Studies On Screening, Fermentation, Purification Of Intracellular Polysaccharides And Biological Activities Of Mutant Irpex Lacteus

Irpex lacteus Fr. is a traditional Chinese medicine, which is mainly used for thetreatment of chronic glomerulonephritis. Its mycelia by fermentation are usually usedas raw materials in clinical application. The active ingredients of Irpex lacteus mainlyconsist of proteins, polysaccharides, adenosine, peptides, saponins and mannitol, etc.However, the fermented products are unstable in the industrial process and theirpharmacological activities remain unknown in Irpex lacteus application. In order tosolve these problems, a series of studies were conducted.In this thesis, chemometrics methods were applied first to optimize thesporogenous media and culture conditions of Irpex lacteus. Then Irpex lacteus wastreated by nitrosoguanidine. A desirability function has been developed to screen themutants. The yields of biomass, intracellular adenosine, intracellular mannitol,intracellular polysaccharide have been simultaneously considered in the desirabilityfunction. In addition, the ITS sequence of the mutational strain was identified. ThenRAPD technique was used to investigate the differences at the molecular level fromIrpex lacteus ILN10and wild type strain. Optimum sporogenous medium comprised:peptone3.78g/L, yeast extract2.44g/L, glucose4.93g/L, MgSO₄·7H₂O0.19g/L andVB10.02g/L. The optimal sporogenous culture conditions were as follows: inculum3%, temperature26℃, rotational speed150r/min and fermentation time4days. Themaximum concentration of spore was2.66×105/mL. A mutant, Irpex lacteus ILN10with high genetic stability was obtained, whose desirability function value was1.68fold higher than that of the wild type Irpex lacteus. ITS sequence of Irpexlacteus ILN10were identified as order Aphyllophorales, family Polyporaceae, genusIrpex Fr.. The whole genome of Irpex lacteus ILN10and wild type strain werescanned using RAPD techniques. The results showed that differential band about400 bp was amplified by primer S83, which proved Irpex lacteus ILN10and wild typestrain were differences in the DNA molecular level.The desirability value and chemometrics methods were also applied to optimizethe fermentation medium of Irpex lacteus ILN10. Then the fermentation characteristicof Irpex lacteus ILN10was studied. Optimum fermentation medium consisted of:lactose26.75g/L, yeast extract23.92g/L,(NH₄)2SO₄0.33g/L,KH₂PO₄0.5g/L,MgSO₄·7H₂O0.5g/L,VB10.15g/L. The average desirability value of threevalidation experiments was0.5225,1.99fold as high as that of the original medium.The fermentation characteristics showed that while Irpex lacteus ILN10beingfermented the intracellular adenosine, intracellular mannitol, intracellularpolysaccharide, utilization of lactose and the pH of the fermentation broth changed indynamics.The polysaccharide fractions were purified from Irpex lacteus ILN10byDEAE52cellulose and Sephadex G100chromatography. Two main fractions,ILN3A and ILN3B, were obtained and the chemical characteristics were analyzed.The average molecular weights of the two fractions were2264and2033kDa,respectively. Both of these polysaccharides consisted primarily of rhamnose, mannoseand glucose, and their ratios were1:6.13:1.96and1:5.04:1.87, respectively.Ultraviolet spectrum indicated the absence of protein and nucleic acid in ILN3A andILN3B. Both infrared spectrum of the purified ILN3A and ILN3B displayed apolysaccharide characteristic absorption peak, such as hydroxyl groups, C H groups,C=O groups, C O C groups and C O H groups. And it also proved the existence ofskeletal modes of pyranose rings. The analysis of periodate oxidation smith showedthat ILN3A had a backbone of (1→2) and (1→4) linked rhamnose, mannose andglucose residues, ILN3B had a backbone of (1→2) and (1→4) linked mannoseresidues and (1→3) linked rhamnose and glucose residues.The MTT method was used to evaluate the antitumor and antinephritis activitiesof Irpex lacteus ILN10polysaccharide fractions. It was found that ILN3A and ILN3Bhad noticeable inhibition effects on HepG2tumor cells. However, when comparedwith those of the wild type strain it had no advantage. In addition, the inhibition ability of ILN3A polysaccharide fraction on HBZT1was higher than otherpolysaccharide fractions, whose IC50value was97.28μg/ml.The nephritis rats induced by cationic bovine serum albumin (C BSA) wereused to study the effect of ILN3A polysaccharide fraction on glomerulonephritis.ILN3A could effectively reduce the level of urine protein (UP), total cholesterol (TC),triglycerides (TG) and serum creatinine (Scr) in nephritis rats. ILN3A could alsoeffectively increase the level of serum urea nitrogen (BUN), serum albumin (Alb) and6keto PGF. The mechanism by which ILN3A provides prevention ofglomerulonephritis may be attributable to inhibition of NF κB mediated cytokinepathway. Through the NF κB inhibition, ILN3A is able to down regulate the releaseof IL2, IL6and TNF α, and up regulate IL2R, thus inhibiting the inflammatoryreaction. The pathological analysis confirmed that all of the ILN3A polysaccharidefraction could mitigate the glomerulonephritis. The effect of ILN3A high dose groupwas most obvious, and the results were most close to LeiGongTeng positive controlgroup. The effect of ILN3A medium dose group was lower, but still close toYiShenKang positive control group. The results indicated that ILN3A polysaccharidefraction can effectively improve the nephritis symptoms, alleviate glomerular immuneinflammation and reduce the kidney damage of nephritis rats.