Studies On The Immunoregulatory Effect And Structure Of EPS From Different Lactobacillus Plantarum Strains

In recent years,a research hotspot about exopolysaccharide(EPS)of lactic acid bacteria with important theoretical significance and potential application value was studied due to EPS possessing unique physical and chemical properties,resulting in abundant biological functions.Lactic acid bacteria including L.plantarum-4,L.plantarum-12,L.plantarum-14,L.plantarum-37 were chosen as the experimental strains.After EPS extracted and purified,the immunoregulatory activity of EPS was studied with RAW264.7.Finally,the primary structure of EPS was studied by physical and chemical method in order to obtain the differences among the four lactic acid bacteria.The main results are displayed as follows:Firstly,the method of phenol-sulfuric acid,Cooamassie brilliant blue,m-hydroxybiphenyl colorimetric and barium chloride turbidimetric were used to determine the content of saccharide,protein,uronic acid and sulfuric acid in the four EPSs.Among them,the saccharide content of EPS-12 displayed the highest(67.16%)value,the sulfate group content of EPS-37 showed a slight higher value than that of the other three EPSs,and the four EPSs all contained about 5%uronic acid and micro protein.Secondly,proteinthe neutral polysaccharides and acidic polysaccharides were extracted using DEAE-Sepharose Fast Flow ion exchange chromatography column,the acid polysaccharides were eluted using Sepharose CL-6B agarose gel column,and the components were named as EPS-4,EPS-12,EPS-14 and EPS-37,respectively.At the same time,according to the standard curve,the molecular weight of the polysaccharides were 2.33×10~5Da,3.58×10~5 Da,2.89×10~5 Da and 4.45×10~5 Da,separately.Forthermore,the four EPSs were applied to the macrophage RAW264.7 with different concentrations.The results of MTT assay showed that excepting for EPS-4(500?g/mL),the other three EPSs had the most obvious effect on the proliferation of macrophages at 750?g/mL,so the concentrations of 250?g/mL,500?g/mL,and 750?g/mL were selected for subsequent experiments.In the neutral red experiment,it was found that a certain difference of the four EPSs enhanced the phagocytic activity of macrophages to some extent,and a difference was in the degree of inhibition of the phagocytic activity of stimulated macrophages using LPS.Nitrate reductase result showed that,with an increase of polysaccharide concentration,the release of NO in macrophages firstly increased and then decreased,and the four polysaccharides could inhibit the excessive release of NO in macrophages induced by LPS resulting in degrees varied.The effects of the kit on the secretion of TNF-?and IL-10 by macrophages were analysed.The secretion of TNF-?and IL-10 by macrophages offered a different in the four EPSs.All the experiments mentioned,an oustanding increase was existing in the effects of EPS-12 at 750?g/mL.The four EPSs had no absorption peaks at 260 nm and 280 nm,which indicating that no nucleic acids or proteins existed in them.Analysis by HPLC,the content of mannose in EPS-4,EPS-14 and EPS-37 was 55%,followed by glucose.Glucose and mannose accounted for85.07%and 9.18%in EPS-12,respectively.The molar ratios of the four polysaccharides were EPS-4:Mannose:rhamnose:glucuronicacid:glucosamine:glucose:galactose:xylose:arabinose=22.63:0.09:1.65:1.87:15.60:2.38:0.27:0.47;EPS-12:Mannose:rhamnose:glucuronicacid:glucosa mine:glucose:galactose=3.91:0.17:0.32:0.57:49.01:1.27;EPS-14:Mannose:rhamnose:glucuron icacid:galacturonicacid:glucosamine:glucose:galactose:xylose:arabinose=24.13:0.09:1.82:1.71:15.17:2.23:0.39:0.53;EPS-37:Mannose:rhamnose:glucuronicacid:glucosamine:glucose:gala ctose:xylose:arabinose=13.91:0.06:1.08:1.22:10.87:1.43:0.15:0.31.Fourier infrared spectrum scans results showed 3400 cm~-11 and 2937 cm~-11 is for the characteristic absorption peaks of the polysaccharide in that four different kinds of EPSs,and584 cm~-11 is for C=O carboxylate vibration peak And an obvious absorption peak around 1230cm~-11 is for S=O bond symmetric stretching vibration characteristic peak,indicating EPS might contain uronic acid and sulfuric acid root,which is consistent with the previous results of polysaccharide chemical composition analysis,at the same time in the 586 cm~-11 appears peak belonging to symmetric stretching vibration caused by pyranose ring.~1H showed that EPS-12contained 6 glycoside bonds,and the other three EPSs all contained 8 glycoside bonds.