Font Size: a A A

Optimization Fermentation, Purification, Stuctureal Elucidation And Bioactivity Of Exopolysaccharides From Paecilomyces Hepiali

Posted on:2015-11-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z W WuFull Text:PDF
GTID:1220330482470983Subject:Bio-engineering
Abstract/Summary:PDF Full Text Request
Paecilomyces hepiali, a parasitic fungus generally found in the most valued traditional Chinese herbs of Cordyceps sinensis (DongChongXiaCao), is one of most important fungal species during the maturation of C. sinensis. Since containing similar chemical constituents to wild C. sinensis, attempts have been made to produce Cordyceps materials as succedaneum of natural C. sinensis with the consideration of the resource rarity and economical reasons by applying artificial cultivation. In the past decades, more attentions have been focused on the structure and bioactivity of the active compounds present in Cordyceps. However, extensive researches were laid on the polysaccharide from mycelium (IPS) or natural C. sinensis, only few studies have conducted on the exopolysaccharide (EPS) in the fermentation broth. Compared to IPS, EPS exhibited more advantages such as higher dissolubility, easier to separate and control in the fermentation process. In this thesis, the fermentation conditions of P. hepiali for higher production of EPS (PHEPS) was optimized, and then the resulting EPS was separated and the structure of purified polysaccharide fractions were identified. Furthermore, their bioactivities including the gut microbes modulation, immuno-regulation and the hepatoprotective activity against alcohol-induced liver injury were also studied. The main results are listed as follows:1. Optimization of submerged fermentation conditions for PHEPSFirstly, the effects of different nutrition factors of carbon sources, nitrogen sources, minerals and vitamins on the growth of mycelia and the production of PHEPS were compared by single factor experiment. The results showed that the eight nutritional factors including sucrose, yeast extract, (NH4)2SO4,KH2PO4, CaCl2, MgSO4, potato extract and malt extract could bring positive effects both on the growth of mycelia and the production of PHEPS. Furthermore, a fractional factorial design (FFD) was employed to screen the significant factors from the eight nutritional factors obtained from single factor experiments and the four factors of sucrose, yeast extract, (NH4)2SO4, KH2PO4 were selected. After that, a central composite design (CCD) was applied to optimize their fittest concentrations in the medium. As results, the maximum production of mycelia (12.98±0.14 g/L) and PHEPS (5.33±0.11 g/L) were achieved under the optimal medium of sucrose 46.08 g/L, yeast extract 4.71 g/L, (NH4)2SO4 5.72 g/L, KH2PO4 1.70 g/L, CaCl2 0.50 g/L, MgSO4 0.50 g/L, potato extract 1% and malt extract 1%.2. Separation, purification, and physicochemical properties of PHEPSIn the present study, the effects of carbon sources on the growth of mycelium, the production of EPS from Paecilomyces hepiali HN1, chemical composition, molecular weight distribution and monosaccharide composition of PHEPS were investigated. As results, the chemical compositions and monosaccharide ratios in PHEPS were significantly varied by the carbon sources used. Feeding with glucose or mannose promoted the biosynthesis of higher molecular weight PHEPS (> 1000 kD), but feeding with lactose or mannose+lactose did not increase the ratio of galactose in PHEPS.The polysaccharide in the fermentation broth produced with sucrose was firstly isolated by ultrafiltration and the polysaccharide molecular ranging from 10 kD to 1000 kD were collected. Then, they were sequentially purified by DEAE-52 cellulose ion-exchange chromatography and Sephadex G-100 size-exclusion chromatography and the three polysaccharide fractions of PHP-1, PHP-2 and PHP-3 were obtained. The molecular weight of them were determined by HPGPC as 1.78×105,2.32 ×105,1.19×105 Da, respectively. The monosaccharide molar ratios of PHP-1, PHP-2 and PHP-3 were determined as (Man:Rib:Rha:Glc:Gal:Ara= 53.62:3.20:3.51:13.10:14.17:2.24), (Man:Rib:Rha:GlcUA: GalUA:Glc:Gal:Ara= 55.26:3.05:2.85:0.73:0.58:14.08:15.52:4.47), (Man:Rib:Rha: GlcUA:GalUA:Glc:Gal:Xyl:Ara= 25.60:3.78:7.30:6.75:4.43:13.76:30.05:1.35:7.84), respectively; Moreover, their contents of neutral sugars, protein, uronic acid, phosphate and UV spectrum were determined and found that there were some differences among them. The structural