Preparation Of Low Molecular Sulphated Polysaccharides From Red Algae And Its Inhibitory Effects On Bacterial And Allergic Diarrhea In Mice

In recent years,due to the abuse of antibiotics,environmental pollution and work pressure,more and more people are suffering from intestinal flora disorder,which leads to the rapid increase of bacterial and allergic diarrhea.In this paper,we extracted and prepared low molecular weight galactosan sulfate from red algae,studied the structure-activity relationship and mechanism of its antibacterial activity,and further explored the relationship between the regulation of intestinal flora and the inhibition of bacterial and allergic diarrhea at the animal level.The main results are as follows:1.Preparation and structure-activity relationship of low molecular weight sulfated polysaccharide.Galactan sulfate was extracted from Eucheuma Serra and Gracilaria by water extraction and alcohol precipitation,and the metal ions were removed by EDTA ion chelation ultrafiltration technology.The galactose content of Eucheuma sulfate polysaccharide?ESP?and Gracilaria sulfate polysaccharide?GSP?was 93.4%and 93.5%respectively,the sulfate content was 30.0%and 15.4%respectively,the total ash content was 8.2%and 6.7%respectively.The viscosity of ESP and GSP decreased to 0.02 dL/g and 0.05 dL/g by degradation at 121?for 1 hour.FTIR analysis showed that the degraded polysaccharide had the characteristic absorption peaks of sulfuric acid group and 3,6-endoether galactose at 1265cm^-1 and 933 cm^-1.The Minimal inhibitory concentration?MIC?of DESP and DGSP with molecular weight less than 6 kDa was determined by Enterotoxigenic Escherichia coli?ETEC-K88?,was 15.0 mg/ml and 25.0 mg/ml respectively,and further purified by DEAE-cellulose 52,the MIC values of DESP and DGSP could reach 8.0 mg/ml and 10.0mg/ml respectively.2.Study on the antibacterial mechanism of low molecular sulfated polysaccharide.ETEC-K88,Staphylococcus aureus and three intestinal probiotics were used to study the effects of DESP and DGSP on different intestinal microorganisms.The results showed that the inhibition rate of DESP and DGSP to ETEC-K88 was 100%,but on the growth of Staphylococcus aureus,Lactobacillus rhamnosus,Bacillus coagulans and Bacillus subtilis had no significant effect.Using 2-AMAC fluorescent labeling technology,it was found that both DESP and DGSP could enter the cells through ETEC-K88 cell membrane and destroy the fluidity and permeability of the cell membrane.Scanning electron microscopy showed that DESP and DGSP made the surface of ETEC-K88 cell membrane crinkle and the surface adhesion of the host cell significantly weakened.3.The effect of low molecular sulfated polysaccharide on bacterial diarrhea.The model of bacterial diarrhea in mice was constructed by streptomycin interfering with intestinal flora:after 36 hours of administration of streptomycin containing 5 g/L in ICR mice,the intestinal flora was disordered,the proportion of bacteroideae in the flora was increased,and the damage of Lactobacillus and Bifidobacterium was serious;then the mice were given daily gavage ETEC-K88 with a dose of 10⁹ CFU/kg showed diarrhea,shivering,curling and other symptoms in mice one day later.It was found that the destruction of small intestinal villi was serious,and the proportion of Enterobacteriaceae and Bacteroideae bacteria in the intestine increased significantly,becoming the dominant flora.DESP can significantly reduce the number of Bacteroides in intestinal flora,and improve the abundance of bacteria such as Rikenellaceae,ruminocaceae and Bifidobacterium.After three days,the serum IgA level of mice in the high-dose group was 1235.75 ng/mL,significantly lower than that in the model group?P<0.01?,and the proportion of Enterobacteriaceae and Bacteroideae in the intestinal flora was significantly lower than that in the model group,while the proportion of Lactobacillus and Bifidobacterium in the flora was close to the normal flora.4.The effect of low molecular sulfated polysaccharide on allergic diarrhea.In ovalbumin?OVA?-induced food allergy model,the levels of IgE,IgA,mMCP-1 and TNF-?in serum of mice were 1399.9 ng/mL,2358.3 ng/mL,3228.8 pg/mL and 992.5 pg/mL respectively.The end of villi of small intestine was ulcerated,the wall of intestine became thinner,and the composition of intestinal microorganism changed.DESP of 100 mg/kg?low dose?,200 mg/kg?middle dose?and 300 mg/kg?high dose?can effectively alleviate the allergic symptoms and reduce the occurrence of intestinal inflammation in mice,and the levels of IgE,IgA,mMCP-1and TNF-?in the serum of high dose group decreased to 698.6 ng/mL,1280.8 ng/mL,1122.6pg/mL and 488.3 pg/mL respectively.From the structure of intestinal flora,the abundance of Lactobacteriaceae in the intestinal flora of sensitized mice increased significantly;while the abundance of Helicobacteraceae decreased;after DESP administration,the proportion of Lactobacillaceae could still be increased,while the abundance of Lachnospiraceae and Helicobacteraceae could be effectively restored,and the proportion of S24-7 could be reduced.