The Biosynthetic Pathway Of Exopolysaccharides From Bifidobacterium Longum And Its Protective Effect On Goldfish Under Cadmium Stress

Bifidobacterium longum is the most ubiquitous Bifidobacterium in the intestinal flora.Individual bifidobacteria can secrete carbohydrate macromolecule compounds outside the cell during the growth and metabolism process,that is,exopolysaccharides（EPS）.However,the biosynthesis and structure of EPS produced by different strains are different.Moreover,the unique heavy metal absorption function of EPS has become a research hotspot in recent years.Therefore,in this paper,firstly,the whole genome of Bifidobacterium longum A17 isolated from the intestine of breast-milk infants was sequenced to study EPS biosynthesis at the genetic level.Secondly,the separation,purification,structural characterization and absorption of heavy metals to protect the health of the fish were conducted on the EPS produced by A17.The main conclusions are as follows:1.The results of the whole genome analysis showed that the Bifidobacterium longum A17 strain does not contain plasmid and consists of a circular chromosome with a chromosome length of 2,501,935 bp and GC content of 61.01%.The results of bioinformatics analysis showed that the genome contained three prerequisite sugar nucleotides（UDP-glucose,UDP-N-acetylglucose and UDP-galactose）and an exopolysaccharides gene cluster with a length of approximately 20 kb.In addition,the results of determination of carbohydrate metabolism capacity showed that strain A17 can metabolize 6 kinds of carbon sources including D-glucose,galactose,D-lactose,D-melibiose,D-sucrose,D-raffinose and the bioinformatics analysis results are consistent.2.After centrifugation,alcohol precipitation and dialysis crude exopolysaccharides are separated from the supernatant of the sugar-producing medium of strain A17.The exopolysaccharides is purified by a gel chromatography column to obtain a single component A17-EPS.The results of UV spectrum analysis showed that A17-EPS is a exopolysaccharide substance without protein and nucleic acid.The molecular weight of A17-EPS determined by GPC was 553,817 Da.In the FT-IR spectrum,A17-EPS showed exopolysaccharide characteristic absorption peaks such as the stretching vibration and bending vibration of hydroxyl,alkyl and carbonyl indicating that A17-EPS is a exopolysaccharide and hasα-type glycosidic bonds.Combining methylation analysis with GC-MS and NMR the analysis speculates that the repeating units of EPS are as follows:（?）3.The lethal effect of different cadmium(Cd²⁺)concentrations on goldfish showed that the optimal Cd²⁺stress concentration in the bucket was 7 mg/L.After adding different doses of A17-EPS to the fish feed,the goldfish under 7 mg/L Cd²⁺stress were tested for abnormal red blood cell nuclei,intestinal digestive enzymes（amylase,lipase and protease）and cadmium content in goldfish muscle.The experimental results showed that the micronucleus rate and nuclear abnormality rate of the A17-EPS high-dose group were significantly lower than those of the positive control group（P<0.01）.In the digestive enzyme results,the amylase activity of the A17-EPS low-dose group was significantly different than that of the positive control group（P<0.05）.The protease activity of the A17-EPS high and medium dose groups was significantly higher than that of the positive control group（P<0.01）.There was no significant difference between the other treatment groups.The muscle cadmium content of the high-dose group was significantly lower than that of the positive control group（P<0.01）,and the muscle cadmium content gradually decreased as the dose of A17-EPS increased,indicating that A17-EPS can reduce fish under cadmium stress accumulation of heavy metals,improve intestinal digestion.