Selection Of Exopolysaccharide-producing Strains From Endophytic Bacteria In Siraitia Grosvenorii And Antioxidant And Hypoglycemic Activity Of Exopolysaccharide

Microbial polysaccharides are extracellular polysaccharides（EPS）that are produced by microorganisms during growth and metabolism and secreted to the outside of cells.Current studies have confirmed that EPS has good antioxidant,immune enhancement and antibacterial functions.Siraitia grosvenorii is mainly distributed in the mountainous areas of Guangxi.The fruit contains glycosides,extracellular polysaccharides,flavonoids and other chemical components,which have the functions of reducing blood sugar and scavenging free radicals.There are many kinds of endophytes in Siraitia grosvenorii.Studies have shown that endophytes can produce biologically active substances with the same or similar functions as the host.Therefore,this study took Siraitia grosvenorii endophytes as the research object,and planned to screen out EPS-producing strains,optimize EPS fermentation conditions and alcohol precipitation conditions,purify alcohol-precipitated EPS,and conducted antioxidant and hypoglycemic activity in vitro studies to broaden the source and application of EPS,and provide reference for the comprehensive utilization of Siraitia grosvenorii resources.The main research contents and results are as following:1.Screening of endophytic bacteria in Siraitia grosvenoriiTheα-naphthol-sulfuric acid method,phenol-sulfuric acid method and DNS method were used to screen EPS-producing strains from the endophytic strains of Siraitia grosvenorii preserved in the laboratory.An EPS producing strain named LHG-3 was selected from 38 strains,and its EPS content reached（0.78±0.02）mg·m L^-1,which was significantly higher than other strains（P<0.05）.2.Strain identificationMorphology,physiology,biochemistry and molecular biology tests were performed on the strain LHG-3.It shows that the strain is Bacillus,which is clustered with Bacillus wiedmannii,with sequence homology of more than 99%.The most certain strain LHG-3 is Bacillus,named Bacillus sp.LHG-3.3.Optimization of fermentation conditions of strain LHG-3The growth curve and EPS production curve of the strain were studied,and the fermentation conditions for EPS production were optimized.The results showed that the optimal fermentation time was 42 h,at which time the content reached（0.76±0.01）mg·m L^-1.The primary and secondary order of the factors affecting the EPS production of the strain is inoculum amount>sucrose added amount>urea added amount>calcium chloride added amount.The best combination is sucrose 2.5%,urea 1.2%,calcium chloride 0.4%,inoculum amount 8%,cultured at p H 7.0 and 30℃for 42 h,the EPS content reaches（1.59±0.03）mg·m L^-1,It is 1.92 times that before optimization,and has good stability.4.Optimization of alcohol precipitation conditions for EPSSingle factor and response surface test optimization of several factors affecting alcohol precipitation.In the final response surface,the optimal EPS alcohol precipitation conditions were 0.768 times the fermentation broth concentration,the centrifugal speed 6504 r·min^-1,the ethanol volume fraction 81.92%,and 4.25 times the volume of ethanol.Under these conditions,the EPS content is（3.29±0.03）mg·m L^-1,which is 2.07 times that before alcohol precipitation.5.Optimization of purification conditions for EPSThe Sevage method was used to remove the protein in the alcohol-precipitated EPS,and the anion exchange column and dextran gel column were used for further purification.The results show that the Sevage method has a good effect on protein removal.After the alcohol-precipitated EPS is subjected to anion exchange column chromatography,two components,EPS-A and EPS-B,are obtained.After the dextran gel column chromatography,it is still a single peak.Description is a homogeneous polysaccharide component.The yields of EPS-A and EPS-B were 30.62%and 19.25%.6.Preliminary identification of EPSThe UV spectrum scanning of EPS-A and EPS-B showed that they did not contain protein and nucleic acid.The results of EPS-A infrared spectroscopy show that the three absorption peaks appearing at a specific wave number indicate that EPS-A containsα-pyranose.The absorption peaks of EPS-A and EPS-B at wavenumbers around 1081.50 cm^-1 respectively indicate that they containα-（1→6）glycosidic bonds and have the basic skeleton and functional groups of a typical polysaccharide structure,indicating that EPS-A and EPS-B are polysaccharide substances.The surface of EPS-A is granular when it is magnified by 1.0 K times,and the surface of the branch part has fine flaky,porous and smooth surface when it is 5.0 K times.When EPS-B is magnified by 1.0 K times,it shows a flake shape.When magnified by 5.0 K times,the surface is smooth and can be used to make biofilm materials.Compared with other materials,it has higher water retention and stability.7.Study on the antioxidant activity in vitro of EPSAnti-oxidation studies in vitro on alcohol-precipitated EPS,EPS-A,EPS-B and VC.It can be seen from the results that several samples have antioxidant activity in vitro,and the EPS purified product has stronger antioxidant capacity and has a dose-concentration effect.Only in the determination of the reducing power,the alcohol-precipitated EPS and EPS-B did not reach a significant level,and the samples of the other groups of tests all reached a significant level（P<0.05）,indicating that the EPS after purification showed a better performance.8.Hypoglycemic activity in vitro of EPSFor EPS-A,EPS-B and acarbose,the hypoglycemic activity in vitro was measured by the hole-punching method and the Bernfeld method.The hole-punching method found that all the test groups had inhibitory activity onα-amylase.Bernfeld method found that the inhibitory rate of the purified polysaccharide onα-amylase increased,and the inhibitory rate of EPS-B was 5.69 times that of the fermentation broth,and the difference between the groups reached a very significant level（P<0.01）.It can be seen that after EPS purification,the inhibitory effect onα-amylase is improved.In summary,this study screened an EPS-producing Siraitia grosvenorii endophytic strain Bacillus sp.LHG-3,obtained the best fermentation conditions and separation and purification conditions for EPS production,and obtained and confirmed EPS-A and EPS-B They are two purified polysaccharides,both of which have hypoglycemic and antioxidant activities in vitro.Among them,EPS-B has strong antioxidant activity and certain in vitro hypoglycemic activity.