| Microbial polysaccharides have been widely studied and spplied due to their unique physical and chemical properties and biological activities.Microbial polysaccharides are biological macromolecules with various types,complex structures,significant microbial diversity and diversity,and most microbial polysaccharide resources and their physical and chemical properties and applications have not been developed and studied in depth.Therefore,screening and isolating novel microorganisms and studying the compositions,structure and biological activity of the polysaccharides produced by them,it had great research significance for developing new microbial polysaccharide resources and obtaining microbial polysaccharides with potential application value and their derived biologically active substances.In this paper,a strain producing Extracellular Polysaccharide(EPS)was screened from nature and identified as Rhizobium sp.While optimizing the conditions of EPS fermentation of Rhizobium,the extracellular crude polysaccharide was isolated and purified and its components were analyzed.In addition,the preparation of Selenium Extracellular Polysaccharide(SEPS)was carried out by the Rhizobium fermentation method,and the structure,morphology and anti-tumor activity of SEPS were studied,and antitumor activity.The Main findings are as follows:(1)The strain was isolated from the roots of dandelion and identified as Rhizobium sp.by morphological observation,fermentation product characteristics and 16S rDNA molecular sequence analysis.Single-factor shake flask fermentation experiments showed that glucose,yeast powder,potato leaching juice and bean sprout juice had significant effects on the fermentation of rhizobium to produce EPS.The above four single-factor response surface test designs showed that the optimal combination of nutrient factors for EPS production was:glucose concentration of 35.59 g/L,yeast powder concentration of 25.58 g/L,potato leaching juice concentration of 30.56%,and bean sprout juice concentration of 20.29%.Under this combination,the yield of EPS from Rhizobium was 15.46 g/L,which increased by 141%compared to before optimization.Based on the optimized conditions of shake flask fermentation,the 10 L fermenter was used to expand the culture,and the yield of EPS was further improved,reaching a maximum of 28.2 g/L.(2)Crude EPS in the fermentation broth was subjected to alcohol precipitation,deproteinization,column chromatography and separation,and then purified to obtain EPS 1-1,EPS 2-1,EPS 3-1,EPS 4-1 and EPS 5-1 5 single polysaccharide components.Each polysaccharide component was analyzed by high performance gel filtration chromatography,ion chromatography and infrared spectroscopy.The results showed that the molecular weights of the five single polysaccharide components were 1.905×107,1.053×107,6.370×106,7.187×106 and 6.155×106 Da,respectively.Among them,EPS 1-1,EPS 2-1 and EPS 5-1 were mainly composed of Gal,Glu and Man,and their molar ratios were 1:4.67:2.35,1:4.69:4.33 and 1:7.72:1.69.In addition,a small amount of Fuc,Rha,and Ara were also contained in the three components.EPS 3-1 was composed of 4 kinds of monosaccharides,and the main monosaccharide molar ratio was Gal,Glu and Man=1:3.42:4.77,and a small amount of Rha;EPS 4-1 consisted of three monosaccharides with a molar ratio of Gal,Glu and Man=1:5.54:1.9.Infrared spectroscopy results showed that all of the five single components were polypyranose formed by ?-type glycosidic linkage,and EPS 1-1 and EPS 3-1 may also contain galactose pyranose.(3)The SEPS of Rhizobium was fermented by shake flask and the addition time and concentration of Na2SeO3 were optimized.The results showed that the concentration of SEPS was up to 4.3 g/L when the concentration was 5 mg/mL Na2SeO3 solution at the 6th hour of fermentation.The selenium content was 792.84 ?g/g.The concentration of Na2SeO3 and the fed medium was further controlled and optimized in the 10 L fermenter.The highest yield of SEPS was 26.8 g/L,which was 523.26%higher than that before optimization.The selenium content was 1205.15?g/g,which was 1.52 times the fermentation yield of shake flasks.Infrared spectroscopy analysis showed that SEPS added two absorption peaks,one at about 1542 cm-1,the O-O bond formed by the combination of polysaccharide chain and SeO32';the other at about 1249 cm-1 may be Se=O.The results showed that selenium binds to EPS successfully by O-O bond or Se=O bond,thereby changing the morphology of EPS.(4)The effects of EPS and SEPS polysaccharides on the proliferation of mouse breast cancer 4T1 cells were compared by in vitro cell experiments.The results showed that EPS and SEPS were cytotoxic to mouse 4T1 cells in a certain concentration range(0?2000?g/mL),and the tumor inhibition rate of SEPS was higher than EPS under the same polysaccharide concentration.When the polysaccharide concentration was 2000 ?g/mL,the cell survival rate of the EPS solution incubation was 52.94%.At this time,the cell survival rate was only 31.16%when the cells were incubated with SEPS solution. |
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