Study On Extraction,degradation And Antioxidant Activity Of Polysaccharide From Grateloupia Livida

Grateloupia livida is a relatively common genus of Grateloupia genus algae,also known as miancai,fozucai?Guangdong?.Grateloupia livida polysaccharides have various biological activities such as anti-oxidation,anti-tumor,lowering blood pressure and so on,but because of its large molecular weight,its role in cells is limited.In this paper,the Grateloupia livida was used as raw material,and its nutrient composition was analyzed and evaluated.The extraction,purification,degradation,separation,physicochemical properties and antioxidant activity of polysaccharides were studied in order to make rational use of Grateloupia livida and provide theoretical reference.The main research contents are as follows:Part 1:Analysis and evaluation of nutrients of Grateloupia livida1.The basic nutrients of Grateloupia livida were determined and analysed by conventional chemical methods.The results showed that Grateloupia livida has high protein,low fat,with the content of crude protein and crude fat was 12.28%,0.18%,respectively.The ash and crude fiber were 13.32%and 1.80%,respectively.2.The amino acid content of Grateloupia livida was determined and analysed by liquid chromatography.The results showed that Grateloupia livida contained 17 kinds of amino acids,including 8 essential amino acids,and 9 non-essential amino acids.The ratio of essential amino acids to total amino acids was 42.58%,the ratio of essential amino acids to non-essential amino acids is 74.15%,which is in line with the ideal protein model recommended by WHO/FAO.Amino acid score?AAS?and chemical score?CS?of amino acids of the Grateloupia livida were revealed that the first limiting amino acid of the Grateloupia livida was tryptophan?Trp?and the second limiting amino acid was lysine?Lys?.The amino acid ratio coefficient score?SRC?of the Grateloupia livida was closer to 100,which indicated that the nutritional value of the Grateloupia livida was higher and the amino acid composition ratio was more balanced.3.The fatty acid content of Grateloupia livida was determined and analysed by gas chromatography.The results showed that Grateloupia livida contained 15 kinds of fatty acids,including octadecadienoic acid,arachidonic acid,eicosapentaenoic acid and docosahexaenoic acid,which have important physiological functions,with the mass fractions of 3.07%,18.97%,0.40%and 1.01%,respectively.4.The content of mineral elements in Grateloupia livida was determined and analysed by ICP-MS.The results showed that Grateloupia livida was rich inmagnesium?Mg?,calcium?Ca?,iron?Fe?,zinc?Zn?and copper.Cu),with the contents of 9267.25,582.00,98.18,45.36 and 3.27 mg/kg,respectively.Grateloupia livida could be used as food source of trace elements.Part ?:Extraction and purification of polysaccharides1.Through single factor test and response surface test,the technological conditions of extraction of polysaccharides from Grateloupia livida by hot water were determined.The optimum technological conditions were as follows:material-liquid ratio 1:37?g/ml?at 100?for 4 h.Under this condition,the yield of polysaccharide was 15.23%.After analysis,the effect of time on the polysaccharide yield was the most significant,followed by the ratio of material to liquid,and the last was temperature.2.Deproteinization of polysaccharides from the Grateloupia livida was studied.The methods of deproteinization,such as Sevag method,TCA method and enzymatic hydrolysis-Sevag method,were compared.Three methods were evaluated based on protein removal rate and polysaccharide loss rate.It was found that the enzymatic hydrolysis-Sevag method had the best deproteinization effect.The protein removal rate was 91.88%and the polysaccharide loss rate was 22.88%.3.Decolorization of polysaccharides from Grateloupia livida was studied.Two decolorization methods of H₂O₂ and D941 macroporous adsorption resin were compared.The decolorization rate and polysaccharide loss rate were used to evaluate the two methods.It was found that D941 macroporous adsorption resin method had the best deproteinization effect.The decolorization rate was 92.83%and the loss rate of polysaccharide was 14.07%.Part ?:Optimization of Polysaccharide DegradationThe degradation of GLP was obtained by microwave assisted with H₂O₂-Vc method to obtain degraded polysaccharide?DGLP?.The three single-factor tests were carried out on microwave temperature,H₂O₂ concentration and time.In order to obtain the highest DPPH free radical scavenging rate of the polysaccharide,the study chose three factors to the response surface experiment with the microwave temperature of 40,50,60?,H₂O₂ concentration of 2,3,4 mM,the degradation time of 20,30,40 min.After the test,It was found that the best process conditions were as followed:the microwave temperature was 54?,the concentration of H₂O₂ was 3 mM and the reaction time was 34 min,the scavenging rate of DPPH radical was 71.39%.Part ?:Separation of Degraded Polysaccharides and Analysis of Their Physicochemical Properties1.The DGLP was separated by a Sephadex G-200 column to obtain two single components,which were named DGLP-1 and DGLP-2,respectively.2.The total suger cntents of GLP,DGLP,DGLP-1 and DGLP-2 were 73.46%?85.09%?92.72%and 68.76%,respectively.The uronic acid contents were 12.99%,11.70%,14.50%and 6.44%,respectively.The sulfate contents were 25.68%,24.31%,25.61%and 25.18%,respectively.The molecular weights of GLP,DGLP,DGLP-1 and DGLP-2 were 23044.3kDa,945.2kDa,158.5kDa and 18.1kDa respectively.The monosaccharides of the four polysaccharides were mainly galactose and they contained the same monosaccharide type,but the contents were slightly different.3.According to the infrared spectroscopy analysis of GLP,DGLP,DGLP-1 and DGLP-2,they all had strong absorption peaks at 3200³500 cm^-1,which were the characteristic peaks of O-H;the absorption peaks of 2919²941 cm^-1 indicated the stretching of C-H;the absorption peaks of 1412 cm^-1 and 1648 cm^-1 represent C=O-C-O stretching;1020 cm^-1 corresponds to C-O-H deformation vibration with similar characteristic peaks of sugar;In addition,two peaks at 1257 cm^-1 and 1074 cm^-1 indicate the presence of sulfate,meaning that the four polysaccharides all contain sulfuric acid groups.Part ?:Antioxidant Activity of Polysaccharides and Their Degradation ProductsDPPH free radical scavenging rate,reducing power,hydroxyl radical scavenging rate and superoxide anion scavenging rate were determined at different concentrations of GLP,DGLP-1 and DGLP-2.The results showed that both GLP,DGLP-1 and DGLP-2 had good antioxidant activity,and DGLP-2 had excellent antioxidant activity in vitro.