Physicochemical Properties And Biological Activity Of Lactobacillus Plantarum Z-1 Extracellular Polysaccharide

Lactic acid bacteria（LAB）are very common and beneficial microbes,which are mostly used in fermented foods.It has a broad application prospect in the food and pharmaceutical industries.The extracellular polysaccharides（EPSs）produced during the metabolism of LABs have antioxidant,antitumour,lipid-lowering,cholesterol-lowering,immunity-enhancing and anti-inflammatory functions,and have a better safety profile than other microbial polysaccharides,so EPSs have wide application prospects in the food and pharmaceutical industries.However,different LABs differ in their ability to produce EPSs,and the biological activities of the yielding EPSs are different.Therefore,it is important to screen the LABs with high EPS-producing capacity.In this study,a strain of Lactobacillus plantarum with high EPS-producing capacity was screened from pickled vegetables,and the fermentation conditions for EPS production were optimized.The polysaccharide obtained from the fermentation broth was isolated and purified;the physico-chemical properties,structural characteristics,antioxidant and anti-inflammatory activities of the purified polysaccharide fractions were investigated.The main results and conclusions of this study are as follows:（1）A strain of Lactobacillus LAB Z-1 with high EPS production,was isolated from cured winter melon and identified as Lactobacillus plantarum by morphological structure,physiological and biochemical analysis and 16S r RNA sequencing.The fermentation conditions were optimized by single-factor experiments and response surface analysis,using the sugar yield of LAB Z-1 as an indicator,and the optimal conditions were determined as follows:incubation temperature of 36.7°C,inoculum size of 2.35%,final concentration of H₂O₂addition of 2.14 m M,and EPS yield increased from 180±0.45 mg/L to 409.52±2.16mg/L.The extracellular polysaccharides（EPS）were purified using DEAE-52 anion-exchange column chromatography and Sephadex G-100 column chromatography to obtain three purified fractions（EPS-0,EPS-1 and EPS-3）.（2）Chemical composition analysis showed that the total sugar content of EPS,EPS-0,EPS-1 and EPS-3 was 41.5±3.18%,37.73±1.83%,36.06±1.12%and 47.19±3.16%respectively;the four polysaccharides contained small amounts of protein,sulphate group and uronic acids.Monosaccharide composition analysis showed that the four polysaccharides were mainly composed of glucose,rhamnose,arabinose and galactose.The molecular weight（Mw）of EPS was 85.4 k Da,Mw of EPS-0 was 25.7 k Da and 1.5 k Da（accounting for 27.90%and 72.10%respectively）,Mw of EPS-1 was 131.3k Da,and Mw of EPS-3 was 93.2k Da,as measured by high performance gel permeation chromatography（HPGPC）.（3）UV-Vis spectroscopy showed that the four fractions contained a small amount of protein but no nucleic acids.FT-IR analysis showed that all four samples had typical characteristic absorption peaks for polysaccharides.¹H NMR spectroscopy showed that all three purified fractions were mainly linked byβ-type glycosidic bonds.Congo red analysis revealed that theλ_maxof EPS and EPS-3 showed a pattern of first increasing and then decreasing,indicating that they might contain an irregular triple helix structure.Scanning electron microscopy（SEM）analysis revealed a dense pore-like structure in each sample.Rheological property analysis showed that the EPS and its fractions exhibited pseudoplasticity（shear thinning）in aqueous solution.Thermogravimetric analysis（TGA）and difference scanning calorimetry（DSC）analysis showed they had good thermal stability.（4）In vitro chemical antioxidant analysis showed that all samples had remarkable free radical scavenging activity,and overall EPS had a better antioxidant capacity than the purified fractions.The analysis of cellular antioxidant activity showed that EPS and its purified fraction could down-regulate ROS and MDA levels and up-regulate SOD and CAT enzymes in hydrogen peroxide-induced oxidative stress RAW 264.7 cells,indicating that they had good antioxidant activity and could reduce the cellular oxidative damage.The results of real-time fluorescence quantitative polymerase chain reaction（RT-qPCR）showed that EPS and its components were able to up-regulate the m RNA expression levels of SOD1 and CAT,and increase the gene expression of enzymes HO-1 and Nrf2 related to oxidative stress in the Nrf-2/HO-1 signaling pathway.Therefore,the effect of antioxidant stress of EPS and its components might be achieved by up-regulating the expression of antioxidant enzyme genes and related genes in Nrf-2/HO-1 signal pathway.（5）A lipopolysaccharide（LPS）-stimulated cellular inflammation model was established to explore the anti-inflammatory activity of EPS and its components.Enzyme linked immunosorbent assay（ELISA）showed that EPS and its components were found to significantly reduce the synthesis and secretion of some pro-inflammatory factors（IL-1β,IL-6,TNF-α）and inflammatory mediators NO in macrophages,and increase the release of the anti-inflammatory factor IL-10.The results of RT-qPCR experiments revealed that EPS and its components could significantly down-regulate the m RNA expression of IL-1β,IL-6,TNF-αand i NOS,and up-regulate m RNA expression of IL-10;and they could also inhibit down-regulate the m RNA expression of TLR4,My D88 and COX-2 Therefore,it is likely that the anti-inflammatory activity of EPS and its components is related to the inhibition on activation of NF-κB/TLR4 signal pathways and COX-2 signal cascade reaction.