Study On The Effect Of Autolysis On Sea Cucumber Polysaccharides And Quantitative Method Of Acidic Polysaccharides

Polysaccharides are an important nutritional ingredient in seafood,and they are also an important index of quality control and nutrition evaluation.Autolysis is the major stress response of sea cucumber and other seafood,and it has not been reported whether or not the active polysaccharides from sea cucumber will be affected in the process of autolysis.It is difficult to quantify acidic polysaccharides,and there are still some drawbacks of the existing methods in accuracy and the operations.Therefore,in this study,the effects of autolysis on polysaccharides of sea cucumber were studied,and a method to quantify acidic polysaccharides was established.The main research contents and results were as follows:(1)The content of sulfated polysaccharides and reducing sugar in sea cucumber samples after autolysis treatment and protease enzymatic hydrolysis were determined by chemical colorimetry,the chemical composition,molecular weight distribution and viscosity of sea cucumber polysaccharides obtained from autolysis treatment and protease enzymatic hydrolysis were compared by IR,1H NMR,HPLC-MS/MS and chemical colorimetry.It had been found that the efficiency to release polysaccharides by autolysis treatment was significantly lower than that by protease enzymatic hydrolysis,Polysaccharides obtained through autolysis treatment have more protein chains,but the degree of destruction of the polysaccharides chain structure by autolysis treatment was lower.Chemical groups of sea cucumber polysaccharides obtained through autolysis treatment(Ap-SCP)were similar to those through protease enzymatic hydrolysis(P-SCP),but there were some differences in monosaccharide composition.The monosaccharide composition and proportion of Ap-SCP were mannose:glucuronic acid:glucose:galactose:fucose = 1.1:1.0:1.7:1.1:8.6,and those of P-SCP were mannose:glucosamine:glucuronic acid:galactosamine:glucose:galactose:fucose = 1.0:1.5:1.7:1.5:3.6:2.4:16.0.The ratio of sea cucumber chondroitin sufate(SC-CHS)and sea cucumber fucan(SC-FUC)was further determined,as 0.30 for Ap-SCP and 0.59 for P-SCP.The molecular weight distribution of P-SCP and Ap-SCP was different.The molecular weight of SC-CHS from P-SCP was 280 kDa,and the molecular weight of SC-FUC from P-SCP was more than 700 kDa.While SC-FUC was the main component of Ap-SCP with its molecular weight of more than 700 kDa.The aqueous solutions of P-SCP and Ap-SCP were all pseudoplastic fluids.However,the viscosity of the aqueous solution of P-SCP was slightly higher than that of Ap-SCP,while the aqueous solution of P-SCP was more sensitive to shear force than Ap-SCP.To conclude,autolysis treatment can release sea cucumber polysaccharides,but the efficiency was lower than protease enzymatic hydrolysis.Moreover,Ap-SCP had higher proportion of SC-FUC,and hysical and chemical properties were different from those of P-SCP.(2)A method to quantify acidic polysaccharides including hyaluronic acid(HA),chondroitin sulfate(CS)and ablone gonad sulfated polysaccharide(AGSP)was established by determining the contents of their disaccharides produced from the acid hydrolysis using hydrophilic interaction chromatography-tandem triple quadrupole mass spectrometry in the positive mode.The instrument was operated with multiple reaction monitoring(MRM)mode.The transitions 379/203 and 356/162 were selected to quantitatively analyze the disaccharides generated from HA,CS and AGSP,respectively.And then the content of acidic polysaccharides could be detected effectively.The results demonstrated that all of the three acidic polysaccharides had excellent linearities in a concentration range of 0.02-2.00 mg/mL with the recoveries for these acidic polysaccharides were all 95%-112%and relative standard deviations(RSDs)were less than 5%.Limits of detection(LODs)were 0.1 ?g/mL-1.2 ?g/mL and limits of quantification(LOQs)were 0.3 ?g/mL-4.1 ?g/mL.It could be inferred that this method was feasible to quantitatively detect HA,CS,AGSP in samples within the detection range.Compared with other quantitative methods,this method was more convenient,faster,and more sensitive and more specific.It showed great significance in the quantitative detection of acidic polysaccharides.