Study On The Mechanism Of Metabolism Of Na₂SeO₃ By Lactobacillus Brevis Based On Transcriptomics

Lactobacillus brevis has a variety of biological functions,such as improving human intestinal flora,regulating blood glucose concentration,lowering cholesterol levels,and enhancing immunizing power.It currently has been using in the production of gamma-aminobutyric acid,beer brewing,fermented foods and food&pharmaceutical industries.Selenium is an essential trace element for human and is closely related to the synthesis of various enzymes in the body,such as thioredoxin reductase,thyroxine-50-deiodinase,and glutathione peroxidase system.Studies have found that the level of selenium in the body is closely related to the incidence of cancer.Currently,selenium supplements on the market are mainly inorganic salts such as sodium selenite and organic selenoproteins.The former has strong toxicity and low bioavailability,although organic selenium has lower toxicity and higher bioavailability compared with inorganic selenium,but its highly cost and the long period of production have became the limitations of the further application.The research on the reduction of sodium selenite by microorganisms has been carried out for many years,and it has been mainly applied in the field of microbial degradation of heavy metals.In recent years,reports on the application of selenium-enriched lactic acid bacteria on the food industry have gradually increased.The common problems all over the researches are that the toxicity of sodium selenite causes the biomass of bacteria to decrease,and the ability of different kinds of lactic acid bacteria to reduce sodium selenite is also very different.A bacterium named L.brevis CGMCC 6683 was used as the research object in this research.First the changes of biomass after its growth in different concentrations of sodium selenite was measured,and the transmission electron microscope and scanning electrons were used to study the morphology of the bacterial cells;then the physiological and biochemical indicators of the L.brevis after the reduction of sodium selenite were measured to detect the membrane damage;finally the high-throughput transcriptome sequencing technology was used to predict the mechanism of sodium selenite metabolism by L.brevis after growing in 5 mM sodium selenite for24 h.Based on the above researching methods,the following results are obtained:1.L.brevis strains were cultured in Na₂SeO₃ containing different concentrations?0 mM,2mM,5 mM,10 mM,15 mM,and 20 mM?for 24 h and the bacterial biomass was determined by plate counting method.The data shows that the biomass of L.brevis decreased from 10⁸ CFU/mL to 10⁴ CFU/mL along with the increasing of concentrations of Na₂SeO₃.2.The cell morphology of selenium-enriched L.brevis were observed by transmission electron microscopy and scanning electron microscopy.It was found that the shrinkage,collapse,and even breakage occurred on the surfaces of bacteria after growth in a medium containing Na₂SeO₃.This kind of changes are more apparently along with the increasing of Na₂SeO₃concentration in the media.The round granular material appear on the surface of the L.brevis cell membrane,which are analyzed by X-ray energy spectrum analyzer that they are zero valence state selenium.3.L.brevis'intracellular ATP concentration,intracellular pH and membrane have changed after growing in the selenium contained media whose concentration were 0 mM,2mM and 5 mM.The membrane integrity of the cells was detected by laser confocal fluorescence microscopy,it was found that in the control group,the fluorescence integrity of the membrane was green,and that the concentration of Na₂SeO₃ was red fluorescence.As the proportion of bacterial cells increased,almost 100%of the cells in the 20 mM experimental group showed red fluorescence.4.Transcriptomic sequencing technology was uesd to analyze the changes of the transcriptome after metabolism of Na₂SeO₃ in L.brevis,a total of 461 genes were significantly differentially expressed.These genes were mainly enriched in the pyruvate metabolic pathway,fatty acid biosynthesis pathway,selenide metabolic pathways and NOD-like receptor signaling pathways,the genes associated with the sulfhydryl oxidoreductase,electron transport and the inorganic salts transported proteins were significantly up-regulated.5.There are two major pathways for the metabolism of Na₂SeO₃ by L.brevis:?1?Na₂SeO₃enters the cell body by the cell membrane transporter,then they are reduced to low molecular mass selenides by substances containing thiol group.These selenides are used as precursors to synthesize selenium-containing amino acids that are further incorporated into the synthesis of selenium-containing proteins;?2?Na₂SeO₃ receives electrons from the electron transport chain of L.brevis and are reduced to elemental selenium.Elemental selenium accumulates on the surface of the bacteria to form elemental selenium particles.