Isolation And Purification Of Exopolysaccharide From Lactobacillus Rhamnosus And Its Immunomodulatory Effects

Lactic acid bacteria are important probiotics in the human intestine and play an important role in maintaining the stabilization and balance of the intestinal microecology.Their secondary metabolites,exopolysaccharides（EPS）,have shown excellent performance in improving food quality with sensory,antioxidant,antitumor,hypolipidemic,and immune enhancing properties.Recently,more and more attention has been paid to the toxic side effects caused by chemosynthetic drugs used for the immunomodulation and treatment,and EPS from lactic acid bacteria have attracted more attention because of their highly efficient immunomodulatory activity,green and safe advantages.However,the application of EPS has been limited due to the following reasons:the culture medium of lactic acid bacteria,such as the raw material of MRS culture,often contains some components that interfere with the analysis of EPS;the low production of EPS using other culture media;and the fact that current studies on the immunomodulatory mechanisms of EPS from lactic acid bacteria are not sufficiently in-depth.In order to sort out these problems,in this study,a strain of Lactobacillus rhamnosus ZFM216with potential high exopolysaccharide producing ability was isolated from fresh milk.A suitable medium and fermentation process have been developed and used for the production of EPS,which do not interfere the quantification of EPS.The obtained EPS has been separated and purified,and its chemical composition and structure are analyzed to reveal its primary structure.The immunomodulatory activity of EPS has been validated through animal experiments.Systematic correlation study has been conducted to reveal the mechanism of immunomodulatory effects from the aspects that EPS affects the microecology of intestinal flora,as well as its antioxidant activity and immunomodulatory effects.The findings will provide a theoretical basis for the development of lactic acid bacteria EPS-related functional foods and drugs.The main findings of the study are summarized as follows:1.The present study has demonstrated that the presence of polysaccharides in MRS would interfere the quantification of EPS secreted by lactic acid bacteria.The polysaccharides in MRS are mainly derived from beef extract,yeast extract,and peptone.After the raw materials were subjected to the removal of polysaccharides,a suitable culture medium（M-MRS）for the production of EPS by L.rhamnosus ZFM216 was developed by single factor test,Plackett-Burman test,Box-Behnken test and response surface optimization.The composition of M-MRS was as follows:7.5 g L^-1 yeast extract（before removal of polysaccharide）,12.5 g L^-1 beef extract（before removal of polysaccharide）,10 g L^-1 peptone（before removal of polysaccharide）21.23g L^-1 maltose,5.51 g L^-1 YNB,2 g L^-1 K₂HPO₄,5 g L^-1 anhydrous sodium acetate,2 g L^-1ammonium citrate,0.58 g L^-1 Mg SO₄·7H₂O,0.25 g L^-1 Mn SO₄·H₂O,1 ml Tween-80)at an initial p H of 6.5.The optimized process for EPS production was as follows:1%inoculum size,37℃culture temperature and 20 h culture time.Under these conditions,the production of EPS of L.rhamnosus ZFM216 reached 496.64±3.15 mg L^-1,which was 76.70%higher than that under the non-optimized condition(281.07±5.90 mg L^-1).2.The EPS of L.rhamnosus ZFM216 was separated and purified by cellulose DEAE-52and Sephadex G-100 column chromatography successively.The neutral polysaccharide component EPS1 and acidic polysaccharide component EPS2 were obtained,with the yield of48.88%and 25.70%,respectively.Ultraviolet spectrum scanning showed that EPS,EPS1 and EPS2 contained almost no protein,nucleic acid and pigment.Chemical composition analysis showed that the total sugar content of EPS,EPS1 and EPS2 was more than 70%,containing a small amount of sulfate;among the three polysaccharides,and EPS contained the most content of uronic acid,EPS2 the next,and no uronic acid was detected in EPS1.The molecular weights of EPS,EPS1 and EPS2 were determined as 32.2,17.6 and 17.3 k Da,respectively.Congo red test showed that EPS and EPS2 had a triple helix structure,and such structure was not found in EPS1.XRD,SEM and AFM analysis showed that EPS1 and EPS2 were amorphous substances;the surface of EPS1 was smooth and porous,while the surface of EPS2 was rough and porous;EPS1 solution presented a linear short rod shape,and EPS2 presented a sphere aggregation state.FT-IR spectrum analysis showed that EPS1 and EPS2 consisted of pyranoses,containing bothαandβtype glycosidic bonds.Monosaccharide composition and NMR analysis showed that EPS1 was a dextran composed of glucose,in which（1→4）-α-Glcp and（1→4,6）-α-Glcp might be the connection mode of the main chain,and the main branch chain was connected by（1→）-α-Glcp,and（1→6）-β-Glcp;EPS2 was composed of glucose,galactose and galacturonic acid and the molar ratio was 48.2:1.5:1.