Screening Of Fucoidanase-Producer Strains And Structure Characterization、Biological Activities Of Enzymatic Hydrolysates

Fucoidan as a macromolecular heteropolysaccharide,it has problems such as large molecular weight,poor solubility,and not easy to be absorbed,which seriously restricts its application.The low molecular weight fucoidan produced from fucoidan as raw material has the advantages of low viscosity,good solubility and easy absorption,and shows good bioavailability.Enzymatic hydrolysis to prepare low molecular weight fucoidan is the most ideal preparation method at present,the enzyme degradation conditions are mild,the catalytic efficiency is high,the molecular weight is easy to control,it will not destroy the structure of the polysaccharide itself,and biocompatible active products can be formed.However,enzymatic degradation also has the disadvantages of high cost and difficulty in industrial production,so it is the focus of current research to establish an efficient enzyme-producing strain screening method and screen a low-cost strain with high enzyme activity.The main research work of this thesis is:（1）A total of 23 fungi were screened from tea by the only carbon source method,and 7strains with obvious production circles were rescreened and detected,and after enzymatic hydrolysis of fucoidan（F）at 50°C for 24 h by fermentation of crude enzyme solution,the enzymatic hydrolysate was detected by high performance gel permeation chromatography（HPGPC）.The low molecular weight fucoidan（LMWF）of less than 10 k Da had the highest relative content of 59.4%from JH-5 strain,and it was used as the starting strain for follow-up experiments.And based on the phylogenetic analysis of the ITS gene sequence,the strain was identified as Aspergillus amstelodami.（2）The results of enzyme production conditions of JH-5 fermentation were optimized showed that the fucoidanase activity was the highest at pH 6.0,30°C,fermentation for 4 d,fucoidan addition of 0.3 g,ammonium sulfate as nitrogen source,and water content of 8 m L,which was 0.354±0.012 U·m L^-1,and the enzyme activity increased by 45.08%after optimization.（3）The enzymatic hydrolysis conditions of JH-5 crude enzyme solution were optimized,and the results showed that the yield of low molecular weight fucoidan polysaccharides was61.70±0.71%at 50°C,substrate concentration of 1.5%and pH 6.0.The reaction system was expanded under optimal enzymatic hydrolysis conditions,and the components below 10 k Da were collected after protein removal,dialysis and salt removal,ultrafiltration fractionation,and stored in vacuum drying dishes after freeze-drying.（4）Characterization of the primary structure of the digest LMWF.The molecular weights of F and LMWF were 277 k Da and 2997 Da,respectively.Monosaccharides are mainly composed of fucose and galactose,and contain small amounts of xylose and glucuronic acid;The sulfate content was 33.0±0.3%and 32.4±0.9%,respectively.The glycosidic bond types are allα-glycosidic bonds;The thermal characteristics measured that the melting temperature of F and LMWF is around 135°C.In addition to the molecular weight,its structural characteristics did not change significantly,and the above experimental results showed that the structure of the crude enzyme liquid hydrolysate of fermentation of this strain was relatively stable,and the active group would not fall off.（5）The results of antioxidant experiments showed that LMWF had significantly better scavenging ability for hydroxyl radicals,DPPH radicals,and total antioxidant capacity than F.The results of in vitro simulated digestion experiments showed that F and LMWF exhibited higher anti-digestive properties during simulated digestion,indicating its potential as a prebiotic.The potential of LMWF to promote the growth of probiotics was evaluated by fermenting and culturing probiotics with media supplemented with different carbon sources,and the results showed that LMWF was able to significantly promote the growth of probiotics and improve their acid production capacity,thus indicating that LMWF can be used as a prebiotic to assist in improving intestinal health.