Preliminary Safety Evaluation On Nano Defatted Soybean Particles

Defatted soybean(DS)is the by-product of soybean oil extraction.As a good vegetable protein feed source,it was commonly used in livestock,poultry and aquaculture.However,there are many anti-nutritional factors contained in DS,which limited the absorption and bioavailability of nutrients.The rapid development of nanotechnology provided new solutions for the full utilization of DS.Nano defatted soybean particles(nDSP)were milled from DS,whose size was reduced to less than100nm.The nDSP have some unique properties superior to DS,but its safety as a new nanoparticle remains uncertain.Therefore,the safety of n DSP were evaluated in presented study.Firstly,the newly nDSP were prepared and characterized comprehensively;then the toxicity of n DSP were evaluated by cell and animal;finally,the distribution of nDSP in gastrointestinal tract and other organs of mice was localized and quantified,based on which the safety of nDSP was evaluated.The main content was addressed as follows.Nano defatted soybean particles were prepared by dry milling in a planetary ball mill,and their sizes were measured by laser particle size analyzer(LPS)and scanning electron microscope(SEM).After 8 h of dry milling,the size of n DSP reached95.29±8.87 nm(LPS)and 78.88±21.01 nm(SEM).The result showed that the n DSP'size was reduced with the milling time increased.And the smaller the size,the more monomodal distribution.During size characterization,the hydrodynamic diameter of all tested nDSP measured by LPS corresponded almost exactly to the Feret diameter obtained by the SEM.Four defatted soybean particles(DSP)with different sizes(100nm,200 nm,500 nm,1?m DSP)were prepared and chosen for safety evaluated.Based on Caco-2 monolayer cell model,the transport rate of four DSP(100 nm,200 nm,500 nm,1?m DSP)was compared.Before transport experiment,CCK-8method was used to detect the cytotoxicity of four different sizes of DSP to Caco-2cells.The results showed that the cell viability was all over 95%,suggesting that DSP had no negative effect on Caco-2 cells.In the Caco-2 cell intestine transepithelial transport experiment,within 4 h,the cumulative transport rate of DSP(100 nm,200 nm,500 nm and 1?m DSP)was 42.9±0.07%/cm~2,43.5±0.01%/cm~2,39.5±0.08%/cm~2,31.7±0.04%/cm~2,respectively.That meant that DSP of lower particle size might have higher transport rate compared to 1?m DSP.The distributionand quantified the content of DSP(100 nm,200 nm,500 nm and1?m DSP)in the gastrointestinal tract were obtained by sacrificing of mice at different time intervals after intragastric gavage administration,and?-Conglycinin in DSP was used as a marker.The quantitative detection of the absorption of DSP in the gastrointestinal tract after different time periods was achieved by sandwich ELISA.At0.5 h,1 h,2 h,and 4 h,the total content of 100 nm DSP were 2.4±0.01 mg,1.9±0.02mg,1.6±0.05 mg,1.4±0.02 mg,respectively;and for 200 nm DSP,they were 3.7±0.45mg,3.32±0.26 mg,2.2±0.21 mg,and 1.9±0.25 mg,respectively;content of 500 nm DSP were 2.0±0.09 mg,2.1±0.04 mg,2.9±0.04 mg,and 1.6±0.2 mg,respectively;data of 1?m DSP were 1.5±0.03 mg,1.9±0.08 mg,2.1±0.14 mg,1.45±0.03 mg,respectively.The distribution of DSP in the gastrointestinal tract was located by immunogold silver staining(IGSS).The small intestine IGSS revealed the distribution of four DSP.They were mainly in the intestinal villi,epithelial cells,and mucosa of the small intestine.Immunoelectron microscopy was used to further investigate the absorption pathway of nDSP in the intestinal tissue of mice.The results of in vivo study showed that nDSP can slightly increase the efficiency of gastrointestinal absorption by possible extra pathways.In addition to the normal intestinal epithelial cells,it can be also absorbed by M-cells in the gut-associated lymphoid tissue(GALT)and a few nDSP seemed to be para-cellular uptake across tight junctions of the epithelial cell layer.Acute toxicity assessment was conducted on 100 nm,500 nm and 1?m DSP.No obvious adverse acute reactions were observed.The distribution of DSP in various organs of mice showed that the concentration of DSP detected in the liver of female mice treated with 100 nm DSP was 11.4%of female mice treated with 500 nm DSP,or9.4%of 1?m DSP;the concentration of DSP detected in the lung of female mice treated with 100 nm DSP and 500 nm DSP was 24.6%and 32.6%of 1?m DSP.The distribution of three particle sizes of DSP in the organs of male mice showed similar pattern.In general,the lethal dose of DSP(100 nm,500 nm,1?m DSP)was higher than10 g/kg body weight,which can be considered as nontoxicity.Compared to the 1?m DSP,100 nm DSP and 500 nm DSP had less undigested macromolecular substances distributed within the organs.