Research On Structure And Probiotic Function Of Exopolysaccharide From Lactobacillus Plantarum WLPL04 And Optimization Of Its Fermentation Processes

Lactic acid bacteria is a kind of probiotics that can improve the microecological balance,inhibit the growth of harmful bacteria and relieve lactose intolerance,and has beneficial effects on the health of the host.The functional study of lactic acid bacteria and its exopolysaccharide?EPS?is currently research hotspots.There are few reports on structural analysis and probiotic function evaluation of Lactobacillus plantarum and its extracellular polysaccharides.Lactobacillus plantarum WLPL04,previously from breast milk,is an EPS producing strain of lactic acid bacteria with good probiotic properties.In this study,structure analysis,inhibition of pathogenic biofilm formation,antagonistics against adhesion of pathogens and antioxidant activity of EPS from WLPL04 were carried out.To increase the EPS production of WLPL04,fermentation conditions were optimized.The full text is divided into five chapters.In the first chapter,lactic acid bacteria and its EPS were reviewed,and the types of EPS of lactic acid bacteria,probiotic function and application aspects were introduced as well.Additionally,a summary and prospect of the research of EPS were proposed.In the second chapter,structural characteristics and probiotic properties of EPS of L.plantarum WLPL04 were evaluated.The molecular weight and structure of EPS of WLPL04 were analyzed by gel permeation chromatography?GPC?,ultraviolet spectroscopy?UV?,fourier transform infrared spectoscopy?FT-IR?and gas chromatography-mass spectrometer?GC-MS?.Moreover,the surface property of EPS was observed by scanning electron microscopy and atomic force microscopy.Finally,the inhibitory effect of EPS on colon cancer cells HT-29,the inhibition of Escherichia coli O157:H7 adhesion to HT-29 cells?competition,exclusion and replacement?and the inhibition of Pseudomonas aeruginosa,E.coli O157:H7,Salmonella typhimurium and Staphylococcus aureus and other pathogens to form the bacterial membrane.The results showed that the relative molecular mass of EPS produced by L.plantarum WLPL04 was 6.67×10⁴ Da,consisting of three kinds of monosaccharides,xylose,glucose and galactose.EPS appeared as a smooth lamellar structure with pores of the microscopic morphology,the formation of different height of the rod-shaped mass,indicating that aggregation phenomenon of EPS was occurred.EPS inhibited the growth of human colon cancer HT-29 cells at a concentration of 800?g/mL and the inhibition rate was 67.26%.EPS inhibited the adhesion of E.coli O157:H7 to HT-29 cells at a concentration of 1.0 mg/m L,the inhibitory rates were20.24%,29.71%and 31.91%,respectively,in the competition,displacement and exclusion experiments.When the EPS concentration was 5.0 mg/m L,the inhibitory rates against Pseudomonas aeruginosa,E.coli O157:H7,Salmonella typhimurium and Staphylococcus aureus were 47.02%,25.82%,35.95%and 48.04%,respectively.The above results suggested that EPS of L.plantarum WLPL04 has good probiotic properties and potential applications.In the third chapter,EPS of WLPL04 and its sulfonated polysaccharide antioxidant activity was studied.Firstly,EPS was sulfonated by sulfur trioxide-pyridine to evaluate the ability of EPS and its sulfonated polysaccharides to scavenge DPPH,hydroxyl radicals and superoxide anion.The cellular model was used to investigate the effect of EPS and its sulfonated polysaccharides.The protective effects on the cells were observed under treating with EPS or sulfated EPS.The activities of SOD,CAT,GSH-Px,MDA and ROS and the expression of antioxidant genes were analyzed.The results showed that the anti-oxidative capacity of EPS after sulfonation was significantly improved,the clearance rate of DPPH,hydroxyl radicals and superoxide anion of EPS?1.0 mg/mL?after sulfonation increased from 22.51±3.05%,63.75±3.84%and 28.99±1.82%to 28.27±2.13%,71.85±0.23%and 32.69±1.05%,respectively.At the same time,sulfated EPS?100?g/m L?had a stronger protective effect on Caco-2 cells when compared with EPS,in which antioxidant-related genes SOD2,GPX2 and MT1A were upregulated.Furthermore,the activity of SOD,CAT,GSH-Px,MDA and ROS increased by 5.58%,30.66%,44.14%,28.22%and 20.06%,respectively.In addition,sulfated EPS showed less cytotoxic effects on Caco-2 cells with IC₅₀ of 2337.71 and 1220.38?g/mL for sulfated EPS and EPS,respectively.Based on the above results,the EPS of L.plantarum WLPL04 has excellent antioxidant activity,sulfonation modification of EPS was one of the effective ways to improve its antioxidant activity.In the fourth chapter,the aim was to optimize the fermentation conditions to improve the production of EPS from L.plantarum WLPL04.The single factor method was adopted,the inoculation amount,temperature,fermentation time,and initial pH were used as the variables,and the best fermentation conditions were at 3%inoculation,37°C,24 h and pH 6.0.The optimal fermentation conditions were determined to be pH 6.0,38°C and 4%inoculation based on the results of response surface methodology.In the fifth chapter,the chemical structure and probiotic function of EPS from L.plantarum WLPL04 were summarized,and the exploration of the molecular mechanism of EPS as probiotic substance was prospected.In summary,chemical structure of EPS of L.plantarum WLPL04 was analysed by using modern instrumental analysis technique,its probiotic function was evaluated in vitro as well.Furthermore,the antioxidant activity of EPS and sulfated EPS were investigated.Finally,the fermentation conditions of WLPL04 were optimized to improve the production of EPS.The whole results provide methods and ideas for the further development of probiotic strains.