| In our previous study, Enterobacter cloacae Z0206, a bacterial strain, can produce large amounts of exopolysaccharides (EPS), was isolated from nature. The culture medium was optimized under laboratory conditions and EPS was found to have antioxidant, immunomodulatory, antiviral, hypoglycemic and especially hypolipidemic bioactivities. In order to establish a set of fermentation process for industrialized production and further clarify the effect of Z0206polysaccharide on lipid metabolism disorder induced by high fat diet. In the present study, we firstly optimized the industrial fermentation conditions and medium composition of EPS produce by Enterobacter cloacae Z0206with corn starch, soybean meal and corn steep power as substrates. The exopolysaccharide was purified with iron exchange chromatography and gel column chromatography, after that the chemical composition and structure of the main components were analyzed by a combination of Infrared Spectroscopy (IR), high Performance Liquid Chromatography (HPLC) and gas chromatography (GC) as well as nuclear magnetic resonance (NMR) and atomic force microscope (AFM). On the base of this, the effects of EPS produced by Enterobacter cloacae Z0206on lipid metabolism disorder in high fat diet induced C57BL/6mice and TNF-a induced3T3-L1were studied. The main results are as bellows:1. Medium composition Optimization of EPS produced by E. cloacae Z0206The industrial fermentation conditions and medium composition of EPS produce by Enterobacter cloacae Z0206were optimized with corn starch, soybean meal and corn steep power as substrates.The optimization of fermentation conditions and the culture medium for the production of EPS from Enterobacter cloacae Z0206was studied using one-factor-at-a-time method, orthogonal matrix method and response surface methodology (RSM) with agroindustrial by-products as the main fermentation substrate. The results of one-factor-at-a-time test showed that the best conditions for title bacterial growth and EPS production were: fermentation temperature30℃, speed250rpm and initial pH7.0. The suitable carbon and complex nitrogen sources were corn starch and soy bean meal, corn steep powder and KNO3respectively. The concentration of inorganic salts and complex nitrogen sources were optimized by orthogonal matrix design. At last a three level, three factor Box-Behnken factorial design was used to optimize the level of three factors significantly affecting the yield of EPS. The optimal medium composition was determined as follows:240.95g/L cornstarch,22.19g/L soybean meal,6.61g/L corn steep powder,3g/L K2HPO43H2O,3g/L KH2PO4and1g/L CaCl2, with a corresponding yield of27.75g/L, which was approximately two times higher than that in the basal medium.2. Purification, chemical composition and structure analysis of EPSOn the basis of optimization of fermentation conditions and culture medium, Z0206polysaccharide was further purified and its chemical composition and structure was analyzed. Crude exopolysaccharides (CEPS) were obtained from the fermentation broth by ethanol precipitation, EPS was obtained through deproteinization, decolorization and dialysis. The deproteinized exopolysaccharides were further purified through a DEAE-52iron exchange chromatography column and a Suprose12gel column. Two main components (EPS-1and EPS-3) were isolated by eluting with distilled water and a gradient of0-0.5mol/L NaCl solution, their yield was40.8%and26.7%respectively.The gel permeation chromatography (GPC) analysis showed that the average molecular weights of EPS-1and EPS-3was2527Da and1.23×106Da respevtively. The neutral sugars, pyruvyl groups and glucuronic acid were analyzed using GC, reversed-phase high-performance liquid chromatography (RP-HPLC) and the carbazole-sulfuric acid method. EPS-1was found to be composed of glucose, and EPS-3was composed of fucose, galactose, glucose, pyruvic acid and glucuronic acid with the relative molar ratio of1.65:1.83:1:1.62:1.20. IR analysis showed that both EPS-1and EPS-3contained-CH2-groups. NMR analysis indicated that EPS-1and EPS-3contained a and β type glycosidic linkages and EPS-3also showed uronic acid carboxyl resonance, methyl groups of the ketal-substituted pyruvate residue and methyl groups of fucose. The average particle size distribution of EPS-1was50.57nm and EPS-3was210.3nm. The photograph of EPS-1and EPS-3fraction were obtained by atomic force microscope and showed that molecules of EPS-1and EPS-3are gathered up with each other in a tight manner. 