Studies On The Preparation Of Low Molecular Weight Fucoidan Extracted From Kjellmaniella Crassofolia And Their Anticoagulant Activities

Many researchers focus their work on Kjellmaniella crassifolia in recent years as it is rich for fucoidan content.Fucoidan is a biologically active substance in brown algae,which has a variety of physiological activities and has attracted widespread attention.Low molecular weight fucoidan is digested easily for the weak and has significant biological functions.In order to obtain low molecular fucoidan oligosaccharide,fucoidan extracted from Kjellmanniella crassifolia was used as the sole carbon source,five strains 2S29 Sediminicola arcticus,0P16 Paenibacillus agaridevorans,2S2 Sinomicrobium oceani,3W24 Vibrio alginolyticus,2P4Lutibacter screened in the early stage by the laboratory work were cultured and flask fermented to prepare crude enzymes.Its OD₆₀₀ of maximum biomass was 1.247,and its enzyme activity(U/m L)could reach 412.5 U/m L.The crude enzymes were then used for hydrolyzing fucoidan and hydrolyzed product was eluted by Sephacryl S-100column.It was found that 2S29 Sediminicola arcticus had better effect on degrading fucoidan sulfate.The fucoidan hydrolysate was prepared when the ratio of enzyme activity:fucoidan was 5000:1(U/g).Four fractions were eluted by Sephacryl S-100 column and which were named as F1,F2,F3 and F4 respectively.The further purification was made by polyacrylamide gel column Bio-gel-P10,and 7 fractions were obtained and which were named as SF4-1?SF4-7.The results of TLC showed that the fucoidan sulfate oligosaccharides F3 and F4 had smaller molecular weights,and the fucoidan enzyme had the effect of degrading the Kjellmanniella crassifolia fucoidan sulfate.The findings obtained by IR spectrum indicated that all fucoidan fractions contained sulfate substitute with absorption peaks of S=O(1250 cm^-1).The absorption peaks at855,853,and 852 cm^-1 were the indication of C-O-S axis.The stretching vibration of coordination,it is inferred that the sulfate group of fucoidan sulfate oligosaccharide is distributed at C4 position,and the carbon configuration may be?-D-pyranose.The anticoagulant activity results of F1,F2,F3 and F4 in vitro showed that with the decrease of the molecular weight of each component of oligosaccharide,the coagulation time and anticoagulant activity decreased,which proved that the molecular weight of fucoidan sulfate oligosaccharide was the main factor affecting the anticoagulant activity.F and its oligosaccharides had significantly prolong the activated partial thromboplastin time(APTT)and thrombin time(TT).It had inhibitory effect on the intrinsic pathway and common pathway,but did not have significantly effect on the prothrombin time(PT).