The Studies on the Purification,Structure Identification and Its’s Immunity Activity of Polysaccharides Isolated from Oat Lao-chao

Author:A Rong

Supervisor:zhang mei li


Degree Year:2019





Lao-chao is a traditional fermented food which has a long history in China and was popular among the folk.At present,the varieties of lao-chao increased to satisfy the needs of different groups of people.Because the low alcohol content,rich nutrition and unique flavor,it has greatly promoted the industrial development of the lao-chao.However,there are few studies focus on bioactivity of lao-chao.This paper studied optimization of manufacturing oat lao-chao,extraction of polysaccharide and removal of its protein,separation and purification of polysaccharide.The separation and purification process based on anion exchange chromatography DEAE-sepharose FF and superdex G-100 gel filtration,molecule size of polysaccharide was determined by high performance gel premeatioin chromatography(HPGPC).The monosaccharide composition analyzed by gas chromatography.The structure of polysaccharide was analyzed by infrared scanning and 1DNMR and 2DHMR.We evaluated the oxidation resistance of polysaccharide and antitumor activity on cancer cell and immunoregulation activities of polysaccharide in vitro.The main results of our study are as follows:1.Lao-chao was fermented with different particle sizes of oats and prosco millet,by measuring total sugar,reducing sugar,polyphenol content,total antioxidant activity,pH value and sensory evaluation,80 mesh’s products were best.The optimum ratio of solid-liquid was 1:3,adding portion of prosco oat and millet was 3:7,and the ratio of starter was 8%.2.Based on the single factor experiment,the optimal alcohol sedimentation process parameters of lao-chao polysaccharides were established by orthogonal experimental design:ethanol concentration was 75%,alcohol precipitation pH was 8,and alcohol precipitation time was 20h.The results of orthogonal test on the basis of single factor showed that the optimal optimization conditions were as follows:ethanol concentration was 85%,alcohol precipitation pH was 8,and precipitation time was 20 h.Under this condition,the extraction amount of polysaccharide was 11.09 g/L.3.The results of the single factor experiment showed that the volume ratio of the best adding portion of polysaccharide solution and sevage agent was 1:1;the ratio of the best chloroform to n-Butanol was 5:1;the best shaking time was 15min;the times of getting rid of protein was 3 times.After the final four-factor and three-level orthogonal experiments,the optimal process conditions were obtained:portion of polysaccharide solution and sevage agent was 3:2,the volume ratio of chloroform to n-Butanol was 5:1,the shaking time was 20minutes.The times of protein removals was 2 times.4.The crude polysaccharide of oat lao-chao was separated and purified by DEAE-Sepharose FF column and superdex G-100 gel purification system.The optimum elution rate was 1.0mL/min,and the optimal elution concentration gradient of NaCl buffer was 0→0.2 mol/L→0.4 mol/L→0.6 mol/L,and the optimal injecting concentration of polysaccharide is 10mg/mL.After the final expansion of the NaCl buffer concentration to 2.0mol/L,three white polysaccharide components LCPS-1,LCPS-2 and LCPS-3 were obtained;After purification by the superdex G-100 polysaccharide gel purification system,only LCPS-2 was obtained.The elution peak showed a single,symmetric normal distribution by HPGPC,a high-purity polysaccharide component was obtained with molecular weight of 49.99 KDa.The monosaccarides compositoin of LCPS-2 was analyzed by GC-MS,arabinose,xylose and galactose are the main components of LCPS-2,a small amount of polysaccharide were glucose and mannose;β-glycosidic bond configuration contained in sugar residue.FT-IR and NMR,LCPS-2 contains fragment of→5)-α-L-Araf-(1→α-L-Araf-(1→3,6)-β-D-Galp-(1→.5.In vitro anti oxidant activity of oat lao-chao polysaccharides showed that the strong and weak order of antioxidant strength was LCPS-2>LCPS-1>crude polysaccharide.Within the experimental range,DPPH free radical scavenging ability,hydroxyl radical scavenging ability and total antioxidant capacity gradually increased during the increasing concentration of polysaccharide.Among polysaccharides of oat lao-chao LCPS-2 had the strongest ability to scavenge DPPH free radicals.When the concentration reached 600μg/mL,the DPPH free radicals,hydroxyl radicals and total antioxidant capacity reached 90%,31%and 20.6 U/mL,respectively.After the selection of LCPS-2,which has the strongest antioxidant capacity,we co-cultured colon cancer cell line Lovo in vitro with LCPS-2,it was found that LCPS-2 had no inhibitory effect on Lovo cell growth.6.Immunomodulatory activity of lao-chao polysaccharides:The immunological activity of LCPS-2 polysaccharide fraction was evaluated by in vitro cellular immunoassay.The results showed that purified polysaccharide LCPS-2 could promote the proliferation of mouse spleen lymphocytes in the range of 50~400 μg/mL.The ability of NO phago cytosis could be enhanced in the experimental range,and the content of NO was significantly increased(P<0.01).The levels of the IFN-y and IL-2 in supertanant of the mouse spleen lymphocyte LCPS-2 group were higher than those in the blank control group(P<0.05)and NK cell killing activity in the supernatant of mouse spleen lymphocyte were also higher than those in the blank control group(P<0.05).The levels of CD3+ subset group was increased significantly when concentration of LCPS-2 was 200 μg/mL(P<0.05),the levels of CD4+and CD8+ subset groups increased not significantly.