3. Effect of Z0206polysaccharide on the lipid metabolism disorder in obese miceOn the base of structure analysis of EPS produced by Enterobacter cloacae Z0206, the effects of CEPS and EPS on the lipid metabolism disorder as well as insulin resistance in high fat induced C57BL/6mice were studied. C57BL/6mice were divided into two groups at the age of6weeks, normal diet (ND) group and high fat diet (HFD) group, feeding normal diet (10%fat kcal%) and high fat diet (45%fat kcal%) respectively for12weeks. Mice in HFD group were then allocated to HFD group, Z0206crude polysaccharide group (HFD+CEPS) and Z0206deproteinization polysaccharide group (HFD+EPS). CEPS and EPS were administered to C57BL/6mice for a period of42days, and the same volume of saline solution were administered to mice in ND and HFD groups. The results showed that administration of CEPS and EPS could significantly decrease the body weight gain, fasting glucose concentration and serum insulin levels, whereas improved the insulin sensitivity index (ISI) and OGTT compared with the HFD group. In addition, CEPS and EPS significantly decreased the liver index, adipose tissue index of subcutaneous fat, retroperitoneal fat and epididymal fat, indicating that CEPS and EPS can inhibit the excessive fat deposition and improve the insulin sensitivity. At the same time, serum free fatty acid (FFA) content, cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL-c) levels were significantly decreased in CEPS and EPS treated mice. Morphology observation shows that CEPS had significant protective effects in the integrity of the organizational structure of liver and the size of adipocyte in epididymal adipose tissue were significantly decreased in CEPS treated mice, which indicated that CEPS ameliorated adipocyte hypertrophy phenomenon in epididymal fat. The results of RT-PCR and western blotting showed that CEPS and EPS could significantly increase the gene expression of insulin-sensitizing factors like adiponectin, vaspin and visftin, while reduce the gene expression of insulin resistance factors like leptin and resistin. At the same time, the expression of PPARy and its target genes, glucose transport protein4(Glut4), insulin receptor substrate1(IRS-1) and Aktl were upregulated in the groups supplemented with CEPS and EPS, which indicated that CEPS and EPS increased the insulin sensitivity of high fat diet induced mice, sequentially regulated the energy metabolism.The above results suggested that CEPS and EPS could enhance the capacity of glucose uptake, relieve fatty degeneration and adipocyte hypertrophy in high fat diet induced mice, thus alleviate insulin resistance. They maintained the lipid metabolism metabolic balance and improved the lipid metabolism disorder by regulating the expression of adipokines and blood lipid.4. The immune mechanism of alleviation effect of Z0206polysaccharide on lipid metabolism disorder.On the basis of the above experiments, the insulin-sensitizing mechanism of Z0206polysaccharide was further studied from the aspect of adipose inflammation using immunohistochemistry, flow cytometry, RT-PCR and Western blotting. The results showed that CEPS and EPS could reduce the F4/80positive macrophages and the ratio of CD11positive cells, and as well as the numbers of crown like structure (CLS) in epididymal adipose tissue compared with the high fat diet induced mice. We also found that treatment with CEPS and EPS markedly reduced the M1macrophages markers, such as F4/80, CDllc and MCP-1, whereas increased the M2macrophages markers, such as CD209, CD163and Mgl2, which suggested that CEPS and EPS induced reduction in the number of adipose tissue macrophages (ATM) as well as the shift toward M2polarity. A gene expression study showed that CEPS and EPS could improve the adipose tissue inflammation via decreasing the expression of TNF-α IL-6and increasing the expression of IL-10. In vitro, the inflammation response in TNF-a induced3T3-L1adipocytes was also attenuated by CEPS and EPS through down-regulating the production of NO and expression of TNF-α、IL-6、IL-1β, while up-regulating the expression of IL-10. CEPS and EPS probably alleviated chronic inflammation in adipose tissue through down-regulation of TLR4expression, suppression of IκBcα phosphorylation, subsequent NF-κB activation, as well as phosphorylation of c-Jun.The above results suggested that Z0206polysaccharide reduced the macrophages infiltration and modulated adipose tissue macrophage polarization. On the other hand, Z0206polysaccharide improved adipose inflammation and prevented the lipid metabolism disorder by inhibiting the TLR4/IκBα/NF-κB pathway and c-Jun pathway... |
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