（1→4）-α-Glcp,（1→4,6）-α-Glcp and（1→4,6）-β-Glap might be the connection mode of the main chain,the main branch chain being connected by（1→）-α-Glcp,（1→6）-β-Glcp and（1→）-β-Glap.3.Animal experiments showed that continuous gavage with EPS produced by L.rhamnosus ZFM216 at a dose of 300 mg kg^-1 BW（high dose）for 11 days was able to obviously elevate the immune function of CTX-induced immunosuppressive mice,being more effective than that of lentinan at 200 mg kg^-1 BW（positive control）.The specific manifestations were as follows:a high dose of EPS significantly increased the body weight of immunosuppressed mice;significantly improved the immune organ index and proliferation of splenic lymphocytes;ameliorated the lesions and morphology of immune organs caused by CTX;promoted the repair and villus growth of damaged intestinal cells;significantly elevated the levels of hemolysin,TNF-α,INF-γand IL-10,the activities of SOD enzymes and CAT enzymes in the serum;significantly reduced the serum levels of MDA;significantly increased the phagocytic index of murine macrophages and reduced the intensity of delayed type allergy;elevated the levels of short chain fatty acids such as acetate,propionate,butyrate,and valerate in the gut.4.The results of diversity analysis of intestinal flora in mice showed that compared with the normal group,in model group,Bacteroidota increased significantly and Firmicutes decreased significantly at the phylum level;Bacteroidia increased significantly,Clostridia decreased significantly at the order level;Bacteroides increased significantly,Trichospirillae,Vibrionales,Clostridia＿UCG-014 decreased significantly at the class level;Muribagulaceae and Prevaliaceae increased significantly,and Treponemaceae,norank＿o＿Clostridia＿UCG-014,Physiobacteriaceae,Ruminococcaceae and Bacteroides all decreased significantly at the family level;bothαandβdiversities of the flora declined and changed significantly.After continuous gavage with EPS（high dose）of L.rhamnosus ZFM216,the level of intestinal flora diversity and community structural composition of mice can recover to the normal level,o＿Oscillospirales,f＿Ruminococcaceae、f＿Hungateiclostridiaceae were significantly different from the model group,which may play an important role in regulating the recovery of flora and maintain the intestinal micro ecological environment.Analysis of the SCFAs environmental factor indicated that Clostridium＿methylpentosum＿group,Ruminocochaceae,Anaerovoracaceae,Christensenellaceae,Hungateiclostriciaceae,Prevotellaceae are positively related to the formation of short chain fatty acids such as intestinal acetic acid,butyric acid and valeric acid.Based on the significant difference of species in the EPS high-dose group,speculated that EPS may be metabolized by the above flora to generate short chain fatty acids such as acetic acid,butyric acid and valeric acid,change the intestinal microenvironment,affect the intestinal flora structure,and play an immune enhancing role.The predictive function correlation analysis found that EPS had significantly enhanced the cytoskeleton,cell movement,transcription,signal transduction mechanism and replication,recombination and repair functions.The above functions were closely related to immune regulation,revealing the possible mechanism of EPS on immune regulation of immunosuppressed mice in terms of influencing intestinal flora.5.Chemical antioxidant assays showed that EPS,EPS1 and EPS2 possessed marked radical（DPPH,hydroxyl and superoxide anion radicals）scavenging ability and reducing power,and the overall performance was EPS>EPS2>EPS1.EPS,EPS1 and EPS2 were found to be non-toxic to macrophage RAW 264.7,and had a certain proliferation-promoting ability.These polysaccharides possessed a good protective effect on H₂O₂ induced oxidative stress cells,significantly reduced the ROS level,increased the activity of SOD and CAT enzymes,and reduced the content of MDA in the oxidative stress cells.At 500μg m L^-1,the cellular antioxidant capacity followed the order:EPS>EPS2>EPS1.Antioxidant mechanism investigation indicated that EPS could significantly increase the expression level of SOD1 and CAT m RNA,and improve the activity of SOD and CAT enzymes.EPS affected the Nrf-2 signal pathway by up regulating the m RNA expression of Nrf-2 and HO-1 in RAW 264.7 cells.6.The results of in vitro immune activity test showed that EPS,EPS1 and EPS2 could effectively enhance the phagocytic activity of macrophages,stimulate macrophages to produce NO and secrete TNF-α、IL-1β、IL-6 cytokine.It has been demonstrated that the immune regulation function of EPS,EPS1 and EPS2 samples was completely derived from polysaccharides themselves,rather than from the interference of endotoxin LPS,their immunoregulatory ability followed the order:EPS>EPS2>EPS1.By detecting the expression of EPS on macrophage immune related genes,it was found that EPS could promote NO release and TNF-α,IL-1βand IL-6 cytokine secretion by up-regulating the m RNA expression of i NOS,TNF-αand IL-1β.Using inhibitor method,Western Blot,immunofluorescence and other technical means,EPS has been demonstrated to realize the immune activation of macrophages RAW264.7 by activating TLR4/MAPK/NF-κB signal pathway.