characteristics of PHP-1, PHP-2 and PHP-3 were elucidated with infrared spectroscopy (IR), methylation, gas chromatography-mass spectrograph (GC-MS), 1D and 2D NMR. Its backbone was composed oâ†'2,6)-a-D-Manp-(1â†', and branching occurred at O-2 position of (1â†'6)-α-D-mannose residue of the backbone with â†'2)-a-D-Manp-(1â†', and terminated with a-D-mannose and β-D-galactose.3. The modulation of PHP-1, PHP-2 and PHP-3 on the gut microbial communityThe digestion of the EPS of PHP-1, PHP-2 and PHP-3 were studied in vitro by employing a simulated gastric and upper gastrointestinal model and analyzing molecular weight variation of digested products at different digestion times. The results indicated that the three polysaccharide fractions can resist the degradation of artificial gastric and upper gastrointestinal digestive juice in the test time of 6 h and 8 h. Furthermore, the transformation process of the three polysaccharide fractions under the metabolism of the gut microbes in vitro and the effect of the polysaccharides on gut microbial community and their metabolites were investigated. It was found that the different gut microbes in the medium containing the polysaccharides could alternate in a rational style, especially the numbers of probiotics of Bifidobacterium spp. and LactobacilluslEnterococcus spp. increased significantly. In addition, the number of Bacteroides-Prevotella spp., Clostridium histolyticum group and Clostridium coccoides-Eubacterium rectale group also increased to some extent. Besides, the degradation process of the polysaccharides and the production of short chain fatty acid (SCFA) were monitored in different fermentation times. The results revealed that the EPS in the medium could be motablised completely by the gut microbes. Meanwhile, the production of SCFA increased rapidly and the ratio of acetic acid to propionic acid and butyric acid decreased significantly, the results demonstated that the polysaccharides in the medium bring a beneficial effect on the production of SCFA.4. Immunomodulatory activity of PHP-3Firstly, the immunomodulatory activities in vitro of PHP-3 were evaluated by using the cell model experiments. It was found that the polysaccharide fraction of PHP-3 had potent in stimulating proliferation of splenocytes, enhancing phagocytosis, nitric oxide production, TNF-α and IL-1β production, and acid phosphatase activity of peritoneal macrophages. The results preliminarily demonstrated the immunostimulatory activity of PHP-3. Then, the immunomodulatory activity of PHP-3 was further investigated by Cyclophosphamide (Cy)-induced immunosuppression mice model. The results showed that the mice of the model group were administered orally with PHP-3 fraction (200 mg/kg) could resist mice spleen index decrease induced by Cy and enhance activity of lysozyme (LZM), the level of hemolysin and the production of TNF-a and IL-1β. These results suggested that PHP-3 possessing the immunomodulatory activity both in non-specific immunity and specific immunity.5. Hepatoprotective activity against alcohol-induced liver injury of PHP-1Hepatoprotective activity against alcohol-induced liver injury of PHP-1 were evaluated by alcohol administration mice model. The results showed that the medication administration team that the mice of model group were administered orally with PHP-1 fraction 200 mg/kg before the dose of 40% alcohol 0.15 mL/g at 2 h everyday for 28 consecutive days could remarkably prevent alcohol-induced elevation of aspartate transaminase (AST), alanine transferase (ALT) and the content of triglyceride (TG) in serum. Meantime, the activity of antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), reduced glutathione peroxidase (GSH-Px), decrease the generation of malondialdehyde (MDA) and the accumulation of TG in liver of the medication administration team were significantly enhanced. The biochemical observations were supplemented by histopathological examination and showed that the volume of lipid droplet in hepatocyte was significantly decreased, meanwhile the degree of pathological change of cellular edema and tissue infiltration were alleviated in the medication administration team. All the results suggested that PHP-1 possessed a relative hepatoprotective effect against alcohol-induced liver injury in mice.
Keywords/Search Tags:Paecilomyces hepiali HN1, Exopolysaccharide, Structure, Modulation on gut microbial community, Immunomodulatory activity, Hepatoprotective activity
PDF Full Text Request
Related items