeNPs treatment,the contents of intracellular reactive oxygen species,extracellular alkaline phosphatase,electrical conductivity,glucose and other indicators in V.alginolyticus were increased significantly with time and SeNPs concentration(P<0.01),indicating that substances inside V.alginolyticus leaked and the integrity of cell wall membrane was destroyed.Scanning electron microscopy observation showed that the morphology of V.alginolyticus cells treated with SeNPs changed,folded or even ruptured,indicating that the cell structure was destroyed.5.The molecular mechanism of SeNPs inhibiting V.alginolyticus.Based on LC-MS non-targeted full-component and targeted lipid metabolomics,the molecular mechanism of SeNPs inhibiting V.alginolyticus was elucidated by identification and analysis of differential metabolites.Thirty three significantly different metabolites were detected by total metabonomics analysis and 82 metabolic pathways were involved.One of the main components of the cell membrane is lipid,and the material basis of SeNPs damaging the cell membrane of V.alginolyticus is further analyzed by targeted lipid metabolomics.A total of 66 significant metabolites were screened by lipid metabolomics.Its molecular mechanism was that the decomposition of phosphonic acid accelerates cell wall cracking,destroys cell wall integrity,changes the lipid composition of cell membrane,reduces the fluidity of cell membrane.Finally,the normal physiological activity of cell membrane was destroyed.