Screening Of Extracellular Polysaccharide-producing Bifidobacterium And Research On Its Biological Characteristics And Yield Optimization

Bifidobacteria is one of the most important normal flora in human intestinal tract,which has extremely important physiological functions.Exoploysaccharides?EPS?are carbohydrate polymers that exist as the outer layer of cells and surround a variety of cell types,from plants to animals,including different microorganisms.Some of the reported health benefits of bifidobacteria are linked to their polymers,which are surrounded by a layer of water-rich exopolysaccharides that protect cells in the environment.However,the reported EPS yield of bifidobacteria is relatively low.In this paper,bifidobacteria with high EPS yield and excellent probiotics was screened from the human gastrointestinal tract,and then the fermentation conditions of EPS were optimized.Firstly,52 strains of bifidobacteria of intestinal origin were isolated by 16S rDNA sequence analysis from feces samples of kashi region,Xinjiang.It was found that 11bifidobacteria produced adhesive wiredrawing in MRS plate and skimmed milk medium.Then11 bifidobacteria were re-screened by phenol-sulfuric acid method,and the results showed that7 bifidobacteria had higher EPS yield,B.longumMFX-1 and MFX-2,B.longum subsp.infantis BF66-16?BF85-36 and BF107-4,B.bifidumBF107-7 and BF52-1,respectively.The EPS yield of all the 7 strains tested reached more than 400 mg/L,especially the EPS yield of B.longum subsp.infantis BF66-16 was reached 513.06 mg/L.Secondly,the probiotic characteristics of 7 bifidobacteria with high EPS yield were analyzed,including the growth curve of the strain,the tolerance at low pH and different bile salt concentrations,survival rate in simulated gastric and intestinal fluids,antibacterial activity,in vitro adhesion characteristics?auto-aggregation and hydrophobicity?and antibiotic resistance.Results show that strains were grew rapidly after 12 h,and the strain grew to 28 h,the cell number reached the highest,at this time the OD 600 value of the bacterial suspension can reach more than 1.8,the number of bacterial strain proliferation and death gradually balanced.The strain has good growth performance,strong vitality and abundant biomass.The culture time of the strain was determined,the strain's resistance to acid and bile salts and the ability to tolerate simulated gastric and intestinal fluid were tested.Results showed that all strains could not grow under pH 2.0.However,the survival rate of the strain was still more than 30%after 4 hours of culture under pH 3.0.The survival rate of MFX-2 and BF66-16 was more than 14%when the concentration of bile salt was 0.3%.In the simulated gastric and intestinal fluid,the survival rate of the strain was still 40%after the simulated gastric fluid survived for 120 min,and the tolerance was strong.Then it was observed that after the strain was transferred to the simulated intestinal fluid for 120 min,only the survival rate of BF66-16 was higher.In the antibacterial test,7 strains of bifidobacteria had differentantibacterial activities against seven common opportunistic pathogens.Then combined with the results of adhesion test and antibiotic tolerance,it was preliminarily speculated that strain BF66-16 could adhere to the gastrointestinal tract and survive in a certain amount to give play to its probiotic characteristics.Therefore,this strain was selected for subsequent studies.Finally,The effect factors of strain BF66-16 EPS production is optimized,the results showed that sucrose 7%?w/v?,tryptone 1.2%?w/v?,beef powder 1%?w/v?,yeast powder,0.7%?w/v?,K₂HPO₄ 0.6%?w/v?,0.6%?w/v?acetic acid sodium,citric acid ammonium 0.3%?w/v?,twain-80-0.2%?v/v?,the initial pH 6.5,temperature 36°C,quantity of 2%?v/v?,the highest strainEPS production.On this basis,the theoretical optimal combination of response values?sucrose 80 g/L,tryptone 14 g/L,pH 6.9?was determined by the steepest climbing test,and the test was designed with this as the center of BBD test,and the quadratic equation was obtained:Y=5.22+0.21A+0.36B+0.21C-0.12AB-0.018AC-0.19BC-0.61A²-0.43B²-0.30C²,The results of regression analysis showed that A,B,C and quadratic terms of each variable?A2,B2,C2?had significant effects on the yield of EPS?P<0.05?.The model predicted that when the sucrose content,tryptone content and initial pH value were 81.5g/L,14.21g/L and 7.00,respectively,the EPS yield of the strain was the maximum.After experimental verification and fermentation process study,the yield of EPS reached 3.13 g/L,which was 6.1 times that of the unoptimized?0.51 g/L?.In summary,7 bifidobacteria with high EPS yield were screened from human intestinal tract in this paper,all of which were above 400 mg/L.The in vitro probiotics of the seven strains were then tested,including tolerance to low acid bile salts and simulated gastric and intestinal fluids,antimicrobial activity,adhesion,and antibiotic resistance.After comprehensive analysis of the test results,it was found that the strain BF66-16 had good probiotics.Then,the fermentation conditions of EPS were optimized,and the yield after optimization?3.13 g/L?was6.1 times higher than that before optimization?0.51 g/L?.The in vitro probiotics of bifidobacteria with high yield EPS were analyzed,which indicated that bifidobacteria EPS had a certain protective effect on bifidobacteria itself,and provided a factual basis for its in-depth development and application as a probiotics.At the same time,the fermentation conditions of bifidobacteria EPS were optimized by response surface method,which improved the yield of EPS to a certain extent,and laid a foundation for the subsequent separation and purification of relatively pure EPS,and the study of its monosaccharide composition,structure and physical and chemical properties.