Screening,Characterization,Antioxidant Function And Mechanism Of Selenium-enriched Bacillus SR14

In this study,a selenium resistant strain SR14 was screened by the way of preliminary screening of selenium containing plate medium and re screening of selenium containing liquid medium.The strain was identified as probiotic Bacillus paralicheniformis,and its ability of reducing sodium selenite,the content of each component of fermentation product and microstructure were analyzed.At the same time,the antioxidant activities of probiotics SR14(BP)and selenium enriched fermentation products(S-BP)against four different free radicals in vitro were studied.On this basis,the oxidative damage model of pig intestinal epithelial cells induced by H2O2 was established,and the effects of BP and S-BP on alleviating oxidative damage were studied and compared.A series of experiments,such as fluorescence analysis,flow cytometry,apoptosis detection and Western blot,were used to explore the possible molecular mechanism of S-BP and BP’s antioxidant function.The main results are as follows:1.Screening of selenium enriched probioticIn this study,10 strains which can tolerate 5 mmol/L sodium selenite were screened from 60 strains by the method of preliminary screening of selenium containing plate medium.They are No.1,No.2,No.5,No.14,No.15,No.16,No.24,No.32,No.41 and No.57,respectively.The results showed that all of the 10 strains could reach 5 However,some strains,such as 16,32 and 41,grew normally in the liquid screening medium of mmol/L sodium selenite,and the red color of the medium was lighter after fermentation,suggesting that the ability of transforming sodium selenite was weaker than other strains;among them,the content of sodium selenite in the liquid screening medium of strain 14 reached 100 At mmol/L,it can still grow normally and transform sodium selenite well.The culture is based on the obvious red color 26 hours after inoculation,and the dark red color of the medium at the end of fermentation(72 hours).According to the speed of the medium turning red and the color after fermentation,it is suggested that strain 14 has the best selenium resistance,named SR14.2.Characterization and identification of SR14In this study,according to the cell morphology and physiological and biochemical characteristics of the strain,and using 16S rDNA and gyrB gene sequencing,identified and screened SR14 strain as Bacillus paralicheniformis.On this basis,through the liquid culture medium containing the gradient concentration of sodium selenite,it was determined that the selenium enriched probiotic SR14 could completely reduce 5 mmol/L sodium selenite after 60 hours of fermentation.Through multiple batches of repeated fermentation,it was determined that the mass of dry matter was 38.2±0.5 g per liter of SR14 selenium enriched fermentation.The content of selenium in the product is 4026.1±120.9 mg/kg,the content of selenium is 1296.3±130.1 mg/kg,and the product does not contain sodium selenite.In addition,field scanning electron microscopy(FESEM),transmission electron microscopy(TEM),Fourier transform infrared spectroscopy(FTIR)and other methods were used to study the micro characteristics of the fermentation products of selenium enriched probiotics SR14.The results showed that the bacteria were not affected by sodium selenite,and were in normal long rod shape.At 2926cm-1 and 1644 cm-1,C=O and COO-were observed.At 860cm-1 and 764cm-1,α-isomer pyranose was observed.The results showed that Se enriched probiotics secreted carbohydrate compounds,wrapped the transformed selenium into spheres and transported it out of the cell.3.Study on antioxidant function of selenium enriched probioticIn this study,the scavenging effects of probiotic SR14(BP)and its selenium enriched fermentation product(S-BP)on superoxide anion free radical,hydroxyl free radical,DPPH free radical and ABTS free radical were studied in vitro.The results show that BP has the ability of scavenging superoxide anion and DPPH radical,and the scavenging rate of hydroxyl radical is close to 100%,but it has no scavenging ability of ABTS radical.S-BP has a stronger ability to scavenge free radicals,and the scavenging ability to superoxide anion,DPPH and ABTS is better than SR14 itself.Meanwhile,the protective effects of S-BP and BP on the oxidative damage of IPEC-J2 cells induced by H2O2 were studied.The results showed that BP or S-BP alone had no effect on the growth of IPEC-J2 cells.When treated with 400 μmol/L H2O2 for 10 hours,the growth of IPEC-J2 cells will be reduced to 60-80%of the normal condition,while when treated with 10 and 20 μmol/L S-BP or 5 × 104 CFU/ml BP in advance for 4 hours,the inhibition of cell growth induced by H2O2 can be significantly alleviated.In addition,the LDH release of IPEC-J2 cells induced by H2O2 was significantly reduced after 4 hours of pretreatment with 10 and 20 μmol/L S-BP or 5 × 104 and 1 × 105 CFU/ml BThese results suggest that S-BP and BP have a strong protective effect on H2O2-induced oxidative damage of IPEC-J2 cells,and the antioxidant capacity of probiotics has been enhanced after selenium enriched fermentation.4.The mechanism of Se enriched probiotic alleviating oxidative stress induced by hydrogen peroxide in porcine intestinal epithelial cellsBased on the preliminary study of the antioxidant function of S-BP and BP,the mechanism of S-BP and BP alleviating the oxidative damage of IPEC-J2 cells induced by H2O2 was further explored,and the potential reasons for the difference of their antioxidant capacity were explored.The results showed that both BP and S-BP could alleviate the increase of MDA production and ROS release in IPEC-J2 cells induced by H2O2.At the same time,by fluorescence observation and flow cytometry analysis of apoptosis,the results showed that compared with the control group,the proportion of apoptotic cells in H2O2 group increased significantly,while the percentage of apoptotic cells in 10 and 20 μmol/L S-BP pretreatment decreased significantly,the results were close to the control grouBP pretreatment with 1 × 105 CFU/ml decreased the proportion of apoptotic cells,but the effect was weaker than that of S-BFurthermore,the effects of S-BP and BP on MAPK related pathway protein expression in oxidative damaged cells were investigated by Western blot and pathway protein inhibitors.The results showed that S-BP significantly increased the phosphorylation level of ERK and p38 MAPK protein compared with H2O2 group,but BP had no significant effect on the phosphorylation level and total protein level of ERK,p38 and JNK protein,suggesting that S-BP activated ERK/p38 MAPK signal pathway to alleviate H2O2-induced oxidative stress,which may be the potential reason why S-BP has better antioxidant function than BP.In conclusion,a selenium resistant bacillus SR14 was screened and identified as Bacillus paralicheniformis.The antioxidant capacity of S-BP and BP was further evaluated.The results showed that S-BP had stronger ability to scavenge free radicals,while BP and S-BP pretreatment could alleviate H2O2 induced MDA production,ROS release and apoptosis of IPEC-J2 cells.In addition,S-BP treatment significantly increased the phosphorylation levels of ERK and p38 MAPK proteins in oxidative stress cells,suggesting that S-BP activates ERK/p38 MAPK signaling pathway to alleviate oxidative stress induced by H2O2.To sum up,one of the best selenium resistant strains SR14 was screened from 60 strains,which was identified as Bacillus paralicheniformis.Furthermore,the contents of each component in SR14 fermentation products were systematically evaluated to characterize the properties of fermentation products and separated selenium.Then,the antioxidant function and potential mechanism of selenium enriched fermentation products and selenium free fermentation products were investigated by free radical test in vitro and oxidative stress model of pig intestinal epithelial cells.