Study On The Bioactivities Of Enzymatic Hydrolysates Of Polysaccaride From Sargassum Fusiforme And Its Purified Fractions

Sargassum is edible brown algae,mainly grown in the temperate north west Pacific coastal area.In addition to its edible value,Sargassum has medicinal value and has been used for thousands of years in the treatment of lymphatic tuberculosis,edema,indigestion,and qi stagnation.The brown algae,such as Sargassum,contains various bioactive components with health benefits,especially acidic polysaccharides there in.Because of the low price and wide distribution of Sargassum,the processing of Sargassum as a functional food or a drug with low side effects has good research prospects.The content of polysaccharides in Sargassum is high,but the bioavailability of crude polysaccharides is relatively low.In order to further study the polysaccharides of Sargassum fusiforme and improve its biological activity,the crude polysaccharide isolated from S.fusiformis was hydrplyzed by pectinase in combination with glucoamylase,and the biological activity of enzymatic hydrolysate of the polysaccharides was studied.The enzymatic hydrolysate of the polysaccharides were separated and purified,and the purified fractions were also investigated for their biological activities,providing the theoretical basis for the development of Sargassum fusiforme functional foods.The main findings are as follows:1.Crude polysaccharides from S.fusiformis(SFP)were obtained by water extraction and alcohol precipitation.Hydrolysis of SFP was performed in the presence of pectinase and glucoamylase to yield enzymatically hydrolyzed S.fusiforme polysaccharide(ESFP).According to the combination of single factor experiment and response surface analysis,the optimal conditions for enzymatic hydrolysis were obtained:reaction temperature 54.2 ?,enzyme concentration 68.4 U/mL,enzyme activity ratio(pectinase:glucoamylase)3.3:1,reaction time 3 hrs.The polysaccharide degradation degree was determined to be 18.57%.2.Comparison of the chemical components of SFP and ESFP indicated that the total sugar,uronic acid and sulfate content increased after degradation,and the protein content did not change obviously.The results of UV and IR spectroscopy showed that the purity of the sample was good and the enzymatic hydrolysis did not result in the destruction of molecular structure and active groups of the polysaccharide.The molecular weights of SFP and ESFP were measured to be 12.56 kDa and 8.68 kDa,respectively.The monosaccharide composition analysis revealed that SFP and ESFP are mainly composed of Fuc,Gal and Man,and a small amount of Xyl and Glu.3.The in vitro antioxidant activity assay of SFP and ESFP showed that the reducing power and free radical(DPPH,OH,·O-)scavenging abilities of ESFP were significantly higher than those of SFP;The results of in vitro bile acid binding activity of SFP and ESFP showed that both SFP and ESFP had good binding ability with sodium cholate and sodium chenodeoxycholate,but the activity of ESFP was significantly better than that of SFP,equivalent to about 50%of cholestyramine.4.The enzymatically hydrolyzed polysaccharide was separated by cellulose DEAE-52 column and further purified and Sephadex G-100 column,obtaining four polysaccharide fractions which are all acidic polysaccharides,namely ESFP1,ESFP2,ESFP3 and ESFP4.The results of UV scanning showed that there were no obvious absorption peaks at 260-280 nm in ESFP1,ESFP2 and ESFP3,indicating that the impurities such as nucleic acid and protein these three components are very low,while ESFP4 has a very low absorption peak at 280 nm,possibly indicating the presence of a small amount of glycoprotein bound to the polysaccharides;The HPGPC results showed that the peaks of ESFP1,ESFP2,ESFP3 and ESFP4 were single peaks,indicating that they were of good purity.The chemical composition analysis of the four purified components showed that the uronic acid contents of ESFP1,ESFP2,ESFP3 and ESFP4 were 10.12%,7.92%,6.72%and 1.76%,respectively;The protein contents were 0.96%,1.04%,1.10%,and 1.18%,respectively.The sulfate content was 8.76%,9.89%,10.03%,and 10.68%,respectively.The molecular weights of ESFP1,ESFP2,ESFP3,and ESFP4 were determined to be 75.5kDa,86.3kDa,76.2kDa,and 86.5kDa.The monosaccharide composition analysis indicated that these four polysaccharide fractions are mainly composed of Fuc,Gal,Man,as well as a small amount of Glu and Xyl.5.1n vitro antioxidant activity assays of ESFP1,ESFP2,ESFP3 and ESFP4:The decreasing order of ferric reduction ability was ESFP4>ESFP3>ESFP2>ESFP1;the scavenging effect on DPPH was ESFP1>ESFP2>ESFP3>ESFP4 and the IC50 was 0.071,0.228,0.375 and 0.703 mg/mL;O2-radical-scavenging ability:ESFP4<ESFP3<ESFP1<ESFP2,and IC50 values were0.432,0.131,0.225,0.371 mg/mL;OH scavenging ability ESFP1>ESFP2>ESFP3>ESFP4,at a concentration of 5mg/ml,the scavenging rates were 85.4%,78.8%,72.4%,20.7%.6.RAW264.7 cells immunoregulatory activity assay:The effects of six samples(SFP,ESFP,ESFP1,ESFP2,ESFP3 and ESFP4)on the proliferation activity of RAW264.7 cells increased first and then decreased with the increase of concentration,and the activity of ESFP was greater than that of SFP.The activity of each fraction of ESFP was ESFP3>ESFP2>ESFP1>ESFP4.The effects of the six samples on the phagocytic activity of RAW264.7 cells increased first and then decreased with the increase of concentration,and the activity of ESFP was greater than that of SFP.The activity of each purified component of ESFP was(according to the peak value)ESFP2>ESFP 1>ESFP3>ESFP4;the ability of the 6 samples in promoting the generation of NO was found to be concentration-dependent,and the activity of ESFP was greater than that of SFP.The activity of each purified component of ESFP was(according to the peak value)ESFP2>ESFP1>ESFP3>ESFP4.The.best component of cellular immune regulation activity is ESFP2,while SFP is the least active.